CPC C07K 16/30 (2013.01) [A61P 35/00 (2018.01); C07K 16/28 (2013.01); C07K 16/2809 (2013.01); C07K 16/2827 (2013.01); C07K 16/2863 (2013.01); C07K 16/2887 (2013.01); C07K 16/468 (2013.01); C07K 2317/31 (2013.01); C07K 2317/52 (2013.01); C07K 2317/55 (2013.01); C07K 2317/56 (2013.01); C07K 2317/569 (2013.01); C07K 2317/62 (2013.01); C07K 2317/624 (2013.01); C07K 2317/64 (2013.01); C07K 2317/66 (2013.01); C07K 2317/73 (2013.01); C07K 2317/75 (2013.01)] | 10 Claims |
1. A multispecific polypeptide construct, the multispecific polypeptide construct comprising a first component comprising a heterodimeric immunoglobulin Fc region and a second component comprising a CD3-binding region wherein:
the first and second components are coupled by a first linker that is a polypeptide of 2-18 amino acids in length composed of at least 50% Glycine residues, wherein the Fc region is amino-terminal to the CD3-binding region;
the CD3-binding region is an anti-CD3 disulfide stabilized Fv antibody fragment (dsFv) comprising a variable heavy chain (VH) and a variable light chain (VL), wherein the VH has the amino acid sequence of SEQ ID NO: 44 or a sequence that exhibits at least 90% sequence identity to SEQ ID NO: 44, and the VL has the amino acid sequence of SEQ ID NO: 72 or a sequence that exhibits at least 90% sequence identity to SEQ ID NO: 72, wherein the VH and VL are each linked to opposite polypeptides of the heterodimeric Fc by the first linker;
each of the Fc polypeptides of the heterodimeric Fc region comprises a knob-into-hole modification or a charge mutation to increase electrostatic complementarity of the Fc polypeptides;
the first component comprises at least one antigen binding domain that is a single domain antibody (sdAb) and binds a tumor associated antigen (TAA), wherein each of the at least one antigen binding domain of the first component is linked amino-terminal to the Fc region by a second linker that is a polypeptide of 2-6 amino acids in length composed of at least 50% Glycine residues; and
the second component further comprises at least one antigen binding domain that is a sdAb and binds to the same TAA as the first component, wherein each of the at least one antigen binding domain of the second component is linked carboxy-terminal to the CD3-binding region by a third linker that is a polypeptide of 2-6 amino acids in length composed of at least 50% Glycine residues,
wherein (i) each of the sdAbs is a camelid VHH or a humanized camelid VHH; and (ii) the CD3-binding region is not able to bind cell surface CD3 as determined by flow cytometry, unless the at least one antigen binding domain is bound to its TAA.
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