| CPC C40B 40/10 (2013.01) [C12N 15/1037 (2013.01); C12N 15/81 (2013.01); C40B 40/08 (2013.01); C40B 50/06 (2013.01)] | 5 Claims |
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1. An antibody library, comprising a first plurality of nucleic acids and a second plurality of nucleic acids,
wherein the first plurality of nucleic acids encodes amino acid sequences of a population of antibody heavy chain variable domains, which collectively comprise a heavy chain framework region 1, a heavy chain framework region 2, a heavy chain framework region 3, a heavy chain framework region 4, one or more heavy chain CDR1s, one or more heavy chain CDR2s, and one or more heavy chain CDR3s located at the CDR1 region, the CDR2 region, and the CDR3 region of an antibody heavy chain variable domain gene, respectively, wherein amino acid sequences of the one or more heavy chain CDRIs, the one or more heavy chain CDR2s, and the one or more heavy chain CDR3s are from naturally occurring human antibodies,
wherein the second plurality of nucleic acids encodes the amino acid sequences of a population of light chain variable domains, which collectively comprise a light chain framework region 1, a light chain framework region 2, a light chain framework region 3, a light chain framework region 4, one or more light chain CDR1s, one or more light chain CDR2s, and one or more light chain CDR3s located at the CDR1 region, the CDR2 region, and the CDR3 region of an antibody light chain variable domain gene, respectively, wherein amino acid sequences of the one or more light chain CDR1s, the one or more light chain CDR2s, and the one or more light chain CDR3s, are from naturally occurring human antibodies,
wherein at least 90% of the one or more heavy chain CDR1s and at least 90% of the one or more heavy chain CDR2s are free of amino acid sequence liabilities, wherein the amino acid sequence liabilities are: (i) a glycosylation site comprising the motif NXS, NXT, or NXC, in which X represents any naturally occurring amino acid residue except for proline; (ii) a deamidation site comprising the motif of NG, NS, NT, NN, NA, NH, ND, NQ, NF, NW or NY; (iii) an isomerization site comprising the motif of DT, DH, DS, DG, DN, DR, DY or DD; (iv) any cysteines; (v) net charge greater than 1; (vi) a tripeptide motif containing at least two residues with aromatic side chains comprising F, H, W or Y; (vii) a polyspecificity site comprising the motif GG, GGG, RR, VG, W, WV, WW, WWW, YY, or WXW, in which X represents any amino acid residue; (viii) a protease sensitive or hydrolysis prone site comprising the motif of DX, in which X is P, G, S, V, Y, F, Q, K, L, or D; (ix) an integrin binding site comprising RGD, RYD, LDV, or KGD; (x) a lysine glycation site comprising KE, EK, or ED; (xi) a metal catalyzed fragmentation site comprising the motif of HS, SH, KT, HXS, or SXH, in which X represents any amino acid residue; (xii) a polyspecificity aggregation site comprising a motif of X.sub.1X.sub.2X.sub.3, wherein each of X.sub.1, X.sub.2, and X.sub.3 is independently selected from the group consisting of F, I, L, V, W and Y; (xiii) a streptavidin binding motif comprises the motif HPQ, EPDW (SEQ ID NO: 49), PWXWL (SEQ ID NO: 50), in which X represents any amino acid residue, GDWVFI (SEQ ID NO: 51), or PWPWLG (SEQ ID NO: 52); (xiv) one or more arginine residues; (xv) a hydrophobic CDR sequence; and/or (xvi) a CDR mutation that reduces binding to protein A said CDR mutation comprising any mutation in the last amino acid of the CDR2, according to the IMGT definition, to A, G, C, D, E, F, G, H, I, L, M, N, P, Q, S, V, W or Y;
wherein at least 90% of the one or more heavy chain CDR1s, at least 90% of the one or more heavy chain CDR2s, and at least 90% of the one or more heavy chain CDR3s are free of non-functional members; wherein functional members are well folded and can form well folded scFvs or Fabs;
wherein at least two of the heavy chain framework regions 1, 2, 3, and 4 are from a human, humanized or partially humanized VH germline sequence framework;
wherein each heavy chain framework region can contain up to five amino acid substitutions, and
wherein the nucleic acid sequences encoding the amino acid sequences of the one or more heavy chain CDR3s are from heavy chain CDR3s of human donor lymphocytes.
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