Method of preparing pH-dependent antibodies

1. A method of preparing an engineered antibody exhibiting pH-dependent binding to its antigen, comprising the steps of:

(a) preparing a panel of engineered variants of a parental antibody which binds to antigen, wherein each of the engineered variants in the panel differs from the parental antibody by substitution of at least one hot-spot amino acid residue with a histidine residue, wherein the at least one hot-spot amino acid residue is selected from the following hot-spot list:

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 each of the engineered variants in the panel comprises an Fc region;

(b) screening the panel of engineered variants for pH dependent binding to the antigen and thereby identifying an engineered antibody exhibiting pH-dependent binding to the antigen.

2. The method of claim 1 , wherein the engineered antibody identified in part (b) has lower affinity for its antigen at acidic pH than at neutral pH.

3. A method of preparing an engineered antibody exhibiting pH dependent binding to its antigen, comprising the steps of:

(a) preparing a panel of engineered variants of a parental antibody which binds to an antigen, wherein each of the engineered variants in the panel differs from the parental antibody by substitution of at least one hot-spot amino acid residue with a histidine residue, wherein the at least one hot-spot amino acid residue is selected from the following hot-spot list:

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 each of the engineered variants in the panel comprises an Fc region;

(b) screening the panel of engineered variants for pH dependent binding to the target antigen and thereby identifying selected amino acid positions at which the presence of histidine confers pH dependent binding to the antigen;

(c) preparing one or more further engineered variants of the parental antibody, wherein each of the variants comprises histidine at two or more selected amino acid positions identified in step (b), wherein each of the engineered variants in the panel comprises an Fc region; and

(d) screening the further engineered variants for pH dependent binding to the antigen; and thereby identifying an engineered antibody exhibiting pH dependent binding to the antigen.

4. The method of claim 3 , wherein the engineered antibody identified in part (d) has lower affinity for its antigen at acidic pH than at neutral pH.

5. A method of preparing an engineered antibody exhibiting pH dependent binding to its antigen, comprising the steps of:

(a) preparing a first panel of engineered variants of a parental antibody which binds to an antigen, wherein each of the engineered variants in the panel differs from the parental antibody by substitution of at least one hot-spot amino acid residue in the VH domain with a histidine residue, wherein the at least one hot-spot amino acid residue is selected from the following hot-spot list:

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

wherein each of the engineered variants in first the panel comprises an Fc region;

(b) screening the first panel of engineered variants for pH dependent binding to the 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;

(c) preparing a second panel of engineered variants of the parental antibody, wherein each of the engineered variants in the panel differs from the parental antibody by substitution of at least one hot-spot amino acid residue in the VL domain with a histidine residue, wherein the at least one hot-spot amino acid residue is selected from the following hot-spot list:

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

wherein each of the engineered variants in the second panel comprises an Fc region;

(d) screening the second panel of engineered variants for pH dependent binding to the 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;

(e) preparing one or more further engineered variants of the parental antibody, wherein each of the variants differs from the parental antibody by substitution of the hot-spot amino acid at one or more of the selected amino acid positions in the VH domain identified in step (b) with histidine and by substitution of the hot-spot amino acid at one or more of the selected amino acid positions in the VL domain identified in step (d) with histidine, wherein each of the engineered variants comprises an Fc region; and

(f) screening the further engineered variants for pH dependent binding to the antigen, thereby identifying an engineered antibody exhibiting pH-dependent binding to the antigen.

6. The method of claim 5 , wherein the engineered antibody identified in part (f) has lower affinity for its antigen at acidic pH than at neutral pH.

7. The method of claim 1 , wherein:

(a) the dissociation rate constant (k d ) for the engineered antibody-antigen interaction at acidic pH is higher than the dissociation rate constant (k d ) for the engineered antibody-antigen interaction at neutral pH;

(b) the dissociation rate constant (k d ) for the engineered antibody-antigen interaction at acidic pH is higher than the dissociation rate constant (k d ) for the parental antibody-antigen interaction at acidic pH; and/or

(c) the equilibrium dissociation constant (K D ) for the engineered antibody-antigen interaction at acidic pH is higher than the equilibrium dissociation constant (K D ) for the engineered antibody-antigen interaction at neutral pH.

