US 2014/0213459 A1 · ANTIBODIES WITH IMPROVED FOLDING STABILITY

Abstract

The present invention relates to methods for improving the folding stability of antibodies, to antibodies with improved folding stability, nucleic acid and vectors encoding such antibodies, and to uses of such antibodies, nucleic acid and vectors.

Claims16

Dependent claims

A method for modifying a parental antibody variable domain comprising a variable heavy (VH) chain domain and a variable light (VL) chain domain, comprising the steps of

(a) establishing a structural model of said parental antibody variable domain based on its amino acid sequence;
(b) identifying in the six CDR regions of the VH and VL chain domains one or more CDR amino acid residues, which are buried in the interface between the VH domain and the VL domain, and which are not determinants of a specific canonical structure;
(c) replacing at least one of the amino acid residues identified in step (b) by a different amino acid residue to generate one or more antibody variable domain variants;
(d) optionally replacing in step (c) one or more additional amino acid residues in the CDR regions and/or in the framework regions of said parental antibody variable domain.

1 more element

A method for modifying a parental antibody variable domain, comprising the step of:

(i) making or causing in a parental Vkappa1 antibody variable domain one or more of the following changes:
(a) in the CDR regions:
(aa) at position L55 a change to an amino acid selected from Y, H, and W, particularly to Y;
(ab) at position L94 a change to an amino acid selected from F, H, I, K, L, M, R, T, V, and Y, particularly L;
(ac) at position L96 a change to an amino acid selected from F and Y, particularly Y;
(ad) at position L32 a change to an amino acid selected from D, F, K, N, Q, S, and Y;
(ae) at position L34 a change to an amino acid selected from A,S, and T, particularly A and S, particularly A;
(af) at position L91 a change to an amino acid selected from A, G, S, and Y, particularly Y;
and/or
(b) in the framework regions:
(ba) at position L1 a change to amino acid A;
(bb) at position L2 a change to amino acid T;
and/or
(bc) at position L70 a change to amino acid E;
or
(ii) making or causing in a parental Vlambda1 antibody variable domain one or more of the following changes:
(a) in the CDR regions:
(aa) at position L34 a change to an amino acid selected from G and S, particularly S;
(ab) at position L96 a change to an amino acid selected from F and Y, particularly to Y;
and/or
(ac) at position L100 a change to amino acid T;
and/or
(iii) making or causing in a parental VH3 antibody variable domain one or more of the following changes:
(a) in the CDR regions:
(aa) at position H50 a change to an amino acid selected from Q, S and T, particularly S and T, particularly S, when said VH3 antibody variable domain is combined with a Vkappa antibody variable domain;
(ab) at position H60 a change to amino acid N;
(ac) at position H63 a change to an amino acid selected from V, I, and F;
(ad) at position H64 a change to amino acid L;
(ae) at position H95 a change to amino acid selected from D, N and T, particularly to D;
(af) at position H102 a change to an amino acid selected from I and V;
(ag) at position H28 a change to amino acid P;
(ah) at position H33 a change to amino acid A;
(ai) at position H52 a change to an amino acid selected from D and S, particularly to D;
(aj) at position H(103 minus 5) a change to amino acid G;
(aj) one or two changes at positions H50 and H95 in order to create a salt bridge, particularly the following salt bridges:
H50:R/H95:E;
and H50:H/H95:E;
(ak) one or two changes at positions H33 and H95 in order to create a salt bridge, particularly the following salt bridges:
H33:R/H95:E;
H33:R/H95:D;
H33:H/H95:D;
and H33:D/H95:H;
and/or
(b) in the framework regions:
(ba) at position H2 a change to an amino acid selected from A and G;
(bb) at position H37 a change to amino acid I;
(bc) at position H48 a change to amino acid I;
and/or
(bd) at position H49 a change to amino acid G.

46 more elements

An antibody variable domain comprising at least one VL or VH domain selected from the group of:

