Granted Patent B2

US 12,516,106 · App. 17/177,236 · Granted Jan 6, 2026

Inventors: Sam Philip Heywood (Slough, GB); Gavin Barry Wild (Slough, GB)

Assignee: UCB BIOPHARMA SRL

C07K16/065C07K1/22C07K16/18C07K16/2878C07K2317/31C07K2317/55C07K2317/62C07K2317/622C07K2317/624

Abstract

Claims (103)

1 . A method for obtaining a human VH3 domain-containing antibody in monomeric form comprising:

a) applying a mixture comprising a human VH3 domain-containing antibody in monomeric and multimeric form to a protein A chromatography material wherein said protein A comprises domain D and/or E, under conditions that allow binding of said antibody to protein A, and

b) recovering the human VH3 domain-containing antibody in monomeric form,

wherein the human VH3 domain-containing antibody does not contain an Fc region and said VH3 domain-containing antibody comprises SEQ ID NOs: 11 and 12 or SEQ ID NOs: 29 and 31; and

wherein said human VH3 domain-containing antibody comprises a multi-specific antibody molecule comprising:

a) a polypeptide chain of formula (I):

VH-CH1-X-V1; and

b) a polypeptide chain of formula (II):

VL-CL-Y-V2;

wherein:

VH represents a heavy chain variable domain;

CH1 represents a domain of a heavy chain constant region, for example domain 1 thereof;

X represents a bond or linker;

Y represents a bond or linker;

V1 represents a dsFv, a sdAb, a scFv or a dsscFv;

VL represents a light chain variable domain;

CL represents a domain from a light chain constant region; and

V2 represents dsFv, a sdAb, a scFv or a dsscFv;

wherein at least one of V 1 or V 2 is a dsFv or dsscFv.

2 . The method according to claim 1 , wherein a first solution is added to the protein A chromatography material after applying the mixture comprising the human VH3 domain-containing antibody in monomeric and multimeric form such that unbound material is removed in the solution.

3 . The method according to claim 1 , wherein an elution buffer is applied to the protein A chromatography material such that the bound antibody is released.

4 . The method according to claim 1 , wherein the eluate recovered from the protein A chromatography is enriched in monomeric antibody over multimeric antibody with respect to the applied mixture.

5 . The method according to claim 1 , wherein said protein A is native recombinant protein A.

6 . The method according to claim 1 , wherein the VH3 domain containing antibody is selected from Fab′, F(ab′) 2 , scFv, Fab-Fv, Fab-scFv, Fab-(scFv) 2 , Fab-(Fv) 2 , Fab-dsFv, diabodies, triabodies, and tetrabodies.

7 . The method according to claim 1 , wherein the VH3 domain-containing antibody comprises at least 2 human VH3 domains.

8 . A method for manufacturing a human VH3 domain-containing antibody comprising:

a) expressing the antibody in a host cell,

b) recovering a mixture containing the antibody, host cells and other contaminants,

c) purifying the antibody using at least a protein A chromatography step wherein said protein A comprises domain D and/or E, and

d) recovering the human VH3 containing antibody,

wherein the human VH3 domain containing antibody does not contain an Fc region and said VH3 domain-containing antibody comprises SEQ ID NOs: 11 and 12 or SEQ ID NOs: 29 and 31; and

wherein said human VH3 domain-containing antibody comprises a multi-specific antibody molecule comprising:

a) a polypeptide chain of formula (I):

VH-CH1-X-V1; and

b) a polypeptide chain of formula (II):

VL-CL-Y-V2;

wherein:

VH represents a heavy chain variable domain;

CH1 represents a domain of a heavy chain constant region, for example domain 1 thereof;

X represents a bond or linker;

Y represents a bond or linker;

V1 represents a dsFv, a sdAb, a scFv or a dsscFv;

VL represents a light chain variable domain;

CL represents a domain from a light chain constant region; and

V2 represents dsFv, a sdAb, a scFv or a dsscFv;

wherein at least one of V 1 or V 2 is a dsFv or dsscFv.

