Rationally designed, synthetic antibody libraries and uses therefor

• US 10,189,894 B2
• Filed: 01/08/2014
• Issued: 01/29/2019
• Est. Priority Date: 09/14/2007
• Status: Active Grant

• Current Assignee: Adimab, LLC
• Original Assignee: Adimab, LLC
• Inventors: Vasquez, Maximiliano, Feldhaus, Michael, Gerngross, Tillman U., Wittrup, K. Dane
• Primary Examiner(s): Boesen, Christian C
• Application Number:
  US14/150,129
• Publication Number:
  US 20140364340A1
• Time in Patent Office: 1,847 Days
• Field of Search: None
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/567 Framework region [FR]
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

The present invention overcomes the inadequacies inherent in the known methods for generating libraries of antibody-encoding polynucleotides by specifically designing the libraries with directed sequence and length diversity. The libraries are designed to reflect the preimmune repertoire naturally created by the human immune system and are based on rational design informed by examination of publicly available databases of human antibody sequences.

Claims

1. An antibody isolated from a library comprising synthetic polynucleotides that encode an antibody heavy chain and an antibody light chain containing at least about 10⁶ unique antibody CDRH3 amino acid sequences, wherein each of the polynucleotides encoding the at least about 10⁶ unique antibody CDRH3 amino acid sequences has an antibody CDRH3 amino acid sequence represented by the following formula:

[X]-[N1]-[DH]-[N2]-[H3-JH], wherein

(i) X is selected from the group consisting of G, D, E and nothing;

(ii) N1 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, IG, KG, GW, FP, EG, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT;

(iii) DH is all of a human CDRH3 DH amino acid sequence, wherein the DH amino acid sequence is selected from the group consisting of ATT, GAT, GIV, IVG, VGA, VLLLWLLL (SEQ ID NO;

     239), ITMIVVVIT (SEQ ID NO;

     240), VQWLV (SEQ ID NO;

     241), VQQLV (SEQ ID NO;

     242), VLRFLEWLLY (SEQ ID NO;

     243), YYDFWSGYYT (SEQ ID NO;

     244), WILYQLLC (SEQ ID NO;

     245), LRL (SEQ ID NO;

     246), TTVT (SEQ ID NO;

     247), VWELL (SEQ ID NO;

     248), VDTAMVT (SEQ ID NO;

     249), WIQLWL (SEQ ID NO;

     250), RILWWLLL (SEQ ID NO;

     251), DIVVVVAAT (SEQ ID NO;

     252), VLLLWFG (SEQ ID NO;

     595), GATT (SEQ ID NO;

     623), GIVG (SEQ ID NO;

     624), IVGA (SEQ ID NO;

     625), VGAT (SEQ ID NO;

     626), GIVGA (SEQ ID NO;

     627), IVGAT (SEQ ID NO;

     628), VGATT (SEQ ID NO;

     629), GIVGAT (SEQ ID NO;

     630), IVGATT (SEQ ID NO;

     631), GIVGATT (SEQ ID NO;

     13), YSG, YSGS (SEQ ID NO;

     632), YSGSY (SEQ ID NO;

     633), YSGSYY (SEQ ID NO;

     14), CSSTSC (SEQ ID NO;

     622), CSSTSCY (SEQ ID NO;

     620), YCSSTSC (SEQ ID NO;

     621), CSSTSCYT (SEQ ID NO;

     617), GYCSSTSC (SEQ ID NO;

     618), YCSSTSCY (SEQ ID NO;

     619), GYCSSTSCY (SEQ ID NO;

     615), YCSSTSCYT (SEQ ID NO;

     616), GYCSSTSCYT (SEQ ID NO;

10), AAM, DIV, IVY, PAA, VPA, VVP, VVV, DIVV (SEQ ID NO;

     634), IVVV (SEQ ID NO;

     635), PAAM (SEQ ID NO;

     636), VPAA (SEQ ID NO;

637, VVPA (SEQ ID NO;

     638), VVVP (SEQ ID NO;

     639), DIVVY (SEQ ID NO;