(i) a Vkappa1 antibody variable domain based on the antibody variable domain of SEQ ID No. 1, comprising one or more of the following changes:
(A) a single amino acid exchange L2:I to L2:T;
or
(B) at least two amino acid changes independently selected from the following group:
(a) in the CDR regions:
(aa) L55:Q to L55:Y;
(ab) L94:T to L941;
and/or
(ac) L96:L to L96:Y;
and/or
(b) in the framework regions:
(ba) L1:D to L1:A;
(bb) L2:I to L2:T;
and/or
(bc) L70:D to L70:E;
and optionally comprising up to 3 additional changes in the framework regions FR1 to FR3 different from those of (i)(A) and/or (B);
provided that the antibody variable domains having the following accession numbers are excluded:
AJ704539, U43767, 4762, 40096, 21224, CS483741, CS483744, U86790, X72459, 4753, 19244, AY043163, L26891, DQ184511, AY686924, 4806, DQ535161, 1S78_C, 1S78_E, and 1L7I_L;
(ii) a Vlambda1 antibody variable domain based on the antibody variable domain of SEQ ID No. 2, comprising the following combination of changes:
(a) in the CDR regions:
(aa) L34:N to L34:S;
and
(ab) L96:V to L96:Y or L96:V to L96:F;
and optionally further comprising up to 3 additional changes in the framework regions FR1 to FR3 different from those of (ii)(a);
(iii) a VH3 antibody variable domain based on the antibody variable domain of SEQ ID No. 3, comprising one or more of the following changes:
(A) a single amino acid exchange selected from the following group:
(a) in the CDR regions:
(aa) H50V:
to H50:T;
(ab) H60A:
to H60:N;
(ac) H63V:
to H63:I
(ad) H63V:
to H63:F;
and
(ae) H64:K to H64:L, provided that H:63 is not D;
(B) at least two amino acid changes independently selected from the following group:
(a) in the CDR regions:
(aa) H50V:
to H50:Q;
(ab) H50V:
to H50:T;
(ac) H60A:
to H60:N;
(ad) H63V:
to H63:I
(ae) H63V:
to H63:F;
(af) H64:K to H64:L, provided that H:63 is not D;
and
(ag) H95:D to H95:
N;
and/or
(b) in the framework regions:
(ba) H2:V to H2:A;
(bb) H37:V to H37:I;
(bc) H48:V to H48:I;
and/or
(bd) H49:S to H49:G;
in both (A) and (B) provided that the antibody variable domains having the following accession numbers are excluded:
AM082547, AM082383, AM080583, AF471288, and AM082399.

59 more elements

A diverse collection of antibody variable domains, wherein said collection comprises one or more diverse collections of amino acid residues at one or more positions in one or more CDR regions, provided that

(aa) none of the following CDR positions is diversified:
Vkappa1:
L96;
Vlambda1:
L96;
VH3:
H50 and H95;
and the following CDR positions are each independently optionally diversified:
Vkappa1:
L55 and L94;
VH3:
H60, H63, and H64;
or
(ab) any of the following CDR positions is not diversified, if it carries one of the following amino acid residues:
Vkappa1:
L55:Y, L94:L, L96:Y;
Vlambda1:
L96:Y;
VH3:
H50:T, H60:N, H63:I, H64:L, and H95:D;
or
(ac) any of the following CDR positions is either not diversified, or it is diversified with a bias towards the following amino acid residues:
Vkappa1:
L55:Y, L94:L, L96:Y;
Vlambda1:
L96:Y;
VH3:
H50:T, H60:N, H63:I, H64:L, and H95:D;
particularly wherein the listed amino acid residues is present to at least 30%, and more particularly to at least 50% in the diversification mixture;
or
(ad) any of the following CDR positions is either not diversified or diversified with the indicated limited diversity only:
Vkappa1:
L55:YHW, L94:FHIKLRY, L96:FY;
Vlambda1:
L96:FY;
VH3:
H50:QT, H60:HNRS, H63:VIF, H64:KL, and H95:DNT.
wherein the antibody variable domain is selected from the group of:
(i) a Vkappa1 antibody variable domain having a sequence identity in the framework regions FR1 to FR3 of at least 90% to the antibody variable domain of SEQ ID No. 1, optionally comprising one or more of the following changes:
(b) in the framework regions:
(ba) L1:D to L1:A;
(bb) L2:I to L2:T;
and/or
(bc) L70:D to L70:E;
(ii) a Vlambda1 antibody variable domain having a sequence identity in the framework regions FR1 to FR3 of at least 90% to the antibody variable domain of SEQ ID No. 2;
and/or
(ii) a VH3 antibody variable domain having a sequence identity in the framework regions FR1 to FR3 of at least 90% to the antibody variable domain of SEQ ID No. 3, optionally comprising one or more of the following changes:
(b) in the framework regions:
(ba) H2:V to H2:A;
(bb) H37:V to H37:I;
(bc) H48:V to H48:I;
and/or
(bd) H49:S to H49:G.

50 more elements

Dependencies are inferred from the claim text.

Facts

Application

14/122,874Filed 29 May 2012

Priority

JP 11004371.827 May 2011

Published

31 Jul 2014

Term ends

Not estimatedThe source records don’t support an estimate.

