Libraries of genetic packages comprising novel HC CDR3 designs
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.
1. A method of diversifying a library, the method comprising mutagenizing 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 method of claim 1 , wherein the mutagenizing comprises error-prone PCR.
3. The method of claim 1 , wherein the mutagenizing comprises wobbling.
4. The method of claim 1 , wherein the mutagenizing comprises dobbling.
5. The method of claim 1 , wherein the mutagenizing introduces on average about 1 to about 10 mutations per HC CDR3.
6. The method of claim 1 , wherein the HC CDR3 is enriched in Tyr (Y) and Ser (S).
7. The method of claim 1 , wherein the library comprises a D region or a fragment of a D region.
8. The method of claim 7 , 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).
9. The method of claim 7 , wherein the D region comprises one or more cysteine (Cys) residues and the one or more Cys residues are held constant.
10. The method of claim 7 , 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.
11. The method of claim 7 , 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.
12. The method 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.