     640), IVVVP (SEQ ID NO;

     641), VPAAM (SEQ ID NO;

     642), VVPAA (SEQ ID NO;

     643), VVVPA (SEQ ID NO;

     644), DIVVVP (SEQ ID NO;

     645), IVVVPA (SEQ ID NO;

     646), VVPAAM (SEQ ID NO;

     647), VVVPAA (SEQ ID NO;

     648), DIVVVPA (SEQ ID NO;

     649), IVVVPAA (SEQ ID NO;

     650), VVVPAAM (SEQ ID NO;

     651), DIVVVPAA (SEQ ID NO;

     652), IVVVPAAM (SEQ ID NO;

     653), DIVVVPAAM (SEQ ID NO;

     11), CSGGSC (SEQ ID NO;

     654), CSGGSCY (SEQ ID NO;

     655), YCSGGSC (SEQ ID NO;

     656), CSGGSCYS (SEQ ID NO;

     657), GYCSGGSC (SEQ ID NO;

     658), YCSGGSCY (SEQ ID NO;

     659), GYCSGGSCY (SEQ ID NO;

     660), YCSGGSCYS (SEQ ID NO;

     661), GYCSGGSCYS (SEQ ID NO;

     16), FGV, GVV, IFG, ITI, TIF, VVI, FGVV (SEQ ID NO;

     662) , GVVI (SEQ ID NO;

     663), IFGV (SEQ ID NO;

     664), ITIF (SEQ ID NO;

     665) , TIFG (SEQ ID NO;

     666), VVII (SEQ ID NO;

     667), FGVVI (SEQ ID NO;

     668), GVVII (SEQ ID NO;

     669), IFGVV (SEQ ID NO;

     670), ITIFG (SEQ ID NO;

     671), TIFGV (SEQ ID NO;

     672), FGVVII (SEQ ID NO;

     673), IFGVVI (SEQ ID NO;

     674), ITIFGV (SEQ ID NO;

     675), TIFGVV(SEQ ID NO;

     676), IFGVVII (SEQ ID NO;

     677), ITIFGVV (SEQ ID NO;

     678), TIFGVVI (SEQ ID NO;

     679), ITIFGVVI (SEQ ID NO;

     680), TIFGVVII(SEQ ID NO;

     681), ITIFGVVII (SEQ ID NO;

     9), ELL, FGE, GEL, LLW, LWF, VLL, WFG, FGEL (SEQ ID NO;

     682), GELL (SEQ ID NO;

     683), LLWF (SEQ ID NO;

     684), LWFG (SEQ ID NO;

     685), VLLW (SEQ ID NO;

     686), WFGE (SEQ ID NO;

     687), FGELL (SEQ ID NO;

     688), LLWFG (SEQ ID NO;

     689), LWFGE (SEQ ID NO;

     690), VLLWF (SEQ ID NO;

     691), WFGEL (SEQ ID NO;

     692), LLWFGE (SEQ ID NO;

     693), LWFGEL (SEQ ID NO;

     694), VLLWFG (SEQ ID NO;

     695), WFGELL (SEQ ID NO;

     696), LLWFGEL (SEQ ID NO;

     697), LWFGELL (SEQ ID NO;

     698), VLLWFGE (SEQ ID NO;

     699), LLWFGELL (SEQ ID NO;

     700), VLLWFGEL (SEQ ID NO;

     701), VLLWFGELL (SEQ ID NO;

     1), GSG, GSY, SGS, SYY, YGS, YYG, YYN, YYY, GSGS (SEQ ID NO;

     702), GSYY (SEQ ID NO;

     703), SGSY (SEQ ID NO;

     704) , SYYN (SEQ ID NO;

     705), YGSG (SEQ ID NO;

     706), YYGS (SEQ ID NO;

     707), YYYG (SEQ ID NO;

     708), GSGSY (SEQ ID NO;

     709), GSYYN (SEQ ID NO;

     710), SGSYY (SEQ ID NO;

     711), YGSGS (SEQ ID NO;

     712), YYGSG (SEQ ID NO;