Classification14 codes

C12N 15/1058
Isolating an individual clone by screening libraries: directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis…
C07K 16/00
Immunoglobulins [ig]
G01N 33/483
Biological material, e.g. blood, urine ; Haemocytometers: physical analysis of biological material
G16B 15/00
Ict specially adapted for analysing two-dimensional [2d] or three-dimensional [3d] molecular structures

Abstract

Claims16

Dependent claims

A method for modifying a parental antibody variable domain comprising a variable heavy (VH) chain domain and a variable light (VL) chain domain, comprising the steps of

(a) establishing a structural model of said parental antibody variable domain based on its amino acid sequence;
(b) identifying in the six CDR regions of the VH and VL chain domains one or more CDR amino acid residues, which are buried in the interface between the VH domain and the VL domain, and which are not determinants of a specific canonical structure;
(c) replacing at least one of the amino acid residues identified in step (b) by a different amino acid residue to generate one or more antibody variable domain variants;
(d) optionally replacing in step (c) one or more additional amino acid residues in the CDR regions and/or in the framework regions of said parental antibody variable domain.

1 more element

A method for modifying a parental antibody variable domain, comprising the step of:

(i) making or causing in a parental Vkappa1 antibody variable domain one or more of the following changes:
(a) in the CDR regions:
(aa) at position L55 a change to an amino acid selected from Y, H, and W, particularly to Y;
(ab) at position L94 a change to an amino acid selected from F, H, I, K, L, M, R, T, V, and Y, particularly L;
(ac) at position L96 a change to an amino acid selected from F and Y, particularly Y;
(ad) at position L32 a change to an amino acid selected from D, F, K, N, Q, S, and Y;
(ae) at position L34 a change to an amino acid selected from A,S, and T, particularly A and S, particularly A;
(af) at position L91 a change to an amino acid selected from A, G, S, and Y, particularly Y;
and/or
(b) in the framework regions:
(ba) at position L1 a change to amino acid A;
(bb) at position L2 a change to amino acid T;
and/or
(bc) at position L70 a change to amino acid E;
or
(ii) making or causing in a parental Vlambda1 antibody variable domain one or more of the following changes:
(a) in the CDR regions:
(aa) at position L34 a change to an amino acid selected from G and S, particularly S;
(ab) at position L96 a change to an amino acid selected from F and Y, particularly to Y;
and/or
(ac) at position L100 a change to amino acid T;
and/or
(iii) making or causing in a parental VH3 antibody variable domain one or more of the following changes:
(a) in the CDR regions:
(aa) at position H50 a change to an amino acid selected from Q, S and T, particularly S and T, particularly S, when said VH3 antibody variable domain is combined with a Vkappa antibody variable domain;
(ab) at position H60 a change to amino acid N;
(ac) at position H63 a change to an amino acid selected from V, I, and F;
(ad) at position H64 a change to amino acid L;
(ae) at position H95 a change to amino acid selected from D, N and T, particularly to D;
(af) at position H102 a change to an amino acid selected from I and V;
(ag) at position H28 a change to amino acid P;
(ah) at position H33 a change to amino acid A;
(ai) at position H52 a change to an amino acid selected from D and S, particularly to D;
(aj) at position H(103 minus 5) a change to amino acid G;
(aj) one or two changes at positions H50 and H95 in order to create a salt bridge, particularly the following salt bridges:
H50:R/H95:E;
and H50:H/H95:E;
(ak) one or two changes at positions H33 and H95 in order to create a salt bridge, particularly the following salt bridges:
H33:R/H95:E;
H33:R/H95:D;
H33:H/H95:D;
and H33:D/H95:H;
and/or
(b) in the framework regions:
(ba) at position H2 a change to an amino acid selected from A and G;
(bb) at position H37 a change to amino acid I;
(bc) at position H48 a change to amino acid I;
and/or
(bd) at position H49 a change to amino acid G.

