Multispecific polypeptide constructs having constrained CD3 binding and methods of using the same

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 V H H or a humanized camelid V H H; 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.

2. The multispecific polypeptide construct of claim 1 , wherein the TAA is selected from the group consisting of 5T4, B7H3, Carbonic anhydrase 9, CD3, CD20, CD38, CD47, CEACAM5 (CEA), cMet, CXCL10, CXCR4, DLL3, EGFR, Folate receptor alpha (FRα), HER2, HGF, IL-6R, IL-23, Mesothelin, MUC1, PD-1, PD-L1, PSMA, TAG-72, TIM-3, Tumor necrosis factor alpha (TNFα), VCAM-1, and VEGFR2.

3. The multispecific polypeptide construct of claim 1 , wherein the TAA is selected from the group consisting of 5T4, B7H3, cMET, EGFR, and Folate receptor alpha (FRα).

4. The multispecific polypeptide construct of claim 1 , wherein the TAA is 5T4.

5. The multispecific polypeptide construct of claim 1 , wherein the TAA is B7H3.

6. The multispecific polypeptide construct of claim 1 , wherein the TAA is EGFR.

7. The multispecific polypeptide construct of claim 1 , wherein the TAA is Folate receptor alpha (FRα).

8. The multispecific polypeptide construct of claim 1 , wherein the first linker, the second linker and the third linker independently comprise amino acid residues selected from the group consisting of Glycine (Gly), Serine (Ser), Alanine (Ala), and Threonine (Thr).

9. The multispecific polypeptide construct of claim 1 , wherein the first linker, the second linker and the third linker independently comprise Glycine (Gly) and Serine (Ser) amino acid residues.

10. A pharmaceutical composition comprising the multispecific polypeptide construct of claim 1 and a pharmaceutically acceptable carrier.