     713), YYYGS (SEQ ID NO;

     714), GSGSYY (SEQ ID NO;

     715), SGSYYN (SEQ ID NO;

     716), YGSGSY (SEQ ID NO;

     717), YYGSGS (SEQ ID NO;

     718), YYYGSG (SEQ ID NO;

     719), GSGSYYN (SEQ ID NO;

     720), YGSGSYY (SEQ ID NO;

     721), YYGSGSY (SEQ ID NO;

     722), YYYGSGS (SEQ ID NO;

     723), YGSGSYYN (SEQ ID NO;

     724), YYGSGSYY (SEQ ID NO;

     725), YYYGSGSY (SEQ ID NO;

     726), YYGSGSYYN (SEQ ID NO;

     727), YYYGSGSYY (SEQ ID NO;

     728), YYYGSGSYYN (SEQ ID NO;

     2), GVI, ITM, MVR, RGV, TMV, VII, VRG, GVII (SEQ ID NO;

     729), ITMV (SEQ ID NO;

     730), MVRG (SEQ ID NO;

     731), RGVI (SEQ ID NO;

     732), TMVR (SEQ ID NO;

     733), VRGV (SEQ ID NO;

     734), ITMVR (SEQ ID NO;

     735), MVRGV (SEQ ID NO;

     736), RGVII (SEQ ID NO;

     737), TMVRG (SEQ ID NO;

     738), VRGVI (SEQ ID NO;

     739), ITMVRG (SEQ ID NO;

     740), MVRGVI (SEQ ID NO;

     741), TMVRGV (SEQ ID NO;

     742), VRGVII (SEQ ID NO;

     743), ITMVRGV (SEQ ID NO;

     744), MVRGVII (SEQ ID NO;

     745), TMVRGVI (SEQ ID NO;

     746), ITMVRGVI (SEQ ID NO;

     747), TMVRGVII (SEQ ID NO;

     748), ITMVRGVII (SEQ ID NO;

     3), DSS, GYY, SGY, SSG, YDS, YYD, DSSG (SEQ ID NO;

     749), GYYY (SEQ ID NO;

     750), SGYY (SEQ ID NO;

     751), SSGY (SEQ ID NO;

     752), YDSS (SEQ ID NO;

     753), YYDS (SEQ ID NO;

     754), YYYD (SEQ ID NO;

     755), DSSGY (SEQ ID NO;

     756), SGYYY (SEQ ID NO;

     757), SSGYY (SEQ ID NO;

     758), YDSSG (SEQ ID NO;

     759), YYDSS (SEQ ID NO;

     760), YYYDS (SEQ ID NO;

     761), DSSGYY (SEQ ID NO;

     762), SSGYYY (SEQ ID NO;

763, YDSSGY (SEQ ID NO;

     764), YYDSSG (SEQ ID NO;

     765), YYYDSS (SEQ ID NO;

     766), DSSGYYY (SEQ ID NO;

     767), YDSSGYY (SEQ ID NO;

     768), YYDSSGY (SEQ ID NO;

     769), YYYDSSG (SEQ ID NO;

     770), YDSSGYYY (SEQ ID NO;

     771), YYDSSGYY (SEQ ID NO;

     772), YYYDSSGY (SEQ ID NO;

     773), YYDSSGYYY (SEQ ID NO;

     774), YYYDSSGYY (SEQ ID NO;

     775), YYYDSSGYYY (SEQ ID NO;

     4), DYG, GDY, YGD, DYGD (SEQ ID NO;

     613), YGDY (SEQ ID NO;

     614), DYGDY (SEQ ID NO;

     12), SYG, YGY, YSY, GYSY (SEQ ID NO;

     776), SYGY (SEQ ID NO;

     777), YSYG (SEQ ID NO;

     778), GYSYG (SEQ ID NO;

     779), YSYGY (SEQ ID NO;

     780), GYSYGY (SEQ ID NO;

     15), SSS, SSW, SWY, SSSW (SEQ ID NO;

     781), SSWY (SEQ ID NO;

