Vh-vl-interdomain angle based antibody humanization

A method for selecting one or more variant antibody Fv fragments derived from a parent antibody Fv fragment comprising the following steps:

• a) generating a multitude of variant antibody Fv fragments by grafting/transferring one or more specificity determining residues from the parent antibody Fv fragment on an acceptor antibody Fv fragment, whereby each variant antibody Fv fragment of the multitude of variant antibody Fv fragments differs from the other variant antibody Fv fragments by at least one amino acid residue,
• (b) determining the VH-VL-orientation for the parent Fv fragment and for each of the variant antibody Fv fragments of the multitude of variant antibody Fv fragments based on a sequence fingerprint of the antibody Fv fragment,
• (c) selecting those variant antibody Fv fragments that have the smallest difference in the VH-VL-orientation compared to the parent antibody's VH-VL-orientation and thereby selecting one or more variant antibody Fv fragments derived from a parent antibody Fv fragment,
• whereby the one or more variant antibody Fv fragments bind to the same antigen as the parent antibody Fv fragment.

A method for producing an antibody comprising the following steps:

• (a) selecting one or more antibodies or antibody Fv fragments comprising the following steps:
• (i) generating a multitude of variant antibodies by grafting/transferring one or more specificity determining residues from a non-human antibody on a human or humanized acceptor antibody or germline antibody sequence, whereby each variant antibody of the multitude of variant antibodies differs from the other variant antibodies by at least one amino acid residue,
• (ii) determining the VH-VL-orientation for the non-human antibody Fv fragment and for each of the variant antibody's Fv fragments of the multitude of variant antibodies by calculating the habitual torsion angle, the four bend angles (two per variable domain), and the length of the pivot axis of VH and VL (HL, HC1, LC1, HC2, LC2, dc) using a random forest model based on a set of VH-VL-interface residues consisting of residues H33, H35, H37, H39, H43, H44, H45, H46, H47, H50, H55, H56, H58, H60, H61, H62, H89, H91, H95, H96, H98, H99, H100x-2, H100x-1, H100x, H101, H102, H103, H105, L32, L34, L36, L38, L41, L42, L43, L44, L45, L46, L49, L50, L53, L55, L56, L85, L87, L89, L91, L93, L94/L95x-1, L95x, L96, L97, L100 (numbering according to Chothia index) of the antibody Fv fragment,
• (iii) selecting those variant antibody Fv fragments that have the smallest average root-mean-square deviation (RMSD) determined for all pairs of corresponding Calpha atoms of the non-human antibody Fv fragment and variant antibody Fv fragment,
• (b) selecting from the one or more antibodies a single antibody based on its binding properties,
• (c) cloning the VH and VL encoding nucleic acids into one or more expression vectors,
• (d) transfecting a cell with the expression vectors obtained in the previous step,
• (e) cultivating the transfected cell and thereby producing the antibody.