9 . A method of separating a human VH 3 domain-containing antibody in monomeric form from the antibody in multimeric form comprising:

a) applying a mixture comprising a human VH 3 domain-containing antibody in monomeric and multimeric form to a protein A chromatography material wherein said protein A comprises domain D and/or E,

b) allowing binding of said antibody to protein A,

c) applying a first elution buffer that selectively disrupts binding of the antibody in monomeric form,

d) recovering the resulting eluate, and optionally

e) applying a second elution buffer that disrupts binding of the antibody in multimeric form and recovering this second eluate,

wherein the human VH3 domain-containing antibody does not contain an Fc region and said VH3 domain-containing antibody comprises SEQ ID NOs: 11 and 12 or SEQ ID NOs: 29 and 31; and

wherein said human VH 3 domain-containing antibody comprises a multi-specific antibody molecule comprising:

a) a polypeptide chain of formula (I):

VH-CH1-X-V1; and

b) a polypeptide chain of formula (II):

VL-CL-Y-V2;

wherein:

VH represents a heavy chain variable domain;

CH1 represents a domain of a heavy chain constant region, for example domain 1 thereof;

X represents a bond or linker;

Y represents a bond or linker;

V1 represents a dsFv, a sdAb, a scFv or a dsscFv;

VL represents a light chain variable domain;

CL represents a domain from a light chain constant region; and

V2 represents dsFv, a sdAb, a scFv or a dsscFv;

wherein at least one of V 1 or V 2 is a dsFv or dsscFv.

10 . A method of separating a human VH3 domain-containing antibody in monomeric form from the antibody in multimeric form comprising:

a) applying a mixture comprising a human VH3 domain-containing antibody in monomeric and multimeric form to a protein A chromatography material wherein said protein A comprises domain D and/or E,

b) allowing binding of the antibody in multimeric form,

c) recovering the antibody in monomeric form in the flow-through, and optionally

d) applying an elution buffer that selectively disrupts binding of the antibody in multimeric form, and

e) recovering the eluate resulting from d);

wherein the human VH3 domain-containing antibody does not contain an Fc region and said VH3 domain-containing antibody comprises SEQ ID NOs: 11 and 12 or SEQ ID NOs: 29 and 31; and

wherein said human VH3 domain-containing antibody comprises a multi-specific antibody molecule comprising:

a) a polypeptide chain of formula (I):

VH-CH1-X-V1; and

b) a polypeptide chain of formula (II):

VL-CL-Y-V2;

wherein:

VH represents a heavy chain variable domain;

CH1 represents a domain of a heavy chain constant region, for example domain 1 thereof;

X represents a bond or linker;

Y represents a bond or linker;

V1 represents a dsFv, a sdAb, a scFv or a dsscFv;

VL represents a light chain variable domain;

CL represents a domain from a light chain constant region; and

V2 represents dsFv, a sdAb, a scFv or a dsscFv;

wherein at least one of V 1 or V 2 is a dsFv or dsscFv.

11 . The method according to claim 1 , wherein both V1 and V2 are a dsscFv or a dsFc.

12 . The method according to claim 8 , wherein both V1 and V2 are a dsscFv or a dsFc.

13 . The method according to claim 9 , wherein both V1 and V2 are a dsscFv or a dsFc.

14 . The method according to claim 10 , wherein both V1 and V2 are a dsscFv or a dsFc.

15 . The method according to claim 8 , wherein a first solution is added to the protein A chromatography material after applying the mixture comprising the human VH 3 domain-containing antibody in monomeric and multimeric form such that unbound material is removed in the solution.

16 . The method according to claim 8 , wherein an elution buffer is applied to the protein A chromatography material such that the bound antibody is released and recovered in an eluate.

17 . The method according to claim 16 , wherein the eluate recovered from the protein A chromatography is enriched in monomeric antibody over multimeric antibody with respect to the applied mixture.

18 . The method according to claim 8 , wherein said protein A is native recombinant protein A.

19 . The method according to claim 9 , wherein a first solution is added to the protein A chromatography material after applying the mixture comprising the human VH3 domain-containing antibody in monomeric and multimeric form such that unbound material is removed in the solution.

20 . The method according to claim 9 , wherein the first eluate recovered from the protein A chromatography is enriched in monomeric antibody over multimeric antibody with respect to the applied mixture.

21 . The method according to claim 9 , wherein said protein A is native recombinant protein A.

22 . The method according to claim 10 , wherein a first solution is added to the protein A chromatography material after applying the mixture comprising the human VH3 domain-containing antibody in monomeric and multimeric form such that unbound material is removed in the solution.

23 . The method according to claim 10 , wherein said protein A is native recombinant protein A.

Assignments (2)

Priority Claims (1)

Continuity (2)

References Cited (30)