     782), YSSS (SEQ ID NO;

     783), GYSSS (SEQ ID NO;

     784), SSSWY (SEQ ID NO;

     785), YSSSW(SEQ ID NO;

     786), GYSSSW (SEQ ID NO;

     787), YSSSWY (SEQ ID NO;

     788), GYSSSWY (SEQ ID NO;

     7) , GWY, GYS, SGW, YSS, GYSS (SEQ ID NO;

     789), SGWY (SEQ ID NO;

     790), SSGW (SEQ ID NO;

     791), YSSG (SEQ ID NO;

     792), GYSSG (SEQ ID NO;

     793), SSGWY (SEQ ID NO;

     794), YSSGW (SEQ ID NO;

     795), GYSSGW (SEQ ID NO;

     796), YSSGWY (SEQ ID NO;

     797), GYSSGWY (SEQ ID NO;

     5), AVA, GIA, IAV, VAG, AVAG (SEQ ID NO;

     798), GIAV (SEQ ID NO;

     799), IAVA (SEQ ID NO;

     800), GIAVA (SEQ ID NO;

     801), IAVAG (SEQ ID NO;

     802), GIAVAG (SEQ ID NO;

     6), AAA, AAG, IAA, AAAG (SEQ ID NO;

     803), GIAA (SEQ ID NO;

     804), IAAA (SEQ ID NO;

     805), GIAAA (SEQ ID NO;

     806), IAAAG (SEQ ID NO;

     807), and GIAAAG (SEQ ID NO;

     8);

(iv) N2 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, IG, KG, GW, FP, EG, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT;

(v) H3-JH is all of a human CDRH3 H3-JH amino acid sequence, wherein the H3-JH amino acid sequence is selected from the group consisting of AEYFQH (SEQ ID NO;

     17), EYFQH (SEQ ID NO;

     583), YFQH (SEQ ID NO;

     584), FQH, QH, H YWYFDL (SEQ ID NO;

     18), WYFDL (SEQ ID NO;

     585), YFDL (SEQ ID NO;

     586), FDL, DL, L, AFDV (SEQ ID NO;

     19), FDV, DV, V, YFDY (SEQ ID NO;

     20), FDY, DY, Y, NWFDS (SEQ ID NO;

     21), WFDS (SEQ ID NO;

     587), FDS, DS, S, YYYYYGMDV (SEQ ID NO;

     22), YYYYGMDV (SEQ ID NO;

     588), YYYGMDV (SEQ ID NO;

     589), YYGMDV (SEQ ID NO;

     590), YGMDV (SEQ ID NO;

     591), GMDV (SEQ ID NO;

     592), MDV, AFDI (SEQ ID NO;

     852), FDI, DI, NWFDP (SEQ ID NO;

     857), WFDP (SEQ ID NO;

     858), FDP, and DP;

wherein the diversity of the polynucleotides encoding the at least about 10⁶ unique antibody CDRH3 amino acid sequences is created by each of the polynucleotides having a CDRH3 sequence that is different from the CDRH3 sequence of every other polynucleotide;

wherein the antibody heavy chain is a variable domain with framework (FRM) and complementary determining regions (CDR) comprising FRMH1-CDRH1-FRM2-CDRH2-FRMH3-CDRH3-FRMH4; and

a VKCDR3 amino acid sequence comprising about 1 to about 10 of the amino acids found at Kabat positions 89, 90, 91, 92, 93, 94, 95, 95A, 96, and 97, in selected VKCDR3 amino acid sequences derived from a particular IGKV or IGKJ germline sequence;

wherein the antibody light chain is a variable domain with framework (FRM) and complementary determining regions (CDR) comprising FRMVK1-VKCDR1-FRMVK2-VKCDR2-FRMVK3-VKCDR3-FRMVK4.

2. The antibody of claim 1, wherein the antibody is expressed in a heterodimeric form.

3. The antibody of claim 1, wherein the antibody is expressed as an antibody fragment.

4. The antibody of claim 1, wherein the antibody is selected from the group consisting of a Fab fragment, a Fab′

fragment, a F(ab′

)₂ fragment, an Fv fragment, a diabody, linear antibody, and a single-chain antibody.

