DK2264163T3 - Fremgangsmåder til modifikation af eukaryotiske celler - Google Patents

Fremgangsmåder til modifikation af eukaryotiske celler Download PDF

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DK2264163T3
DK2264163T3 DK10010741.6T DK10010741T DK2264163T3 DK 2264163 T3 DK2264163 T3 DK 2264163T3 DK 10010741 T DK10010741 T DK 10010741T DK 2264163 T3 DK2264163 T3 DK 2264163T3
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Andrew J Murphy
George D Yancopoulos
Aris Economides
Margaret Karow
Lynn Macdonald
David Valenzuela
Sean Stevens
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Regeneron Pharma
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Claims (15)

1. En fremgangsmåde til helt at erstatte et endogent variabelt genlocusområde af immunoglobulin med et orthologt humant genlocus, eller til delvis at erstatte en eller flere V- og J- eller V-, D- og J-gensegmenter deraf med orthologe humane V- og J- eller V-, D- og J-gensegmenter, i en embryonal stamcelle (ES) i en mus for at danne et modificeret immunoglobulinlocus, der kan rearrangere til at producere kimære antistoffer, der indeholder humane variable områder og mus konstante områder, hvilken fremgangsmåde omfatter: a) opnåelse af et stort, klonet genomisk fragment, der er større end 20kb, indeholdende orthologe humane V- og J- eller V-, D- og J-gensegmenter; b) anvendelse af bakteriel homolog rekombination til genetisk modifikation af det store, klonede genomiske fragment ifølge a) for at skabe en stor targeting-vektor til anvendelse i den embryonale stamcelle; c) indføring af den store targeting-vektor ifølge b) i den embryonale stamcelle til hel eller delvis erstatning af det endogene variable genlocusområde af immunoglobulin i cellen; og d) påvisning af modifikation af en allel i det endogene variable genlocusområde af immunoglobulin for at identificere celler, der har undergået homolog rekombination medførende hel eller delvis substitution af det endogene variable genlocusområde af immunoglobulin.
2. Fremgangsmåde ifølge krav 1, hvor: (I) den pågældende fremgangsmåde yderligere omfatter: e) opnåelse af et stort, klonet genomisk fragment indeholdende en del af det orthologe humane genlocus, der indeholder V- og J- eller V-, D- og J -gensegmenter, og som adskiller sig fra fragmentet ifølge (a); f) anvendelse af bakteriel homolog rekombination til genetisk modifikation af det klonede genomiske fragment ifølge e) for at danne en stor targetering-vektor til anvendelse i den embryonale stamcelle; g) indføring af den store targeting-vektor ifølge f) i den embryonale stamcelle ifølge trin d) til hel eller delvis erstatning af det endogene, variable genlocus af immunoglobulin i cellen; og h) påvisning af modifikationen af en allel ved det endogene, variable genlocusområde af et immunoglobulin for at identificere celler, der har undergået homolog rekombination til hel eller delvis substitution af det endogene variable genlocusområde af immunoglobulin; eventuelt, hvor trinene (d) til (h) gentages, indtil det endogene variable genlocusområde af immunoglobulin erstattes helt med et orthologt humant genlocus; eller (II) det variable genlocus af immunglobulin omfatter et locus valgt fra gruppen bestående af: a) et variabelt genlocus af kappa-let-kæden; b) et variabelt genlocus af lambda-let-kæden; og c) et variabelt genlocus af den tunge kæde.
