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US 2022/0177555 A1Published application
METHOD OF PREPARING pH-DEPENDENT ANTIBODIES
Assigneeargenx BVBA
InventorsChristophe BLANCHETOT, Erik HOFMAN, Johannes DE HAARD, Jacobus Cornelis RASSER
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Abstract
Methods for preparing engineered antibodies exhibiting improved pH-dependent antigen binding are disclosed. The methods are based on introduction of histidine residues at a subset of defined amino acid positions within the antibody CDRs. The set of amino acid positions selected for histidine substitution is derived from a heat-map of histidine occurrence within the CDRs of functional antibodies from a natural antibody repertoire. The methods provide a simpler and less time-consuming approach to the identification of pH-dependent antibody variants.
Claims27
A method of preparing an engineered antibody exhibiting pH-dependent binding to its antigen, comprising preparing the engineered antibody by substituting at least one amino acid residue of a parental antibody with a histidine residue, wherein at least one amino acid residue selected from the following hot-spot list is substituted with histidine:
- VH CDR1
- H31, H32, H33, H35
- VH CDR2
- H50, H52, H52a, H53, H56, H58, H59, H62, H63
- VH CDR3
- H95, H96, H97, H98, H99, H100, H100a, H100b, H100c,
- H100d, H100e, H100f, H100h, H100i, H100j, H100l,
- H101, H102
- VL CDR1
- L27, L27d, L29, L30, L31, L32, L34
- VL CDR2
- L51, L52, L53, L54, L55
- VL CDR3
- L89, L90, L91, L92, L93, L94, L95a, L95b, L95c, L96
- FR
- L49, L87
- and at least one amino acid residue from the following cold-spot list is not substituted with histidine:
- VH CDR1
- H34, H35a, H35b, H35c
- VH CDR2
- H51, H52b, H52c, H54, H55, H57, H60, H61, H64, H65
- VH CDR3
- H100g, H100k, H100m, H100n
- VL CDR1
- L24, L25, L26, L27a, L27b, L27c, L27e, L28, L33
- VL CDR2
- L50, L51a, L51b, L51c, L51d, L56
- VL CDR3
- L95, L95d, L95e, L95f, L97
- wherein said engineered antibody exhibits pH-dependent binding to its antigen.
27 more elements
A method of preparing an engineered antibody exhibiting pH-dependent binding to its antigen, comprising the steps of:
- (a) providing a parental antibody which binds to an antigen;
- (b) preparing a panel of engineered variants of said parental antibody, wherein each of said engineered variants in said panel differs from said parental antibody by substitution of at least one amino acid residue with a histidine residue, wherein at least one amino acid residue selected from the following hot-spot list is substituted with histidine:
- VH CDR1
- H31, H32, H33, H35
- VH CDR2
- H50, H52, H52a, H53, H56, H58, H59, H62, H63
- VH CDR3
- H95, H96, H97, H98, H99, H100, H100a, H100b, H100c,
- H100d, H100e, H100f, H100h, H100i, H100j, H100l,
- H101, H102
- VL CDR1
- L27, L27d, L29, L30, L31, L32, L34
- VL CDR2
- L51, L52, L53, L54, L55
- VL CDR3
- L89, L90, L91, L92, L93, L94, L95a, L95b, L95c, L96
- FR
- L49, L87
- and at least one amino acid residue from the following cold-spot list is not substituted with histidine:
- VH CDR1
- H34, H35a, H35b, H35c
- VH CDR2
- H51, H52b, H52c, H54, H55, H57, H60, H61, H64, H65
- VH CDR3
- H100g, H100k, H100m, H100n
- VL CDR1
- L24, L25, L26, L27a, L27b, L27c, L27e, L28, L33
- VL CDR2
- L50, L51a, L51b, L51c, L51d, L56
- VL CDR3
- L95, L95d, L95e, L95f, L97
- (c) screening said panel of engineered variants for pH dependent binding to said antigen and thereby identifying an engineered antibody exhibiting pH-dependent binding to said antigen.
29 more elements
16-22. (canceled)
A method of preparing an engineered antibody exhibiting pH dependent binding to its antigen, comprising the steps of:
- (a) identifying a parental antibody which binds to an antigen;
- (b) preparing a panel of engineered variants of said parental antibody, wherein each of said engineered variants in said panel differs from said parental antibody by substitution of at least one amino acid residue with a histidine residue, wherein at least one amino acid residue selected from the following hot-spot list is substituted with histidine:
- VH CDR1
- H31, H32, H33, H35
- VH CDR2
- H50, H52, H52a, H53, H56, H58, H59, H62, H63
- VH CDR3
- H95, H96, H97, H98, H99, H100, H100a, H100b, H100c,
- H100d, H100e, H100f, H100h, H100i, H100j, H100l,
- H101, H102
- VL CDR1
- L27, L27d, L29, L30, L31, L32, L34
- VL CDR2
- L51, L52, L53, L54, L55
- VL CDR3
- L89, L90, L91, L92, L93, L94, L95a, L95b, L95c, L96
- FR
- L49, L87
- and at least one amino acid residue from the following cold-spot list is not substituted with histidine:
- VH CDR1
- H34, H35a, H35b, H35c
- VH CDR2
- H51, H52b, H52c, H54, H55, H57, H60, H61, H64, H65
- VH CDR3
- H100g, H100k, H100m, H100n
- VL CDR1
- L24, L25, L26, L27a, L27b, L27c, L27e, L28, L33
- VL CDR2
- L50, L51a, L51b, L51c, L51d, L56
- VL CDR3
- L95, L95d, L95e, L95f, L97
- (c) screening said panel of engineered variants for pH dependent binding to said target antigen and thereby identifying selected amino acid positions at which the presence of histidine confers pH dependent binding to said antigen;
- (d) preparing one or more further engineered variants of said parental antibody, wherein each of said variants comprises histidine at two or more selected amino acid positions identified in step (c);
- and
- (e) screening said further engineered variants for pH dependent binding to said antigen;
- and thereby identifying an engineered antibody exhibiting pH dependent binding to said antigen.