5. An antibody isolated from a library comprising synthetic polynucleotides that encode an antibody heavy chain and an antibody light chain containing at least about 10⁶ unique antibody CDRH3 amino acid sequences, wherein each of the polynucleotides encoding the at least about 10⁶ unique antibody CDRH3 amino acid sequences has an antibody CDRH3 amino acid sequence represented by the following formula:

[X]-[N1]-[DH]-[N2]-[H3-JH], wherein

(i) X is selected from the group consisting of G, D, E and nothing;

(ii) N1 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, IG, KG, GW, FP, EG, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT;

(iii) DH is all of a human CDRH3 DH amino acid sequence, wherein the DH amino acid sequence is selected from the group consisting of ATT, GAT, GIV, IVG, VGA, VLLLWLLL (SEQ ID NO;

     239), ITMIVVVIT (SEQ ID NO;

     240), VQWLV (SEQ ID NO;

     241), VQQLV (SEQ ID NO;

     242), VLRFLEWLLY (SEQ ID NO;

     243), YYDFWSGYYT (SEQ ID NO;

     244), WILYQLLC (SEQ ID NO;

     245), LRL (SEQ ID NO;

     246), TTVT (SEQ ID NO;

     247), VWELL (SEQ ID NO;

     248), VDTAMVT (SEQ ID NO;

     249), WIQLWL (SEQ ID NO;

     250), RILWWLLL (SEQ ID NO;

     251), DIVVVVAAT (SEQ ID NO;

     252), VLLLWFG (SEQ ID NO;

     595), GATT (SEQ ID NO;

     623), GIVG (SEQ ID NO;

     624), IVGA (SEQ ID NO;

     625), VGAT (SEQ ID NO;

     626), GIVGA (SEQ ID NO;

     627), IVGAT (SEQ ID NO;

     628), VGATT (SEQ ID NO;

     629), GIVGAT (SEQ ID NO;

     630), IVGATT (SEQ ID NO;

     631), GIVGATT (SEQ ID NO;

     13), YSG, YSGS (SEQ ID NO;

     632), YSGSY (SEQ ID NO;

     633), YSGSYY (SEQ ID NO;

     14), CSSTSC (SEQ ID NO;

     622), CSSTSCY (SEQ ID NO;

     620), YCSSTSC (SEQ ID NO;

     621), CSSTSCYT (SEQ ID NO;

     617), GYCSSTSC (SEQ ID NO;

     618), YCSSTSCY (SEQ ID NO;

     619), GYCSSTSCY (SEQ ID NO;

     615), YCSSTSCYT (SEQ ID NO;

     616), GYCSSTSCYT (SEQ ID NO;

10), AAM, DIV, IVY, PAA, VPA, VVP, VVV, DIVV (SEQ ID NO;

     634), IVVV (SEQ ID NO;

     635), PAAM (SEQ ID NO;

     636), VPAA (SEQ ID NO;

637, VVPA (SEQ ID NO;

     638), VVVP (SEQ ID NO;

     639), DIVVY (SEQ ID NO;

     640), IVVVP (SEQ ID NO;

     641), VPAAM (SEQ ID NO;

     642), VVPAA (SEQ ID NO;

     643), VVVPA (SEQ ID NO;

     644), DIVVVP (SEQ ID NO;

     645), IVVVPA (SEQ ID NO;

     646), VVPAAM (SEQ ID NO;

     647), VVVPAA (SEQ ID NO;

     648), DIVVVPA (SEQ ID NO;

     649), IVVVPAA (SEQ ID NO;

     650), VVVPAAM (SEQ ID NO;

     651), DIVVVPAA (SEQ ID NO;

     652), IVVVPAAM (SEQ ID NO;

     653), DIVVVPAAM (SEQ ID NO;

     11), CSGGSC (SEQ ID NO;

     654), CSGGSCY (SEQ ID NO;

     655), YCSGGSC (SEQ ID NO;

