Single domain antibody libraries with maximized antibody developability characteristics

1. A VHH antibody library, comprising:

nucleic acids encoding a framework region 1, a framework region 2, a framework region 3, and a framework region 4; and

a plurality of nucleic acids encoding a population of VHH domains comprising one or more CDR1s, one or more CDR2s, and one or more CDR3s located at the CDR1 region, the CDR2 region, and the CDR3 region of a VHH gene, respectively;

wherein the nucleic acid sequences encoding the amino acid sequences of the one or more CDR1s and the one or more CDR2s are from naturally-occurring antibodies of a mammalian species;

wherein at least 90% of the one or more CDR1s and at least 90% of the one or more 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 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; 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 95% of the one or more CDR1s, at least 95% of the one or more CDR2s, and at least 95% of the one or more CDR3s are free of non-functional members; wherein functional members are well-folded and can form well-folded VHHs;

wherein the framework regions 1, 2, 3, and 4 are from a single therapeutic antibody VHH; wherein the therapeutic antibody VHH is selected from the group consisting of Caplacizumab, Envafolimab, Gontivimab, Isecarosmab, Ozoralizumab, Sonelokimab, and Vobarilizumab;

wherein each 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 CDR3s are from heavy chain CDR3s of human donor lymphocytes.

2. The VHH antibody library of claim 1 , wherein the nucleic acid sequences encoding the amino acid sequences of the CDR1s, the CDR2s, and the CDR3s are synthesized.

3. The VHH antibody library of claim 1 , wherein the CDR1 comprises human sequences.

4. The VHH antibody library of claim 3 , wherein the CDR2 comprises human sequences.