LIBRARIES OF GENETIC PACKAGES COMPRISING NOVEL HC CDR3 DESIGNS

• US 20110118147A1
• Filed: 03/13/2009
• Published: 05/19/2011
• Est. Priority Date: 03/13/2008
• Status: Active Grant

• Current Assignee: Takeda Pharmaceutical Company Limited
• Original Assignee: Dyax Corporation (Shire plc)
• Inventors: Ladner, Robert C.
• Granted Patent: US 9,873,957 B2
• Time in Patent Office: Days
• US Class Current: 506/14
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C07K 16/1275 from Streptococcus (G)
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• 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

Provided are compositions and methods for preparing and identifying antibodies having CDR3s that vary in sequence and in length from very short to very long which in certain embodiments may bind to a carbohydrate moiety or the active site of an enzyme. Libraries coding for antibodies with the CDR3s are also provided. The libraries can be provided by modifying a pre-existing nucleic acid library.

Claims

1. A focused library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, wherein the vectors or genetic packages comprise variegated DNA sequences that encode a heavy chain (HC) CDR3 selected from the group consisting of:

(a) a HC CDR3 that is about 3 or about 4 or about 5 amino acids in length;

(b) a HC CDR3 that is about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34 or about 35 amino acids in length (e.g., about 23 to about 35 amino acids in length); and

c) a HC CDR3 that is from about 6 to about 20 amino acids in length,

wherein the HC CDR3 comprises amino acids from a diversified D region or fragment thereof or an extended JH region.

2. The library of claim 1, wherein the HC CDR3 is enriched in Tyr (Y) and Ser (S).

3. The library of claim 1, wherein the library comprises a D region or a fragment of a D region.

4. The library of claim 3 wherein the D region is selected from the group consisting of D2-2(RF 2), D2-8(RF 2), D2-15(RF 2), D2-21(RF 2), D3-16(RF 2), D3-22 (RF 2), D3-3 (RF-2), D3-9 (RF 2), D3-10 (RF 2), D1-26 (RF 3), D4-11 (RF 2), D4-4 (RF 2), D5-5 (RF 3), D5-12 (RF 3), D5-18 (RF 3), D6-6 (RF1), D6-13 (RF 1), and D6-19 (RF 1).

5. The library of claim 3 wherein the D region comprises one or more cysteine (Cys) residues and the one or more Cys residues are held constant.

6. The library of claim 3, wherein the HC CDR3 comprises one or more filling codons between FR3 and the D region and each filling codon is individually NNK, TMY, TMT, or TMC.

7. The library of claim 3, wherein the HC CDR3 comprises one or more filling codons between the D region and JH and each filling codon is individually NNK, TMY, TMT, or TMC.

8. The library of claim 1, wherein the library further comprises a HC CDR1, HC CDR2, or a light chain and comprises diversity in the HC CDR1, HC CDR2, or light chain.

15. A method of diversifying a library, the method comprising mutagenizing the library of claim 1.

16. The method of claim 15, wherein the mutagenizing comprises error-prone PCR.

17. The method of claim 15, wherein the mutagenizing comprises wobbling.

18. The method of claim 15, wherein the mutagenizing comprises dobbling.

19. The method of claim 15, wherein the mutagenizing introduces on average about 1 to about 10 mutations per HC CDR3.

9. A library comprising a HC CDR3 that is 3, 4, or 5 amino acids in length, wherein the CDR3 comprises amino acids from a JH region or from a D region joined to the FR4 portion of a JH region.

10. The library of claim 9, wherein the HC CDR3 is from a D region joined to the FR4 portion of a JH region and comprises a trimer, a tetramer, or a pentamer, wherein the trimer, tetramer, or pentamer does not comprise a cysteine residue.

11. The library of claim 9, wherein the HC CDR3 is from a D region joined to the FR4 portion of a JH region and comprises a trimer, a tetramer, or a pentamer, wherein the trimer, tetramer, or pentamer does not comprise a stop codon.

12. The library of claim 9, wherein the D region comprises a TAG codon and the TAG codon is replaced by a codon selected from the group consisting of TCG, TTG, TGG, CAG, AAG, TAT, and GAG.

13. The library of claim 9, wherein the D region comprises a TAA codon and the TAA codon is replaced by a codon selected from the group consisting of TCA, TTA, CAA, AAA, TAT, and GAA.

14. The library of claim 9, wherein the D region comprises a TGA codon and the TGA codon is replaced by a codon selected from the group consisting of TGG, TCA, TTA, AGA, and GGA.