Trivalent trispecific antibody constructs

A trivalent trispecific binding molecule, comprising:

• (I) a first, a second, a third, a fourth, and a fifth polypeptide chain, wherein:
• (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, a domain E, a domain N and a domain O,
• wherein the domains are arranged, from N-terminus to C-terminus, in an N—O-A-B-D-E orientation, and
• domain A has a variable region domain amino acid sequence, domain B has a constant region domain amino acid sequence, domain D has a CH2 amino acid sequence, domain E has a constant region domain amino acid sequence, domain N has a variable region domain amino acid sequence, and domain O has a constant region domain amino acid sequence;
• (b) the second polypeptide chain comprises a domain F and a domain G,
• wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and
• wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence;
• (c) the third polypeptide chain comprises a domain H, a domain I, a domain J, and a domain K,
• wherein the domains are arranged, from N-terminus to C-terminus, in a H—I-J-K orientation, and
• wherein domain H has a variable region domain amino acid sequence, domain I has a constant region domain amino acid sequence, domain J has a CH2 amino acid sequence, and K has a constant region domain amino acid sequence;
• (d) the fourth polypeptide chain comprises a domain L and a domain M,
• wherein the domains are arranged, from N-terminus to C-terminus, in a L-M orientation, and
• wherein domain L has a variable region domain amino acid sequence and domain M has a constant region domain amino acid sequence;
• (e) the fifth polypeptide chain comprises a domain P and a domain Q,
• wherein the domains are arranged, from N-terminus to C-terminus, in a P-Q orientation, and
• wherein domain P has a variable region domain amino acid sequence and domain Q has a constant region domain amino acid sequence,
• (f) the first and the second polypeptides are associated through an interaction between the A and the F domains and an interaction between the B and the G domains;
• (g) the third and the fourth polypeptides are associated through an interaction between the H and the L domains and an interaction between the I and the M domains;
• (h) the first and the fifth polypeptides are associated through an interaction between the N and the P domains and an interaction between the O and the Q domains to form the binding molecule;
• (i) the first and the third polypeptides are associated through an interaction between the D and the J domains and an interaction between the E and the K domains to form the binding molecule;
• (j) the amino acid sequences of domain N, domain A, and domain H are different,
• (k) the second and the fifth polypeptide chains are identical and the fourth polypeptide chain is different, or the fourth and the fifth polypeptide chains are identical and the second polypeptide chain is different, and
• (l) the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen,
• the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and
• the interaction between the N domain and the P domain form a third antigen binding site specific for a third antigen;
• optionally, wherein
• (i) the second and the fifth polypeptide chains are identical and the fourth polypeptide chain is different from the second and the fifth polypeptide chains,
• the amino acid sequences of domain O and domain B are identical, and the amino acid sequence of domain I is different from domains O and B;
• or
• (ii) the fourth and the fifth polypeptide chains are identical and the second polypeptide chain is different from the second and the fifth polypeptide chains,
• the amino acid sequences of domain O and domain I are identical, and the amino acid sequence of domain B is different from domains O and I;
• or
• (I) a first, a second, a third, a fourth, and a sixth polypeptide chain, wherein:
• (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E,
• wherein the domains are arranged, from N-terminus to C-terminus, in an A-B-D-E orientation, and
• domain A has a variable region domain amino acid sequence, domain B has a constant region domain amino acid sequence, domain D has a CH2 amino acid sequence, and domain E has a constant region domain amino acid sequence;
• (b) the second polypeptide chain comprises a domain F and a domain G,
• wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and
• wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence;
• (c) the third polypeptide chain comprises a domain H, a domain I, a domain J, a domain K, a domain R, and a domain S
• wherein the domains are arranged, from N-terminus to C-terminus, in a R—S—H—I-J-K orientation, and
• wherein domain H has a variable region domain amino acid sequence, domain I has a constant region domain amino acid sequence, domain J has a CH2 amino acid sequence, domain K has a constant region domain amino acid sequence, domain R has a variable region domain amino acid sequence, and domain S has a constant region domain amino acid sequence;
• (d) the fourth polypeptide chain comprises a domain L and a domain M,
• wherein the domains are arranged, from N-terminus to C-terminus, in a L-M orientation, and
• wherein domain L has a variable region domain amino acid sequence and domain M has a constant region domain amino acid sequence;
• (e) the sixth polypeptide chain comprises a domain T and a domain U,
• wherein the domains are arranged, from N-terminus to C-terminus, in a T-U orientation, and
• wherein domain T has a variable region domain amino acid sequence and domain U has a constant region domain amino acid sequence,
• (f) the first and the second polypeptides are associated through an interaction between the A and the F domains and an interaction between the B and the G domains;
• (g) the third and the fourth polypeptides are associated through an interaction between the H and the L domains and an interaction between the I and the M domains;
• (h) the first and the sixth polypeptides are associated through an interaction between the R and the T domains and an interaction between the S and the U domains to form the binding molecule;
• (i) the first and the third polypeptides are associated through an interaction between the D and the J domains and an interaction between the E and the K domains to form the binding molecule;
• (j) the amino acid sequences of domain R, domain A, and domain H are different,
• (k) the second and the sixth polypeptide chains are identical and the fourth polypeptide chain is different, or the fourth and the sixth polypeptide chains are identical and the second polypeptide chain is different,
• (l) the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen,
• the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and
• the interaction between the R domain R and the T domain form a third antigen binding site specific for a third antigen;
• optionally, wherein
• (i) the fourth and the sixth polypeptide chains are identical and the fourth polypeptide chain is different from the second and the sixth polypeptide chains, the amino acid sequences of domain S and domain I are identical, and the amino acid sequence of domain B is different from domains S and I;
• or
• (ii) the second and the sixth polypeptide chains are identical and the fourth polypeptide chain is different from the second and the sixth polypeptide chains, the amino acid sequences of domain S and domain B are identical, and the amino acid sequence of domain I is different from domains S and B.