3. En fremgangsmåde til helt at erstatte et endogent variabelt genlocusområde af immunoglobulin med et orthologt humant genlocus, eller til delvis at erstatte en eller flere V- og J- eller V-, D- og J-gensegmenter deraf med orthologe humane V- og J- eller V-, D- og J-gensegmenter til dannelse af et modificeret immunoglobulin locus, der kan rearrangere til at producere kimære antistoffer, der indeholder humane variable områder og mus konstante områder, hvilken fremgangsmåde omfatter: a) dannelse af et LTVEC omfattende et stedspecifikt rekombinationssite, en nedstrøms homologiarm indeholdende området umiddelbart op til, men ikke inklusiv, J-segmenterne af det pågældende variable genlocusområde af immunoglobulin, og en opstrøms homologiarm inden for det variable genlocus; b) dannelse af et LTVEC omfattende et stedspecifikt rekombinationssite, en opstrøms homologiarm indeholdende området, der støder op til det mest distale V-gensegment, men ikke indeholder nogle V-gensegmenter af det variable genlocusområde af immunglobulin, og en nedstrøms homologiarm inden for det variable genlocus; c) indføring af LTVECerne ifølge (a) og (b) i en mus embryonal stamcelle (ES) for at danne stedspecifikke rekombinationssteder, der flankerer det endogene variable genlocusområde; d) påvisning af en modifikation af en allel i det endogene variable genlocus af immunoglobulin for at identificere celler i trin c) i hvilke de stedspecifikke rekombinationssteder flankerer det endogene, variable genlocus af immunoglobulin; e) dannelse af en vektor, der indeholder de stedspecifikke rekombinationssekvenser, der flankerer alle, eller en eller flere af V- og J- eller V-, D- og J-gensegmenterne af det orthologe humane genlocus; og f) indføring af vektoren ifølge (e) i en celle ifølge trin (c), således, at det endogene variable genlocusområde af immunoglobulin erstattes helt eller delvis ved rekombination med det orthologe humane genlocus.
4. Et genetisk modificeret, variabelt genlocusområde af immunoglobulin, der kan opnås ved fremgangsmåden ifølge kravene 1 til 3; eller en genetisk modificeret mus embryonal stamcelle omfattende det pågældende, genetisk modificerede variable genlocusområde af immunoglobulin; eller en ikke-human organisme, såsom en mus, omfattende det pågældende, genetisk modificerede variable genlocusområde af immunoglobulin.
5. En mus embryonal stamcelle ifølge krav 4, hvor: a) et mus tung-kæde-locus af det variable område erstattes helt eller delvist med et humant tung-kæde-genlocus af det variable område; eller b) et mus kappa-let-kæde-locus af det variable område erstattes helt eller delvis med et humant kappa-let-kæde-locus af det variable område; eller c) et mus lambda-let-kæde-locus af det variable område erstattes helt eller delvist med et humant lambda-let-kæde-locus af det variable område; eller d) de tunge og lette kæde-loci af det variable område erstattes helt med deres humane homologer eller orthologer.
6. En mus fremstillet fra mus embryonale stamcellen ifølge krav 4 eller 5; eller en transgen mus med et genom, der udelukkende omfatter humane tunge og lette kæde variable lociområder operabelt forbundet til udelukkende endogene mus konstante lociområder, således, at musen producerer et serum, der indeholder et antistof omfattende en human variabel region og en mus konstant region som respons på antigen stimulering; eller en transgen mus med et genom, der omfatter human tung og / eller let kæde variable lociområder operativt koblet til endogene mus konstante lociområder, således, at musen producerer et serum, der indeholder et antistof omfattende et humant variabelt område og et mus konstant område som respons på antigen stimulering.
7. En fremgangsmåde til fremstilling af et humant antistof, omfattende: a) udsættelse af en mus ifølge krav 4 eller 6 for antigen stimulering, således, at musen producerer et antistof mod antigenet; b) isolering af DNAet, der koder de variable områder af de tunge og lette kæder af antistoffet; c) operabel forbindelse af DNAet, der koder de variable områder af (b) med DNA, der koder human tung og let kæde konstante områder, i en celle, der er i stand til at udtrykke aktive antistoffer; d) dyrkning af cellen under sådanne betingelser, at cellen udtrykker det humane antistof; og e) udvinding af antistoffet.
8. Fremgangsmåden ifølge krav 7, hvor: (I) cellen er en CHO-celle; eller (II) det pågældende DNA i trin (b) isoleres fra et hybridom dannet af milten af mus udsat for antigenstimulering i trin (a); eller (III) det pågældende DNA isoleres ved PCR.
