Patent ID: 11965030
Assignee: SANOFI
Field: Biotechnology (Chemistry)
Classification: CPC A  C | IPC C

Claim 3:
4. A multispecific antigen-binding protein comprising four polypeptide chains, that form three antigen-binding sites, wherein
a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL1  [I],

a second polypeptide chain comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH1-1-hinge-CH2-CH3  [II],

a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-2-hinge-CH2-CH3  [III], and

a fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL2  [IV],

wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL1 is a first immunoglobulin light chain constant domain;
CL2 is a second immunoglobulin light chain constant domain;
CH1-1 is a first immunoglobulin CH1 heavy chain constant domain;
CH1-2 is a second immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains;
Fc comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and
L1, L2, L3, and L4 are amino acid linkers,
wherein VH1 is paired with VL1, VH2 is paired with VL2, and CH1-1 is paired with CL1,
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair,
wherein one or more cysteine residues are engineered into the one or more of VH1/VL1, VH2/VL2, and VH3/VL3 pairs to form one or more disulfide bonds,
wherein at least one or both of VL1 and VH1 pair and of VL2 and VH2 pair comprises opposite charged mutations that facilitate pairing, said opposite charged mutations comprising (1) a mutated residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutated residue in the VL region at Kabat position 38 selected from E, D, K, R, or H,
wherein the mutated residue in the VH region has an opposite charge from the mutated residue in the VL region, and
wherein one or both of the CH1-1 and CL1 domain pair and the CL2 and CH1-2 domain pair comprise mutations that facilitate pairing,
wherein when both of CL1 and CH1-1 pair and of CL2 and CH1-2 pair comprise mutations to facilitate pairing, the mutations in CH1-1 and CL1 are different than the mutations in CH1-2 and CL2, wherein:
i) CH1-1 comprises a T192E mutation and CL1 comprises N137K and S114A mutations and/or CH1-2 comprises a T192E mutation and CL2 comprises N137K and S114A mutations:

ii) CH1-1 comprises L143Q and S188V mutations and CL1 comprises V133T and S176V mutations and/or CH1-2 comprises L143Q and S188V mutations and CL2 comprises V133T and S176V mutations;
iii) CH1-1 comprises T192E, L143Q and S188V mutations and CL1 comprises N137K, S114A, V133T and S176V mutations and/or CH1-2 comprises T192E, L143Q and S188V mutations and CL2 comprises N137K, S114A, V133T and S176V mutations;
iv) CH1-1 comprises a K221E mutation and CL1 comprises a E123K mutation and/or CH1-2 comprises a K221E mutation and CL2 comprises a E123K mutation;
v) CH1-1 comprises T192E and K221E mutations and CL1 comprises N137K, S114A and E123K mutations and/or CH1-2 comprises T192E and K221E mutations and CL2 comprises N137K, S114A and E123K mutations;
vi) CH1-1 comprises a K228D mutation and CL1 comprises a D122K mutation and/or CH1-2 comprises a K228D mutation and CL2 comprises a D122K mutation;
vii) CH1-1 comprises K221E and K228D mutations and CL1 comprises D122K and E123K mutations and/or CH1-2 comprises K221E and K228D mutations and CL2 comprises D122K and E123K mutations;
viii) CH1-1 comprises a L143E, a L143D, a L143K, a L143R, or a L143H mutation, and CL1 comprises a S176E, a S176D, a S176K, a S176R, or a S176H mutation and/or CH1-2 comprises a L143E, a L143D, a L143K, a L143R, or a L143H mutation, and CL2 comprises a S176E, a S176D, a S176K, a S176R, or a S176H mutation; and/or
ix) CH1-1 comprises a L124E, a L124D, a L124K, a L124R, or a L124H mutation, and CL1 comprises a V133E, a V133D, a V133K, a V133R, or a V133H mutation and/or CH1-2 comprises a L124E, a L124D, a L124K, a L124R, or a L124H mutation, and CL2 comprises a V133E, a V133D, a V133K, a V133R, or a V133H mutation,
wherein the mutations are according to Kabat numbering.