Multi-specific antibodies and methods of making and using thereof

A tetra-specific antibody monomer having a N-terminal and a C-terminal, comprising in tandem from the N-terminal to the C-terminal,

• a first scFv domain at the N-terminal comprising
• (i) 3 complementarity determining regions (CDRs) of SEQ ID NO:
• 26, and 3 CDRs of SEQ ID NO:
• 28;
• or
• (ii) 3 CDRs of SEQ ID NO:
• 30, and 3 CDRs of SEQ ID NO:
• 32,
• a second scFv domain comprising
• (i) 3 CDRs of SEQ ID NO:
• 2, and 3 CDRs of SEQ ID NO:
• 4:
• or
• (ii) 3 CDRs of SEQ ID NO:
• 6, and 3 CDRs of SEQ ID NO:
• 8,
• a Fab domain comprising
• (i) 3 CDRs of SEQ ID NO:
• 14, and 3 CDRs of SEQ ID NO:
• 16 ;
• (ii) 3 CDRs of SEQ ID NO:
• 18, and 3 CDRs of SEQ ID NO:
• 20;
• or
• (iii) 3 CDRs of SEQ ID NO:
• 22, and 3 CDRs of SEQ ID NO:
• 24,
• a Fc domain, and
• a third scFv at the C-terminal comprising
• 3 CDRs of SEQ ID NO:
• 10, and 3 CDRs of SEQ ID NO:
• 12,
• wherein the first scFv domain, the second scFv domain, the Fab domain, and the third scFv domain each has a binding specificity against a different antigen,
• wherein the antigen is a tumor antigen, an immune signaling antigen, or a combination thereof, and
• wherein the first scFv domain, the second scFv domain, the Fab domain, and the third scFv domain have a binding specificity against CD19, CD3, 4-1BB, and PD-L1, respectively.