9. En fremgangsmåde til at danne, i en mus embryonal stamcelle (ES), et endogent genlocus flankeret nedstrøms af et stedspecifikt rekombinationssted, omfattende: a) at danne et LTVEC omfattende det stedspecifikke rekombinationssted, en nedstrøms homologiarm indeholdende et område, der flankerer 3'-enden af det endogene genlocusområde og en opstrøms homologiarm inden for locuset; b) indføring af LTVEC ifølge (a) i mus ES-cellen; og c) påvisning af en modifikation af en allel på det endogene genlocus, således at man kan identificere de celler ifølge b) i hvilke det endogene genlocus flankeres nedstrøms af det stedspecifikke rekombinationssted.
10. En metode til at danne, i en mus embryonal stamceller (ES), et endogent genlocus flankeret opstrøms af et stedspecifikt rekombinationssted omfattende: a) at danne et LTVEC omfattende det stedspecifikke rekombinationssted, en opstrøms homologiarm indeholdende et område, der flankerer 5'-enden af det endogene genlocusområde og en nedstrøms homologiarm inden for locuset; b) indføring af LTVEC ifølge (a) i mus ES-cellen; og c) påvisning af en modifikation af en allel på det endogene genlocus, således, at man kan identificere de celler ifølge b) i hvilke det endogene genlocus flankeres opstrøms af det stedspecifikke rekombinationssted.
11. En metode til at danne, i en mus embryonal stamcelle (ES), et endogent genlocus flankeret af stedspecifikke rekombinationssteder, omfattende: a) at danne et LTVEC omfattende det stedspecifikke rekombinationssted, en nedstrøms homologiarm indeholdende et område, der flankerer 3'-enden af det endogene genlocusområde og en opstrøms homologiarm inden for locuset; b) at danne et LTVEC omfattende det stedspecifikke rekombinationssted, en opstrøms homologiarm indeholdende et område, der flankerer 5'-enden af det endogene genlocusområde og en nedstrøms homologiarm inden for locuset; c) indføring af LTVECer ifølge (a) og (b) i mus ES-cellen; og d) påvisning af en modifikation af en allel på det endogene genlocus, således, at man kan identificere de celler ifølge c) i hvilke de stedspecifikke recombinationssteder flankeres af det endogene genlocus.
12. En metode til at danne, i en mus embryonal stamcelle (ES), et endogent variabelt genlocus af immunoglobulin flankeret af stedspecifikke rekombinationssteder omfattende: a) at danne et LTVEC omfattende et stedspecifikt rekombinationssted, en nedstrøms homologiarm indeholdende området umiddelbart op til, men ikke inklusiv, J-segmenterne af det variable genlocusområde af et immunglobulin, og en opstrøms homologiarm inden fordet variable genlocus; b) indføring af LTVEC ifølge (a) i mus ES-cellen; og c) påvisning af en modifikation af en allel på det endogene genlocus, således, at man kan identificere de celler ifølge b), hvor de stedspecifikke recombinationssteder flankerer den nedstrøms ende af det endogene variable genlocus af et immunglobulin.
13. En metode til at danne, i en mus embryonal stamcelle (ES), et endogent variabelt genlocus af immunoglobulin flankeret af stedspecifikke rekombinationssteder omfattende: a) at danne et LTVEC omfattende et stedspecifikt rekombinationssted, en opstrøms homologiarm indeholdende området umiddelbart op til det mest distale V-gensegment, men ikke indeholdende nogle V-gensegmenter af det variable genlocusområde af immunglobulinet, og en nedstrøms homologiarm inden for locuset; b) indføring af LTVEC ifølge (a) i mus ES-cellen; og c) påvisning af en modifikation af en allel på det variable genlocus, således, at man kan identificere de celler ifølge b) i hvilke de stedspecifikke recombinationssteder flankerer den opstrøms ende af det endogene variable genlocusområde af et immunglobulin.
14. En metode til at danne, i en mus embryonal stamcelle (ES), et endogent variabelt genlocus af et immunoglobulin flankeret af stedspecifikke rekombinationssteder, omfattende: a) dannelse af et LTVEC omfattende et stedspecifikt rekombinationssted, en nedstrøms homologiarm indeholdende området umiddelbart op til, men ikke inklusiv, J-segmenterne af det variable genlocusområde af immunoglobulin, og en opstrøms homologiarm inden for locuset; b) dannelse af et LTVEC omfattende et stedspecifikt rekombinationssted, en opstrøms homologiarm indeholdende det område, der støder op til det mest distale V-gensegment, men ikke indeholder nogle V-gensegmenter af det variable genlocusområde af immunglobulinet, og en nedstrøms homologiarm inden for det variable genlocus; c) indføring af LTVECerne ifølge (a) og (b) i en mus ES celle; og d) påvisning af en modifikation af en allel på det endogene variable genlocus af immunoglobulin, således, at man kan identificere de celler ifølge c) i hvilke de stedspecifikke rekombinationssteder flankerer det endogene variable genlocusområde af immunoglobulin;
15. Et endogent variabelt genlocus af immunoglobulin som (i) er flankeret af et stedspecifikt rekombinationssted; eller (ii) er flankeret af stedspecifikke rekombinationssteder; eller en mus ES-celle omfattende det pågældende, endogene variable genlocus af et immunoglobulin; eller en mus frembragt af den pågældende mus ES-celle. Figure 1
Figure 2
PIGITRB 3 A 10 20 ' 30 ' 40 .50 60 CCCCOOGCTT CCTGTTCTAA TAAGAATACC TCCTAGGTCC CCCATGGGCT AACCTCATGT GGGGCCCGAA GGACAAGATT ATTCTTATGG AGGATCCAGG SGGTACCCGA TTGGAGTAGA 70 80 SD 100 · 110 120 TTGGTACTCA ACAGGGGTCT TCTTTA.TGAG ‘ CTTCGGACCA 5CTCTTTTGA TGTGGCAGGG AACCATGAGT TGTCCCCAGA AGAAATACTC GAAGCCTGGT CGAGAAAACT ACACCGTCCC 130 140 150 160 ' 170 1BD ACTGACDCTG GGTG.GGGAÅO CCACTCAGTG CATGACCCCA SCTGGTTCAC CACATATACC TGACTGQGAC CCÅCCCCTTC SGTGAGTCAC GTACTGGGGT CGACCAA6TG GTGTATATGG 190 200 210 22□ 230 ACATACTTTT CTTGCAGQTC -TGGGACACAG C ATG CCC CGG GGC CCA GTG GCT GCC • TGTATGAAAA GAACGTCCAG ACCCTGTGTC G TAC GGG GCC CDG GOT CAC CGA CGG Mat Pro Arg· Gly Pro Val Ala Aia> '240 25D 250 270 280 TTA CTC C TG CTG. ATT CTC CAT GGA GCT TGG A5C TGC C TG GAC CTC ACT AAT GAG GAC GAC TAA GAG GTA CCT CGA ACC TCG ACG GAC CTG GAG TGA Lieu Leu Læu Lau Ile lieu Kis Gly Ala Trp Ser Cys Leu Asp Lsu T\hr> 290 300 310 320 330 TGC TAC ACT GAC TAC CTC TGG ACC ATC ACC TGT GTC CTG GAG ACA CGG ACG ATG TGA CTG ATG GAG ACC TGG TAG TGG ACA CAG GAC CTC TGT GCC Cys Tyr Thx Asp Tyr Len Trp Thr Ile Thr Cys Val Leu Glu Thr Arg> 340 350 360 370 A.GC CCC AAC CCC AGC ATA CTC AGT'CTC