Rationally designed, synthetic antibody libraries and uses therefor

• US 8,877,688 B2
• Filed: 03/13/2009
• Issued: 11/04/2014
• 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): Calamita, Heather
• Assistant Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US12/404,059
• Publication Number:
  US 20100056386A1
• Time in Patent Office: 2,062 Days
• Field of Search: None
• US Class Current: 506/17
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• 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: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• 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, with or without DH segments derived from other species, and are based on rational design informed by examination of publicly available databases of antibody sequences.

Claims

1. A library comprising synthetic polynucleotides that encode an antibody heavy chain or alternative scaffold 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 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, 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, and WGT;

(iii) DH is all of a non-human germline CDRH3 DH amino acid sequence from a vertebrate species;

(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, 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, and WGT; and

(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), and MDV;

wherein the diversity of the polynucleotides encoding the at least about 10⁶ unique antibody CDRH3 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

wherein the alternative scaffold is selected from the group consisting of fibronectin, the β

-sandwich, lipocalin, EETI-IFAGRP, BPTIFLAC1-D1/IT1-D2, thioredoxin, protein A, ankyrin repeats, γ

B-crystallin/ubiquitin, CTLD3 and (LDLR-A module)₃.

2. The library of claim 1, wherein the vertebrate species is selected from the group consisting of Mus musculus, Camelus sp., Llama sp., Camelidae sp., Raja sp., Ginglymostoma sp., Carcharhinus sp., Heterodontus sp., Hydrolagus sp., Ictalurus sp., Gallus sp., Bos sp., Marmaronetta sp., Aythya sp., Netta sp., Equus sp., Pentalagus sp., Bunolagus sp., Nesolagus sp., Romerolagus sp., Brachylagus sp., Sylvilagus sp., Oryctolagus, sp., Poelagus sp., Ovis sp., Sus sp., Gadus sp., Salmo sp., Oncorhynchus sp, Macaca sp., Rattus sp., Pan sp., Hexanchus sp., Heptranchias sp., Notorynchus sp., Chlamydoselachus sp., Heterodontus sp. Pristiophorus sp., Pliotrema sp., Squatina sp., Carcharia sp., Mitsukurina sp., Lamma sp., Isurus sp., Carcharodon sp., Cetorhinus sp., Alopias sp., Nebrius sp., Stegostoma sp., Orectolobus sp., Eucrossorhinus sp., Sutorectus sp., Chiloscyllium sp., Hemiscyllium sp., Brachaelurus sp., Heteroscyllium sp., Cirrhoscyllium sp., Parascyllium sp., Rhincodon sp., Apristurus sp., Atelomycterus sp., Cephaloscyllium sp., Cephalurus sp., Dichichthys sp., Gallus sp., Halaelurus sp., Haploblepharus sp., Parmaturus sp., Pentanchus sp., Poroderna sp., Schroederichthys sp., Scyliorhinus sp., Pseudotriakis sp., Scylliogaleus sp., Furgaleus sp., Hemitriakis sp., Mustelus sp., Triakis sp., Iago sp., Galeorhinus sp., Hypogaleus sp., Chaenogaleus sp., Hemigaleus sp., Paragaleus sp., Galeocerdo sp., Prionace sp., Sciolodon sp., Loxodon sp., Rhizoprionodon sp., Aprionodon sp., Negaprion sp., Hypoprion sp., Carcharhinus sp., Isogomphodon sp., Triaenodon sp., Sphyrna sp., Echinorhinus sp., Oxynotus sp., Squalus sp., Centroscyllium sp., Etmopterus sp., Centrophorus sp., Cirrhigaleus sp., Deania sp., Centroscymnus sp., Scymnodon sp., Dalatias sp., Euprotomicrus sp., Isistius sp., Squaliolus sp., Heteroscymnoides sp., Somniosus sp. and Megachasma sp.

3. The library of claim 1, wherein the CDRH3 amino acid sequences are expressed as part of full-length heavy chains.

4. A library of vectors comprising the polynucleotide library of claim 1.

5. A population of cells comprising the vectors of claim 4.

6. The cells of claim 5, wherein the cells are yeast cells.

7. The cells of claim 5, wherein the cells are S. cerevisiae.

8. A kit comprising a library comprising synthetic polynucleotides that encode an antibody heavy chain or alternative scaffold 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 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, 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, and WGT;

(iii) DH is all of a non-human germline CDRH3 DH amino acid sequence from a vertebrate species;

(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, 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, and 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), and MDV;

wherein the diversity of the polynucleotides encoding the at least about 10⁶ unique antibody CDRH3 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

wherein the alternative scaffold is selected from the group consisting of fibronectin, the β

-sandwich, lipocalin, EETI-IFAGRP, BPTIFLAC1-D1/IT1-D2, thioredoxin, protein A, ankyrin repeats, γ

B-crystallin/ubiquitin, CTLD3 and (LDLR-A module)₃.