8. The method of claim 1 , wherein at least two hot-spot amino acid residues are selected from the hot-spot list.

9. The method of claim 1 , wherein at least three hot-spot amino acid residues are selected from the hot-spot list.

10. The method of claim 1 , wherein at least four hot-spot amino acid residues are selected from the hot-spot list.

11. The method of claim 1 , wherein one or more of the following residues is also substituted with histidine: H100g, H100k, H100m, H100n, L95, L95d, L95e, L95f, and L97.

12. The method of claim 1 , wherein at least two amino acid residues selected from the cold-spot list are not substituted with histidine.

13. The method of claim 1 , wherein at least three amino acid residues selected from the cold-spot list are not substituted with histidine.

14. The method of claim 1 , wherein none of the amino acid residues selected from the cold-spot list for VHCDR1, VHCDR2, VLCDR1 and VLCDR2 are substituted with histidine.

15. The method of claim 1 , wherein the parental antibody or the CDRs thereof are derived from a camelid species.

16. The method of claim 3 , wherein:

(a) the dissociation rate constant (k d ) for the engineered antibody-antigen interaction at acidic pH is higher than the dissociation rate constant (k d ) for the engineered antibody-antigen interaction at neutral pH;

(b) the dissociation rate constant (k d ) for the engineered antibody-antigen interaction at acidic pH is higher than the dissociation rate constant (k d ) for the parental antibody-antigen interaction at acidic pH; and/or

(c) the equilibrium dissociation constant (K D ) for the engineered antibody-antigen interaction at acidic pH is higher than the equilibrium dissociation constant (K D ) for the engineered antibody-antigen interaction at neutral pH.

17. The method of claim 3 , wherein at least two hot-spot amino acid residues are selected from the hot-spot list.

18. The method of claim 3 , wherein at least three hot-spot amino acid residues are selected from the hot-spot list.

19. The method of claim 3 , wherein at least four hot-spot amino acid residues are selected from the hot-spot list.

20. The method of claim 3 , wherein one or more of the following residues is also substituted with histidine: H100g, H100k, H100m, H100n, L95, L95d, L95e, L95f, and L97.

21. The method of claim 3 , wherein at least two amino acid residues selected from the cold-spot list are not substituted with histidine.

22. The method of claim 3 , wherein at least three amino acid residues selected from the cold-spot list are not substituted with histidine.

23. The method of claim 3 , wherein none of the amino acid residues selected from the cold-spot list for VHCDR1, VHCDR2, VLCDR1 and VLCDR2 are substituted with histidine.

24. The method of claim 3 , wherein the parental antibody or the CDRs thereof are derived from a camelid species.

25. The method of claim 5 , wherein:

(a) the dissociation rate constant (k d ) for the engineered antibody-antigen interaction at acidic pH is higher than the dissociation rate constant (k d ) for the engineered antibody-antigen interaction at neutral pH;

(b) the dissociation rate constant (k d ) for the engineered antibody-antigen interaction at acidic pH is higher than the dissociation rate constant (k d ) for the parental antibody-antigen interaction at acidic pH; and/or

(c) the equilibrium dissociation constant (K D ) for the engineered antibody-antigen interaction at acidic pH is higher than the equilibrium dissociation constant (K D ) for the engineered antibody-antigen interaction at neutral pH.

26. The method of claim 5 , wherein at least two hot-spot amino acid residues are selected from the hot-spot list.

27. The method of claim 5 , wherein at least three hot-spot amino acid residues are selected from the hot-spot list.

28. The method of claim 5 , wherein at least four hot-spot amino acid residues are selected from the hot-spot list.

29. The method of claim 5 , wherein one or more of the following residues is also substituted with histidine: H100g, H100k, H100m, H100n, L95, L95d, L95e, L95f, and L97.

30. The method of claim 5 , wherein at least two amino acid residues selected from the cold-spot list are not substituted with histidine.

31. The method of claim 5 , wherein at least three amino acid residues selected from the cold-spot list are not substituted with histidine.

32. The method of claim 5 , wherein none of the amino acid residues selected from the cold-spot list for VHCDR1, VHCDR2, VLCDR1 and VLCDR2 are substituted with histidine.

33. The method of claim 5 , wherein the parental antibody or the CDRs thereof are derived from a camelid species.