ACC TGG C AA GAT GAA TAT GAG TCG GGG TTG GGG TCG TAT GAG TCA GAG TGG ACC GTT CTA CTT ATA CTC Sar Pro Asn Pro Ser Ile Lsu Sar Lau Thr Trp Gin Asp Giv Tyr Glu> - 380 390 400 410 420 GAA CTT CAG GAC C AA GAG ACC TTC TGC AGC CTA· CAC AÅG TCT GGC C.SC CTT GAA GTC CTG GTT CTC TGG AAG, ACG ’TCG' GAT. GTG TTC AGA CCG GTG’ Glu Leu Gin Asp Gin Glu Thr Phe Cys :Ser Leu His Lys Sar Gly' Hiss- 43 0 440 4.50 460 470 AAC .ACC ACA CAT ATA TGG TAC ACG TGC CAT ATG CGC TTG TCT C AA TTC TTG TGG TGT GTA TAT ACC ATG TGC ACG GTA TAC GCG AAC AGA GTT AAG Asn Thr Thr· His Ile Trp Tyr Thr Cys His Met Arg Lau Ser Gin Phs> 4B0 490 500 51D 520 CTG TCC GAT GAA GTT TTC ATT GTC AAC GTG ACG GAC CÅG TCT GGC AAC . GAC AGG CTA CTT' C AA AAG TAA CAG TTG CAC TGC CTG GTC AGA CCG TTG Leu Sar Asp Gin Val Phe Ile Val Asn Val Thr-Asp Gin Sax Qly Asn> ,330 540 , 550 560 570 AAC TCC C AA GAG TGT GGC AGC TTT GTC CTG GCT GAG AGC ATC AAG CCA TTG AGG GTT CTC ACA CCG TCG AAA CAG GAQ CGA CTC TCG TAG TTC GGT Asn Ser Gin Glu Cys Giv Ser She Val Leu Ala Glu Ser Ile Lys Pro> PIGCRS 33 . 580 590 . 600 .Sio· GCT CCC CCC TTG AAC GTG ACT G TG GCC TTC TCA GGA CSC TAT SAT ATC CGA GGG GGG AAC TTG CAC TGÅ ' CAC CGG AAG ÅGT CCT GCG ATA CTA TAG. Ala Pro Pro leu Asa Val Thr Val Ala Phe Ssr Sly Arg Tyr Asp Ile> 62D 530 540 550 ggo TCC TGG GAC TCA GOT TAT GAC GAA' CCC TCC AAC TAC GTG CTG ÅGA GGC AGG ACC C TG AGT CGA ATA C TG CTT GGG .AGG TTG ATG CAC GAC TCT CCG Ser' Trp' Asp Ser Ala Tyr Asp G lu Pro Ssr Asn Tyr Val Lea Arg Gly> 670 680 690 700 710 ÅAG CTA CAA TAT GAG CTG CAG TAT CGG .AAC CTC AGA GAC CCC .TAT GCT TTC SAT QTT „ATA CTC GAC GTC ATA GCC 'TTG GAG TCT DTG GGG ATA CGA Lys Lsu Gin Tyr Glu Leu Gin Tyr· Arg Asn Lau Arg Asp Pro Tyr Ala> 720 730 740 750 ?£0 GTG ÅGS CCS GTG ACC AAG C TG ATC TCA GTG GAC TCÅ AGA AAC GTC TCT CAC TCC GGC CAC CGG TTC GAC TAG AGT CAC C TG ÅGT TCT TTG CAG AGA Val Arg Pro Val Thr Lys Lau Ils Ssr Val Asp Ser Arg „isn Val Sar? 770 7 BO 790 8DD . 810 CTT CTC CCC GAA GÅG TTC CAC AAA GAT TCT AGC TAC CAG CTG CAG ATG GAA GAG 3GA CTT CTC AÅG GTG TTT CTA AGA TCG ATG GTC GAC GTC TAC Lsu Lsu. Pro Giu G lu Piae His Lys Asp Ser Ser Tyr Gin Lsu Gin Met> ' 820 S30 840 B50 CGG GCA GCG CCT CAG CCA GGC ACT TCÅ TTC AGG GGG ACC TGG AGT GAG GCC CGT CGC GGA GTC GGT CCG TGA AGT AAG TCC CCC TGG ACC TCA CTC Arg Ala Ala Pro Gin Pro Gly Thr Ser Phe Arg Qly Thr Torp Ser Glu> 860 ' 870 880 890 300 TOG AGT GÅC CCC GTC ATC TTT CAG ACC CAG GCT GGG GAG CCC GAG GCA ACC TCA CTG GGG CAG TAG. AAA GTC TGG GTC CGA CCC CTC GGG CTC CGT Trp Ser Asp Pro Val .Ils ?he Gin Thr Gin Ala. Gly slu Pro Gin Ala> 910 920 930 940 - . S5G GGC TGG GAC CCT