     656), CSGGSCYS (SEQ ID NO;

     657), GYCSGGSC (SEQ ID NO;

     658), YCSGGSCY (SEQ ID NO;

     659), GYCSGGSCY (SEQ ID NO;

     660), YCSGGSCYS (SEQ ID NO;

     661), GYCSGGSCYS (SEQ ID NO;

     16), FGV, GVV, IFG, ITI, TIF, VVI, FGVV (SEQ ID NO;

     662) , GVVI (SEQ ID NO;

     663), IFGV (SEQ ID NO;

     664), ITIF (SEQ ID NO;

     665) , TIFG (SEQ ID NO;

     666), VVII (SEQ ID NO;

     667), FGVVI (SEQ ID NO;

     668), GVVII (SEQ ID NO;

     669), IFGVV (SEQ ID NO;

     670), ITIFG (SEQ ID NO;

     671), TIFGV (SEQ ID NO;

     672), FGVVII (SEQ ID NO;

     673), IFGVVI (SEQ ID NO;

     674), ITIFGV (SEQ ID NO;

     675), TIFGVV(SEQ ID NO;

     676), IFGVVII (SEQ ID NO;

     677), ITIFGVV (SEQ ID NO;

     678), TIFGVVI (SEQ ID NO;

     679), ITIFGVVI (SEQ ID NO;

     680), TIFGVVII(SEQ ID NO;

     681), ITIFGVVII (SEQ ID NO;

     9), ELL, FGE, GEL, LLW, LWF, VLL, WFG, FGEL (SEQ ID NO;

     682), GELL (SEQ ID NO;

     683), LLWF (SEQ ID NO;

     684), LWFG (SEQ ID NO;

     685), VLLW (SEQ ID NO;

     686), WFGE (SEQ ID NO;

     687), FGELL (SEQ ID NO;

     688), LLWFG (SEQ ID NO;

     689), LWFGE (SEQ ID NO;

     690), VLLWF (SEQ ID NO;

     691), WFGEL (SEQ ID NO;

     692), LLWFGE (SEQ ID NO;

     693), LWFGEL (SEQ ID NO;

     694), VLLWFG (SEQ ID NO;

     695), WFGELL (SEQ ID NO;

     696), LLWFGEL (SEQ ID NO;

     697), LWFGELL (SEQ ID NO;

     698), VLLWFGE (SEQ ID NO;

     699), LLWFGELL (SEQ ID NO;

     700), VLLWFGEL (SEQ ID NO;

     701), VLLWFGELL (SEQ ID NO;

     1), GSG, GSY, SGS, SYY, YGS, YYG, YYN, YYY, GSGS (SEQ ID NO;

     702), GSYY (SEQ ID NO;

     703), SGSY (SEQ ID NO;

     704) , SYYN (SEQ ID NO;

     705), YGSG (SEQ ID NO;

     706), YYGS (SEQ ID NO;

     707), YYYG (SEQ ID NO;

     708), GSGSY (SEQ ID NO;

     709), GSYYN (SEQ ID NO;

     710), SGSYY (SEQ ID NO;

     711), YGSGS (SEQ ID NO;

     712), YYGSG (SEQ ID NO;

     713), YYYGS (SEQ ID NO;

     714), GSGSYY (SEQ ID NO;

     715), SGSYYN (SEQ ID NO;

     716), YGSGSY (SEQ ID NO;

     717), YYGSGS (SEQ ID NO;

     718), YYYGSG (SEQ ID NO;

     719), GSGSYYN (SEQ ID NO;

     720), YGSGSYY (SEQ ID NO;

     721), YYGSGSY (SEQ ID NO;

     722), YYYGSGS (SEQ ID NO;

     723), YGSGSYYN (SEQ ID NO;

     724), YYGSGSYY (SEQ ID NO;

     725), YYYGSGSY (SEQ ID NO;

     726), YYGSGSYYN (SEQ ID NO;

     727), YYYGSGSYY (SEQ ID NO;

     728), YYYGSGSYYN (SEQ ID NO;

