Multispecific polypeptide constructs containing a constrained cd3 binding domain and a receptor binding region and methods of using the same

A multispecific polypeptide construct, the multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3-binding region, wherein:

• the CD3 binding region is an anti-CD3 antibody or antigen binding fragment that is an Fv antibody fragment comprising a variable heavy chain region (VH) and a variable light chain region (VL);
• the Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide and the VH and VL of the anti-CD3 antibody or antigen binding fragment are linked to opposite polypeptides of the heterodimeric Fc;
• the first and second components are coupled by a polypeptide linker, wherein the Fc region is positioned N-terminal to the CD3-binding region;
• one or both of the first and second components comprises at least one antigen binding domain that binds a tumor associated antigen (TAA);
• and
• one or both of the first and second components comprises at least one co-stimulatory receptor binding region (CRBR) that binds a co-stimulatory receptor.

A multispecific polypeptide construct, the multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3-binding region, wherein:

• the CD3 binding region is an anti-CD3 antibody or antigen binding fragment that is an Fv antibody fragment comprising a variable heavy chain region (VH) and a variable light chain region (VL);
• the Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide and the VH and VL of the anti-CD3 antibody or antigen binding fragment are linked to opposite polypeptides of the heterodimeric Fc;
• the first and second components are coupled by a polypeptide linker, wherein the Fc region is positioned N-terminal to the CD3-binding region;
• one or both of the first and second components comprises at least one antigen binding domain that binds a tumor associated antigen (TAA);
• and
• one or both of the first and second components comprises at least one inhibitory receptor binding region (IRBR) that binds an inhibitory receptor.

A multispecific polypeptide construct, the multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3-binding region, wherein:

• the CD3 binding region is an anti-CD3 antibody or antigen binding fragment that is an Fv antibody fragment comprising a variable heavy chain region (VH) and a variable light chain region (VL);
• the Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide and the VH and VL of the anti-CD3 antibody or antigen binding fragment are linked to opposite polypeptides of the heterodimeric Fc;
• the first and second components are coupled by a polypeptide linker, wherein the Fc region is positioned N-terminal to the CD3-binding region;
• one or both of the first and second components comprises at least one antigen binding domain that binds a tumor associated antigen (TAA);
• one or both of the first and second components comprises at least one co-stimulatory receptor binding region (CRBR) that binds a co-stimulatory receptor;
• and
• one or both of the first and second components comprises at least one inhibitory receptor binding region (IRBR) that binds an inhibitory receptor.

A multispecific polypeptide construct, comprising in order from N-terminus to C-terminus:

• (a) a co-stimulatory receptor binding region (CRBR) that binds a co-stimulatory receptor and/or an antigen binding domain that binds a tumor-associated antigen (TAA);
• an immunoglobulin Fc region;
• a linker;
• a CD3 binding region that binds CD3 (CD3c);
• and
• a co-stimulatory receptor binding region (CRBR) that binds a co-stimulatory receptor and/or an antigen binding domain that binds to a tumor-associated antigen (TAA);
• or
• (b) an inhibitory receptor binding region (IRBR) that binds a inhibitory receptor and/or an antigen binding domain that binds a tumor-associated antigen (TAA);
• an immunoglobulin Fc region;
• a linker;
• a CD3 binding region that binds CD3 (CD3c);
• and
• an inhibitory receptor binding region (IRBR) that binds a inhibitory receptor and/or an antigen binding domain that binds to a tumor-associated antigen (TAA);
• wherein the multispecific polypeptide construct comprises at least one CRBR or IRBR and at least one antigen binding domain.

A multispecific polypeptide construct, wherein the multispecific construct comprises in order, from N-terminus to C-terminus:

• one of an inhibitory receptor binding region (IRBR) that binds a inhibitory receptor or a co-stimulatory receptor binding region (CRBR) that binds a costimulatory receptor, and/or an antigen binding domain that binds a tumor-associated antigen (TAA);
• an immunoglobulin Fc region;
• a linker;
• a CD3 binding region that binds CD3 (CD3c);
• and
• the other of the IRBR or the CRBR, and/or an antigen binding domain that binds to a tumor-associated antigen (TAA);
• wherein the multispecific polypeptide construct comprises at least one IRBR, at least one CRBR, and at least one antigen binding domain.

A multispecific polypeptide construct, wherein the multispecific construct comprises in order, from N-terminus to C-terminus:

• (a) an immunoglobulin Fc region;
• a linker;
• a CD3 binding region that binds CD3 (CD3c);
• and
• an antigen binding domain that binds a tumor-associated antigen (TAA) and a co-stimulatory receptor binding region (CRBR) that binds a co-stimulatory receptor;
• or
• (b) an antigen binding domain that binds a tumor-associated antigen (TAA) and a co-stimulatory receptor binding region (CRBR) that binds a co-stimulatory receptor;
• an immunoglobulin Fc region;
• a linker;
• and
• a CD3 binding region that binds CD3 (CD3c).

A multispecific polypeptide construct, wherein the multispecific construct comprises in order, from N-terminus to C-terminus:

• (a) an immunoglobulin Fc region;
• a linker;
• a CD3 binding region that binds CD3 (CD3c);
• and
• an antigen binding domain that binds a tumor-associated antigen (TAA) and an inhibitory receptor binding region (IRBR) that binds an inhibitory receptor;
• or
• (b) an antigen binding domain that binds a tumor-associated antigen (TAA) and an inhibitory receptor binding region (IRBR) that binds a inhibitory receptor;
• an immunoglobulin Fc region;
• a linker;
• and
• a CD3 binding region that binds CD3 (CD3c).