CAC ATG CTG CTG CTC CTG GCT GTC TTG ATC ATT GTC CCG ACC CTG GGA GTG TAC GAC G.AC GAG GAC CGA CAG AAC TAG ΤΆΆ CAG Gly Trp Asp Pro Kis Met Leu Lsu Leu Leu Ala Val Leu Ile Ils Val> 960 970 ' 980 920 100O CTG GTT TTC A.TG GGT CTG AÅG ATC CAC CTG OCT TSS AGG CTA TGG AAA GAC CAA AAG TAC CCA GAC TTC TAG GTG GAC GGA ACC TCC GAT ACC TTT Lau Val Pia Mat Gly Lsu Lys Ile His Lau Pro Trp Arg Ley Trp Lys? 'lOlO . 1020 1030 1040 1050 AÅG ATA TGG GCA CCA GTG CCC ÅCC CCT GAG ÅGT TTC TTC· CAG CCC CTG TTC TAT ACC CGT GGT CAC GGG TGG GGA CTC TCA AAG AAG GTC GGG GAC Lys Ile Trp Ala Pro Val Pro Thr Pro Gin Ssr Phe Phe Gin Pro Lsu> FSGOTlS 3 C 1060 1070 108-0 1050 TAC AGG GAG CAC. AGC GGG AAC PTC AAG ΆΑΑ TGG STT AAT ACC CCT TTC ATG TCC CTC G TG TCG CCC TTG AAG TTC TTT ACC CAA TTA TGO GGA AAG Tyr Arg Glu His Bar Gly .Asn Plis Lys Lys Trp Val Asn Thr Pro Phe> 1100 1110 1120 1130 1140 ACG GCC TCC AGC ATA GAG TTG GTG CCA CAG AGT TCC ACA ACA ACA TCA TG C CGG AGG TCG TAT CTC AAC CAC GGT GTC TCA AGG TGT TGT TGT AGT Thr Ala Ser Ser Ila Glu Lau Val Pro Gin Ser Ser Thr Thr Thr Ser> 1150 , 1160 1170 1130 . 1190 GCC TTA CAT C TG TCA TTG TAT CCA GCC' AAG' GAD AAG AAG TTC CCG GGG CGG AAT GTA GAC AGT AAC ATA GGT. CGG TTC CTC 'TTC TTC AAG GGC CCC Ala Leu His Leu Ser Leu Tyr Pro Ala Lys Glu -.Lys Lys Phs Pro Gly* 1200 1210 1220 1230 124D CTQ CCG GGT CTG G AA GAG CAA CTG GAG TGT GAT GGA ATG TCT GAG CCT GAC GGC CCA GAC C TT CTC GTT GAC CTC ACA CTA CCT TAC AGA CTC GGA Leu Pro Gly Leu Glu Glu Gin Leu Glu Cys Asp Gly Met Ser Glu Pro* 1250 1260 1270 . 1280 1290 GOT CAC TGG TGC ATA ATC COG TTG GCA GCT GGC CAA GCG GTC TCA GCC CCA GTG ACC ACG TAT TAG GGG AAC- CGT CGA CCG GTT CGC CAG AGT CGG Gly His Trp Cys Ile Xla Pro Leu Ala Ala Gly Gin Ala Val Ser Ala> 13 00 1310 1320 1330. . . TAC AGT GAG GAG AGA GAC CGG CCA TAT GGT CTG GTG TCC ATT GAC ACA ATG TCA CTC CTC TCT CTG GCC GGT ATA CCA GAC CAC AGG TAA CTG TGT ' Tyr Ser Glu Glu Arg .Asp Arg Pro Tyr Gly Lau Val Ser Ile Asp Thr> 1340 ' 1350 1360 1370 1360 GTG ACT GTG GGA GAT GCA GAG GGC CTG TGT GTC TGG CCC TGT AGC TGT , CAC TGA CAC* CCT CTA CGT CTC CCG GAC ACA. CAG ACC GGG ACA TCG ACA Val .Thr Val Gly Asp Ala 'Glu Gly Leu Cys Val Tip Pro Cys Ser Cys> 1380 1400 1410 1420 .1430 . GAG GAT GAT GGC TAT CCA GCC ATG AAC CTG GAT GCT GGC AGA GAG TCT CTC CTA CTA CCG ATA GGT CGG TAC TTG GAC CTA CGA CCG TCT CTC AGA Glu Asp Asp Gly' Tyr Pro Ala Met Asn Lau Asp Ala Gly Arg Glu 3sr> 1440 1450 . 1460 1470 14B0 GGT CCT AAT TCA GAG GAT CTG CTC TTG GTC ACA GAC CCT GCT TTT CTG CCA GGA TTA AGT CTC CTA GAC GAS AAC CAG TGT CTG GGA CGA AAA GAC Gly Pro Asn Ser Glu .Asp Lau Lsu Len Val Thr Asp Pro Ala Phs Lsu> 1450 1500 1S10 ’ 1520 . 