     2), GVI, ITM, MVR, RGV, TMV, VII, VRG, GVII (SEQ ID NO;

     729), ITMV (SEQ ID NO;

     730), MVRG (SEQ ID NO;

     731), RGVI (SEQ ID NO;

     732), TMVR (SEQ ID NO;

     733), VRGV (SEQ ID NO;

     734), ITMVR (SEQ ID NO;

     735), MVRGV (SEQ ID NO;

     736), RGVII (SEQ ID NO;

     737), TMVRG (SEQ ID NO;

     738), VRGVI (SEQ ID NO;

     739), ITMVRG (SEQ ID NO;

     740), MVRGVI (SEQ ID NO;

     741), TMVRGV (SEQ ID NO;

     742), VRGVII (SEQ ID NO;

     743), ITMVRGV (SEQ ID NO;

     744), MVRGVII (SEQ ID NO;

     745), TMVRGVI (SEQ ID NO;

     746), ITMVRGVI (SEQ ID NO;

     747), TMVRGVII (SEQ ID NO;

     748), ITMVRGVII (SEQ ID NO;

     3), DSS, GYY, SGY, SSG, YDS, YYD, DSSG (SEQ ID NO;

     749), GYYY (SEQ ID NO;

     750), SGYY (SEQ ID NO;

     751), SSGY (SEQ ID NO;

     752), YDSS (SEQ ID NO;

     753), YYDS (SEQ ID NO;

     754), YYYD (SEQ ID NO;

     755), DSSGY (SEQ ID NO;

     756), SGYYY (SEQ ID NO;

     757), SSGYY (SEQ ID NO;

     758), YDSSG (SEQ ID NO;

     759), YYDSS (SEQ ID NO;

     760), YYYGS (SEQ ID NO;

     761), DSSGYY (SEQ ID NO;

     762), SSGYYY (SEQ ID NO;

763, YDSSGY (SEQ ID NO;

     764), YYDSSG (SEQ ID NO;

     765), YYYDSS (SEQ ID NO;

     766), DSSGYYY (SEQ ID NO;

     767), YDSSGYY (SEQ ID NO;

     768), YYDSSGY (SEQ ID NO;

     769), YYYDSSG (SEQ ID NO;

     770), YDSSGYYY (SEQ ID NO;

     771), YYDSSGYY (SEQ ID NO;

     772), YYYDSSGY (SEQ ID NO;

     773), YYDSSGYYY (SEQ ID NO;

     774), YYYDSSGYY (SEQ ID NO;

     775), YYYDSSGYYY (SEQ ID NO;

     4), DYG, GDY, YGD, DYGD (SEQ ID NO;

     613), YGDY (SEQ ID NO;

     614), DYGDY (SEQ ID NO;

     12), SYG, YGY, YSY, GYSY (SEQ ID NO;

     776), SYGY (SEQ ID NO;

     777), YSYG (SEQ ID NO;

     778), GYSYG (SEQ ID NO;

     779), YSYGY (SEQ ID NO;

     780), GYSYGY (SEQ ID NO;

     15), SSS, SSW, SWY, SSSW (SEQ ID NO;

     781), SSWY (SEQ ID NO;

     782), YSSS (SEQ ID NO;

     783), GYSSS (SEQ ID NO;

     784), SSSWY (SEQ ID NO;

     785), YSSSW(SEQ ID NO;

     786), GYSSSW (SEQ ID NO;

     787), YSSSWY (SEQ ID NO;

     788), GYSSSWY (SEQ ID NO;

     7) , GWY, GYS, SGW, YSS, GYSS (SEQ ID NO;

     789), SGWY (SEQ ID NO;

     790), SSGW (SEQ ID NO;

     791), YSSG (SEQ ID NO;

     792), GYSSG (SEQ ID NO;

     793), SSGWY (SEQ ID NO;

     794), YSSGW (SEQ ID NO;

     795), GYSSGW (SEQ ID NO;

     796), YSSGWY (SEQ ID NO;

     797), GYSSGWY (SEQ ID NO;

