Single domain antibody libraries with maximized antibody developability characteristics

A method for producing an antibody library, comprising:

• (a) providing a first plurality of nucleic acids encoding a population of naturally-replicated CDR1 fragments;
• (b) providing a second plurality of nucleic acids encoding a population of naturally-replicated CDR2 fragments;
• (c) providing a third plurality of nucleic acids encoding a population of naturally-replicated CDR3 fragments;
• (d) providing a nucleic acid gene encoding a common VHH domain comprising heavy chain framework region 1, heavy chain framework region 2, heavy chain framework region 3 and heavy chain framework region 4, and inserting the first plurality of nucleic acids, the second plurality of nucleic acids, and the third plurality of nucleic acids into the CDR1 region, the CDR2 region, and the CDR3 region, respectively, of the common VHH domain, thereby producing a population of nucleic acids encoding a VHH domain library;
• wherein at least 90% of the population of heavy chain CDR1s, and at least 90% of the population of heavy chain CDR2s are completely free of members comprising one or more of:
• (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;
• (hi) an isomerization site comprising the motif of DT, DH, DS, DG, DN, DR, DY, or DD;
• (iv) any cysteine residues;
• (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 poly specificity 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 X1X2X3,wherein each of X1, X2, and X3 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;
• (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 95% of the population of heavy chain CDR1s, the population of heavy chain CDR2s, and the population of heavy chain CDR3s are completely free of nonfunctional members;
• wherein framework 1, framework 2, framework 3, and framework 4 are from a single VHH domain from a therapeutic comprised of one or more VHH domains;
• and
• wherein each framework region can contain up to 5 amino acid substitutions.