Single domain antibody libraries with maximized antibody developability characteristics

1. A method for producing a VHH library, comprising:

(a) providing a first plurality of nucleic acids encoding amino acid sequences of one or more CDR1 fragments replicated from naturally occurring antibodies;

(b) providing a second plurality of nucleic acids encoding amino acid sequences of one or more CDR2 fragments replicated from naturally occurring antibodies;

(c) providing a third plurality of nucleic acids encoding amino acid sequences of one or more CDR3 fragments replicated from naturally occurring antibodies;

(d) providing a nucleic acid gene encoding amino acid sequences of a common VHH domain comprising a framework region 1, a CDR1 region, a framework region 2, a CDR2 region, a framework region 3, a CDR3 region, and a framework region 4; and

(e) assembling 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;

the VHH domain library comprising:

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 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 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 partially humanized VH germline sequence or 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 method of claim 1 , wherein the first plurality of nucleic acids, the second plurality of nucleic acids, and the third plurality of nucleic acids is produced by a process comprising:

(a) obtaining amino acid sequences of the heavy chain CDR1 regions, heavy chain CDR2 regions and heavy chain CDR3 regions of a population of naturally occurring antibodies;

(b) excluding from (a) the heavy chain CDR1 amino acid sequences, the heavy chain CDR2 amino acid sequences, and the heavy chain CDR3 amino acid sequences amino acid sequences that comprise any or all of (i) to (xvi) to obtain sequence liability-free heavy chain CDR1 sequences, sequence liability-free heavy chain CDR2 sequences and sequence liability-free heavy chain CDR3 sequences; and

(c) synthesizing the first plurality of nucleic acids that encode the sequence liability-free heavy chain CDR1 regions, the second plurality of nucleic acids that encode the sequence liability-free heavy chain CDR2 regions, and the third plurality of nucleic acids that encode the sequence liability-free heavy chain CDR3 regions.

3. The method of claim 1 , wherein the human donor lymphocytes are B-cells.

4. The method of claim 1 , wherein the processes for producing the first plurality of nucleic acids, the second plurality of nucleic acids, and the third plurality of nucleic acids further comprise isolating functional members from the liability-free heavy chain CDR1 and CDR2 regions, and from the CDR3 regions, wherein:

(i) the functional members of the sequence liability-free heavy chain CDR1 and CDR2 regions or the functional members of the CDR3 regions are isolated by expressing antibodies comprising the sequence liability-free heavy chain CDR1 and CDR2 regions, and the CDR3 regions in host cells in a manner that the antibodies are displayed on surface of the host cells, isolating the host cells that display antibodies on their surface, and using the CDR1, CDR2, and CDR3 regions in the displayed antibodies, which are functional members of the CDR1, CDR2, and/or CDR3 regions; or

(ii) the functional members of the sequence liability-free heavy chain CDR1 and CDR2 regions, and the CDR3 regions are isolated by expressing antibodies comprising the sequence liability-free heavy chain CDR1 and CDR2 regions, and the CDR3 regions in fusion with a folding reporter, which optionally is β-lactamase or green fluorescent protein, or fragments thereof, to obtain members with improved folding.

5. The method of claim 1 , wherein the mammalian species is human or camelid.