1530 TCT TGT GGC TGT GTC TCA GGT AGT GGT CTC AGG. CTT GGG GGC TCC CCA AGA ACA CCG ACA CAG AGT CCA TCA CCA GAG TCC GAA CCC CCG AGG GGT Ser Cys Gly Cys Val Ser Gly Sar Gly Leu Arg Leu Gly Sly Ser Pro* Pig-urs 3D 1540 155-0 1550 1570 GGC AGC CIA C TG GAD AGG TTG AGG C TG TCA TTT GCA AAG GAA GGG GAC-C CG TCG GAT GAC CTQ TCC AAC TCC GAC AGT AAA CGT TTC C TT CCC C TG G ly Ser Læm 1» au Asp Arg Leu Arg L-stt Ser Plis Ala Lys Glu Gly Asp> 1580 1590 1600 1510 1620 TGG ACA Ga GAC CCA ACC TG G AGA ACT- GGG TCC CCA GGA 5GG GGC TCT ACC TGT' C GT C TG GGT TGG .ACC TCT TGÅ CCC AGG GGT CCT CCC CCG AGA Trp Tiiy Ala Asp Pro Thr Tarp Arg Tar Gly Ssr Pro Gly Gly Gly Sera- 1630 · 1640 1650 . 1560 1670 , GAG AGT GAA GCA GGT TCC CCC CCT GGT CTG GAC ATS GAC ACA TTT GAC CTC TCA C TT 'CCT CCÅ AGG GGG GGA CCA GAC CTG TAC 'CTG .TGT' AAA CTG Glu Ssr Glu Ala Gly Ser Pro Pro Gly L>eu Asp .iset Asp Thr" ?ls Asp> 1580' . 1690 ·_ 1700 1710 1720 AGT GGC TTT GCA 'GGT TCA GAC TGT GGC AGC CCC GTG GAG ACT GAT GAA TCA CCG AAA CGT CCA AGT CTG ACA CCG TOG GGG CAC CTC TGA CTA C TT Ser Gly Phe Ala Gly Ser Asp Cys Gly Ssr Pro Val Glu Thr Asp Glu>' 1730 1740 1750 1760 1770 GGA CCC CCT C GÅ ACC TAT CTC CGC CAS TGG GTG GTC AGS ACC CCT CCÅ CCT GGG GGA GCT TCG ATA GAG GOS GTC ACC CAC CAG TCC TGG GGA GGT Gly Pro Pro Arg Ser Tyr Lau Arg Gin Trp Val Val Arg Thr Pro' Pro> 1780 1790 1-300 CCT GTG GAC AGT GGA GCC CAG AGC AGC TAG GGA CAC CTG TCA CCT CGG GTC TCG TCG ATC Pro Val Asp Ssr Gly Åla Gin Sar „Ssr ***> Figure 4A Human Ig heavy chain locus (total length ^IMb, not drawn to scale):
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US20130130388A1 (en) 2013-05-23
DE14163642T1 (de) 2014-10-09
JP5345463B2 (ja) 2013-11-20
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US20110283376A1 (en) 2011-11-17
JP2004524841A (ja) 2004-08-19
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US20140017781A1 (en) 2014-01-16
DE14172437T1 (de) 2014-11-27
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EP2786657B1 (en) 2018-02-07
ES2660749T3 (es) 2018-03-26
US10526630B2 (en) 2020-01-07
CZ305619B6 (cs) 2016-01-13
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US20140023637A1 (en) 2014-01-23
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US20140020124A1 (en) 2014-01-16
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US9528136B2 (en) 2016-12-27
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US20110258710A1 (en) 2011-10-20
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CA2438390A1 (en) 2002-08-29
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