     5), AVA, GIA, IAV, VAG, AVAG (SEQ ID NO;

     798), GIAV (SEQ ID NO;

     799), IAVA (SEQ ID NO;

     800), GIAVA (SEQ ID NO;

     801), IAVAG (SEQ ID NO;

     802), GIAVAG (SEQ ID NO;

     6), AAA, AAG, IAA, AAAG (SEQ ID NO;

     803), GIAA (SEQ ID NO;

     804), IAAA (SEQ ID NO;

     805), GIAAA (SEQ ID NO;

     806), IAAAG (SEQ ID NO;

     807), and GIAAAG (SEQ ID NO;

     8);

(iv) N2 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, IG, KG, GW, FP, EG, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT;

(v) H3-JH is all of a human CDRH3 H3-JH amino acid sequence, wherein the H3-JH amino acid sequence is selected from the group consisting of AEYFQH (SEQ ID NO;

     17), EYFQH (SEQ ID NO;

     583), YFQH (SEQ ID NO;

     584), FQH, QH, H YWYFDL (SEQ ID NO;

     18), WYFDL (SEQ ID NO;

     585), YFDL (SEQ ID NO;

     586), FDL, DL, L, AFDV (SEQ ID NO;

     19), FDV, DV, V, YFDY (SEQ ID NO;

     20), FDY, DY, Y, NWFDS (SEQ ID NO;

     21), WFDS (SEQ ID NO;

     587), FDS, DS, S, YYYYYGMDV (SEQ ID NO;

     22), YYYYGMDV (SEQ ID NO;

     588), YYYGMDV (SEQ ID NO;

     589), YYGMDV (SEQ ID NO;

     590), YGMDV (SEQ ID NO;

     591), GMDV (SEQ ID NO;

     592), MDV, AFDI (SEQ ID NO;

     852), FDI, DI, NWFDP (SEQ ID NO;

     857), WFDP (SEQ ID NO;

     858), FDP, and DP;

wherein the diversity of the polynucleotides encoding the at least about 10⁶ unique antibody CDRH3 amino acid sequences is created by each of the polynucleotides having a CDRH3 sequence that is different from the CDRH3 sequence of every other polynucleotide;

wherein the antibody heavy chain is a variable domain with framework (FRM) and complementary determining regions (CDR) comprising FRMH1-CDRH1-FRMH2-CDRH2-FRMH3-CDRH3-FRMH4; and

a VKCDR3 amino acid sequence of at least about 80% identity to an amino acid sequence represented by the following formula;

[VK_Chassis]-[L3-VK]-[X]-[JK*], wherein;

(a) VK_Chassis is an amino acid sequence selected from the group consisting of about Kabat amino acid 1 to about Kabat amino acid 88 encoded by IGKV1-05, IGKV1-06, IGKV1-08, IGKV1-09, IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-16, IGKV1D-17, IGKV1D-43, IGKV1D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-40, IGKV2D-26, IGKV2D-29, IGKV2D-30, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07, IGKV3D-11, IGKV3D-20, IGKV4-1, IGKV5-2, IGKV6-21, and IGKV6D-41, or a sequence of at least about 80% identity to any of them;

(b) L3-VK is the portion of the VKCDR3 encoded by the IGKV gene segment; and

(c) X is any amino acid residue; and

(d) JK* is an amino acid sequence selected from the group consisting of amino acid sequences encoded by IGJK1, IGJK2, IGJK3, IGJK4, and IGJK5, wherein the first residue of each IGJK amino acid sequence is not present;

wherein the antibody light chain is a variable domain with framework (FRM) and complementary determining regions (CDR) comprising FRMVK1-VKCDR1-FRMVK2-VKCDR2-FRMVK3-VKCDR3-FRMVK4.

6. The antibody according to claim 5 wherein the antibody is expressed in heterodimeric form.

7. The antibody according to claim 5, wherein the antibody is a full-length antibody, a Fab fragment, a Fab′

fragment, a F(ab′

)₂ fragment, an Fv fragment, a diabody, a linear antibody, and a single-chain antibody.