46 more elements

An antibody variable domain comprising at least one VL or VH domain selected from the group of:

(i) a Vkappa1 antibody variable domain based on the antibody variable domain of SEQ ID No. 1, comprising one or more of the following changes:
(A) a single amino acid exchange L2:I to L2:T;
or
(B) at least two amino acid changes independently selected from the following group:
(a) in the CDR regions:
(aa) L55:Q to L55:Y;
(ab) L94:T to L941;
and/or
(ac) L96:L to L96:Y;
and/or
(b) in the framework regions:
(ba) L1:D to L1:A;
(bb) L2:I to L2:T;
and/or
(bc) L70:D to L70:E;
and optionally comprising up to 3 additional changes in the framework regions FR1 to FR3 different from those of (i)(A) and/or (B);
provided that the antibody variable domains having the following accession numbers are excluded:
AJ704539, U43767, 4762, 40096, 21224, CS483741, CS483744, U86790, X72459, 4753, 19244, AY043163, L26891, DQ184511, AY686924, 4806, DQ535161, 1S78_C, 1S78_E, and 1L7I_L;
(ii) a Vlambda1 antibody variable domain based on the antibody variable domain of SEQ ID No. 2, comprising the following combination of changes:
(a) in the CDR regions:
(aa) L34:N to L34:S;
and
(ab) L96:V to L96:Y or L96:V to L96:F;
and optionally further comprising up to 3 additional changes in the framework regions FR1 to FR3 different from those of (ii)(a);
(iii) a VH3 antibody variable domain based on the antibody variable domain of SEQ ID No. 3, comprising one or more of the following changes:
(A) a single amino acid exchange selected from the following group:
(a) in the CDR regions:
(aa) H50V:
to H50:T;
(ab) H60A:
to H60:N;
(ac) H63V:
to H63:I
(ad) H63V:
to H63:F;
and
(ae) H64:K to H64:L, provided that H:63 is not D;
(B) at least two amino acid changes independently selected from the following group:
(a) in the CDR regions:
(aa) H50V:
to H50:Q;
(ab) H50V:
to H50:T;
(ac) H60A:
to H60:N;
(ad) H63V:
to H63:I
(ae) H63V:
to H63:F;
(af) H64:K to H64:L, provided that H:63 is not D;
and
(ag) H95:D to H95:
N;
and/or
(b) in the framework regions:
(ba) H2:V to H2:A;
(bb) H37:V to H37:I;
(bc) H48:V to H48:I;
and/or
(bd) H49:S to H49:G;
in both (A) and (B) provided that the antibody variable domains having the following accession numbers are excluded:
AM082547, AM082383, AM080583, AF471288, and AM082399.

59 more elements

(aa) none of the following CDR positions is diversified:
Vkappa1:
L96;
Vlambda1:
L96;
VH3:
H50 and H95;
and the following CDR positions are each independently optionally diversified:
Vkappa1:
L55 and L94;
VH3:
H60, H63, and H64;
or
(ab) any of the following CDR positions is not diversified, if it carries one of the following amino acid residues:
Vkappa1:
L55:Y, L94:L, L96:Y;
Vlambda1:
L96:Y;
VH3:
H50:T, H60:N, H63:I, H64:L, and H95:D;
or
(ac) any of the following CDR positions is either not diversified, or it is diversified with a bias towards the following amino acid residues:
Vkappa1:
L55:Y, L94:L, L96:Y;
Vlambda1:
L96:Y;
VH3:
H50:T, H60:N, H63:I, H64:L, and H95:D;
particularly wherein the listed amino acid residues is present to at least 30%, and more particularly to at least 50% in the diversification mixture;
or
(ad) any of the following CDR positions is either not diversified or diversified with the indicated limited diversity only:
Vkappa1:
L55:YHW, L94:FHIKLRY, L96:FY;
Vlambda1:
L96:FY;
VH3:
H50:QT, H60:HNRS, H63:VIF, H64:KL, and H95:DNT.
wherein the antibody variable domain is selected from the group of:
(i) a Vkappa1 antibody variable domain having a sequence identity in the framework regions FR1 to FR3 of at least 90% to the antibody variable domain of SEQ ID No. 1, optionally comprising one or more of the following changes:
(b) in the framework regions:
(ba) L1:D to L1:A;
(bb) L2:I to L2:T;
and/or
(bc) L70:D to L70:E;
(ii) a Vlambda1 antibody variable domain having a sequence identity in the framework regions FR1 to FR3 of at least 90% to the antibody variable domain of SEQ ID No. 2;
and/or
(ii) a VH3 antibody variable domain having a sequence identity in the framework regions FR1 to FR3 of at least 90% to the antibody variable domain of SEQ ID No. 3, optionally comprising one or more of the following changes:
(b) in the framework regions:
(ba) H2:V to H2:A;
(bb) H37:V to H37:I;
(bc) H48:V to H48:I;
and/or
(bd) H49:S to H49:G.

50 more elements

Dependencies are inferred from the claim text.

Antibodies with improved folding stability

Life of this patent

Abstract

Claims16

Antibodies with improved folding stability

Life of this patent

Abstract

Claims16