Bispecific T cell activating antigen binding molecules

A T cell activating bispecific antigen-binding molecule, comprising:

• (a) a first Fab molecule which specifically binds to a first antigen;
• (b) a second Fab molecule which specifically binds to a second antigen, and wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other;
• (c) a third Fab molecule which specifically binds to the first antigen;
• and
• (d) an Fc domain composed of a first and a second subunit capable of stable association;
• wherein the first antigen is CEA and the second antigen is CD3;
• wherein the third Fab molecule is identical to the first Fab molecule;
• wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule and the third Fab molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain;
• wherein the first Fab molecule and the third Fab molecule comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
• 22 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:
• 23;
• and
• wherein the second Fab molecule comprises a heavy chain variable region comprising the HCDR 1 of SEQ ID NO:
• 4, the HCDR 2 of SEQ ID NO:
• 5, the HCDR 3 of SEQ ID NO:
• 6, and a light chain variable region comprising the LCDR 1 of SEQ ID NO:
• 8, the LCDR 2 of SEQ ID NO:
• 9, and the LCDR 3 of SEQ ID NO:
• 10.

A T cell activating bispecific antigen-binding molecule, comprising:

• (a) a first Fab molecule which specifically binds to a first antigen;
• (b) a second Fab molecule which specifically binds to a second antigen, and wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other;
• (c) a third Fab molecule which specifically binds to the first antigen;
• and
• (d) an Fc domain composed of a first and a second subunit capable of stable association;
• wherein the first antigen is CEA and the second antigen is CD3;
• wherein the third Fab molecule is identical to the first Fab molecule;
• wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule and the third Fab molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain;
• wherein the first Fab molecule and the third Fab molecule comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
• 22 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:
• 23;
• wherein the second Fab molecule comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
• 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:
• 7;
• and
• wherein:
• (a) the Fc domain is a human IgG1 Fc domain;
• (b) the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises amino acid substitutions Y3490, T366S, L368A, and Y407V;
• and
• (c) each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A, and P329G (numberings according to Kabat EU index).

A T cell activating bispecific antigen-binding molecule comprising:

• (a) a first antigen-binding moiety and a third antigen-binding moiety, each of which specifically binds to CEA;
• wherein each of the first antigen-binding moiety and the third antigen-binding moiety is a conventional Fab molecule comprising:
• (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
• 22;
• and
• (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:
• 23, and wherein in the constant domain CL the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat), and wherein in the constant domain CH1 the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index);
• (b) a second antigen-binding moiety which specifically binds to CD3;
• wherein the second antigen-binding moiety is a crossover Fab molecule wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other, wherein the second antigen-binding moiety comprises:
• (i) a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:
• 3, wherein the amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:
• 3 comprises the HCDR1 of SEQ ID NO:
• 4, the HCDR2 of SEQ ID NO:
• 5, and the HCDR3 of SEQ ID NO:
• 6;
• and
• (ii) a light chain variable region comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:
• 7, wherein the amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:
• 7 comprises the LCDR1 of SEQ ID NO:
• 8, the LCDR2 of SEQ ID NO:
• 9, and the LCDR3 of SEQ ID NO:
• 10;
• and
• (c) an Fc domain composed of a first and a second subunit capable of stable association, wherein the Fc domain is a human IgG1 Fc domain, wherein the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, the second subunit of the Fc domain comprises the amino acid substitutions Y3490, T366S, L368A, and Y407V, and wherein each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A, and P329G (numberings according to Kabat EU index),
• wherein the second and the third antigen-binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety.