Sequence-based high throughput method generating camelids antibodies to cover broad epitopes with high-resolution

A method generating a camelid antibody specific for an antigen, comprising:

• a) enriching and proliferating B-cells from immunized camelids specific to the antigen,
• b) generating antibody NGS libraries comprising VHH2, VHH3, VH1 and VL1 chain sequences from said antigen-specific B-cells,
• c) grouping sequences of VHH2, VHH3, VH1 and VL1 in the NGS libraries by lineages,
• d) pairing VH1/VL1 lineage according to anchor binders generated by single B-cell sorting and heterohybridoma approaches,
• e) subgrouping the lineages and lineage pairs in step d), respectively, by VHH unique features selected from a group consisting of
• i. FR2 hydrophilic region,
• ii. extended CDR1,
• iii. extra disulfide bond between CDR1-CDR3 or FR2-CDR3,
• iv. extra disulfide bond within CDR3,
• v. long CDR3 (12 amino acids or more),
• vi. extra disulfide bond within CDR1,
• vii. non-classic VHH which have the same V and J germlines as conventional IgG1,
• viii. non-classic VHH which have predetermined sequence signatures,
• ix. predetermined novel canonical binding loop structure,
• x. convergent motif or sequence signature,
• xi. long CDR2 (14-17 amino acids);
• xii. absence of 3 or more continuing positive charges in CDR3;
• xiii. absence of positive charge at N-terminus;
• xiv. even number of cysteines in the sequence;
• and
• xv. 2-4 amino acid motifs in the CDR regions, wherein the motif is identified from the 3D structures of a ligand/receptor complex;
• f) ranking lineages and lineage pairs from step c) and step d) by one or more lineage priority factors,
• g) selecting a representative sequence or sequence pair from a lineage of VHH2 or VHH3 and a lineage pair of VH1/VL1 respectively with a top ranking from step f) in the NGS library,
• h) testing an antibody comprising the selected sequence pair or sequence from step
• g) to determine if the antibody binds to the antigen or portion thereof.