33 more elements
25-31. (canceled)
A method of preparing an engineered antibody exhibiting pH dependent binding to its antigen, comprising the steps of:
- (a) identifying a parental antibody which binds to an antigen;
- (b) preparing a first panel of engineered variants of said parental antibody, wherein each of said engineered variants in said panel differs from said parental antibody by substitution of at least one amino acid residue in the VH domain with a histidine residue, wherein at least one amino acid residue selected from the following hot-spot list is substituted with histidine:
- VH CDR1
- H31, H32, H33, H35,
- VH CDR2
- H50, H52, H52a, H53, H56, H58, H59, H62, H63,
- VH CDR3
- H95, H96, H97, H98, H99, H100, H100a, H100b, H100c,
- H100d, H100e, H100f, H100h, H100i, H100j, H100l, H101, H102
- and at least one amino acid residue from the following cold-spot list is not substituted with histidine:
- VH CDR1
- H34, H35a, H35b, H35c
- VH CDR2
- H51, H52b, H52c, H54, H55,
- H57, H60, H61, H64, H65
- VH CDR3
- H100g, H100k, H100m, H100n
- (c) screening said first panel of engineered variants for pH dependent binding to said antigen;
- and thereby identifying one or more selected amino acid positions in the VH domain at which the presence of histidine confers pH dependent binding;
- (d) preparing a second panel of engineered variants of said parental antibody, wherein each of said engineered variants in said panel differs from said parental antibody by substitution of at least one amino acid residue in the VL domain with a histidine residue, wherein at least one amino acid residue selected from the following hot-spot list is substituted with histidine:
- VL CDR1
- L27, L27d, L29, L30, L31, L32, L34
- VL CDR2
- L51, L52, L53, L54, L55
- VLCDR3
- L89, L90, L91, L92, L93, L94, L95, L95a,
- L95b, L95c, L95d, L95e, L95f, L96, L97
- and at least one amino acid residue selected from the following cold-spot list is not substituted with histidine:
- VL CDR1
- L24, L25, L26, L27a, L27b, L27c, L27e, L28, L33
- VL CDR2
- L50, L51a, L51b, L51c, L51d, L56
- VL CDR3
- L95, L95d, L95e, L95f, L97
- (e) screening said second panel of engineered variants for pH dependent binding to said antigen;
- and thereby identifying one or more selected amino acid positions in the VL domain at which the presence of histidine confers pH dependent binding;
- (f) preparing one or more further engineered variants of said parental antibody, wherein each of said variants differs from said parental antibody by substitution of the amino acid at one or more of the selected amino acid positions in the VH domain identified in step (c) with histidine and by substitution of the amino acid at one or more of the selected amino acid positions in the VL domain identified in step (e) with histidine;
- and
- (g) screening said further engineered variants for pH dependent binding to said antigen, thereby identifying an engineered antibody exhibiting pH-dependent binding to said antigen.
36 more elements
(canceled)
(canceled)
36-42. (canceled)
An engineered antibody which exhibits pH dependent binding to its antigen wherein at least one amino acid in the CDRs of the engineered antibody is a histidine residue, characterised in that at least one amino acid residue selected from the following hot-spot list is a histidine residue:
- VH CDR1
- H31, H32, H33, H35
- VH CDR2
- H50, H52, H52a, H53, H56, H58, H59, H62, H63
- VH CDR3
- H95, H96, H97, H98, H99, H100, H100a, H100b, H100c,
- H100d, H100e, H100f, H100h, H100i, H100j, H100l,
- H101, H102
- VL CDR1
- L27, L27d, L29, L30, L31, L32, L34
- VL CDR2
- L51, L52, L53, L54, L55
- VL CDR3
- L89, L90, L91, L92, L93, L94, L95a, L95b, L95c, L96
- FR
- L49, L87
- and at least one amino acid residue selected from the following cold-spot list is not a histidine residue:
- VH CDR1
- H34, H35a, H35b, H35c
- VH CDR2
- H51, H52b, H52c, H54, H55, H57, H60, H61, H64, H65
- VH CDR3
- H100g, H100k, H100m, H100n
- VL CDR1
- L24, L25, L26, L27a, L27b, L27c, L27e, L28, L33
- VL CDR2
- L50, L51a, L51b, L51c, L51d, L56
- VL CDR3
- L95, L95d, L95e, L95f, L97
26 more elements
45-55. (canceled)
Dependencies are inferred from the claim text.