Bifunctional proteins and their uses

Abstract

This specification provides compositions and methods for treating muscular dystrophy by administering an effective amount of a composition comprising a bifunctional protein having a first binding site that binds to muscle-specific molecules and a second functional domain that activates Notch signaling, thereby spatiotemporally activating Notch signaling in muscle tissue to boost satellite cell regeneration in situ, and thus improving muscle repair / regeneration, thereby improving muscle function as a treatment for various muscular dystrophy and sarcopenia. Also provided are an engineered DLL4 extracellular domain and a protein construct comprising the same.

Description

[Technical Field]

[0001] Cross-reference of related applications This patent application claims priority to U.S. Provisional Patent Application No. 63 / 639,356, filed on 26 April 2024, and to U.S. Provisional Patent Application No. 63 / 537,382, filed on 8 September 2023, the contents of which, in whole, are incorporated herein by reference.

[0002] Reference to electronic sequence lists The contents of the electronic sequence listing (340742000140SEQLIST.xml; size: 457,221 bytes; and creation date: September 5, 2024) are incorporated herein by reference in their entirety.

[0003] Field of Invention The present invention relates to a bifunctional protein construct comprising a first binding site that specifically binds to muscle-specific molecules and a second binding site that specifically binds to and activates the Notch receptor. The present invention also relates to a method for producing such a bifunctional protein construct and the use of such a bifunctional protein construct for treating muscle-related diseases (e.g., muscular dystrophy and sarcopenia). Furthermore, an engineered DLL4 extracellular domain (ECD) and a protein construct comprising the same are provided. [Background technology]

[0004] Muscular dystrophy (MD) is a diverse group of muscle diseases with many subtypes that present with varying degrees of muscle weakness and degeneration. MD is generally associated with defects in genes that encode proteins important for muscle wholeness or function. Degenerative MD is typically caused by defects in structural proteins directly or indirectly related to the dystrophin-associated glycoprotein complex (DGC), which is essential for maintaining muscle cell membrane wholeness.

[0005] A direct link between muscular dystrophy (MD) and Notch signaling in humans has been established in limb-girdle muscular dystrophy (LGMD R21), which is caused by biallelic mutations in protein O-glucosyltransferase 1 (POGLUT1), reduced Notch signaling, and satellite cell loss (Servian-Morilla et al., 2016, EMBO Mol Med. 8(11):1289-1309; Servian-Morilla et al., 2020, Acta Neuropathol. 139(3):565-582). All references cited herein, including patent applications, patent publications, and Genbank Accession numbers, are incorporated herein by reference in the same manner as each individual reference is specifically and individually indicated to be incorporated by reference in whole. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Servian-Morilla et al.,2016,EMBO Mol Med.8(11):1289-1309 [Non-Patent Document 2] Servian-Morilla et al.,2020,Acta Neuropathol.139(3):565-582 [Overview of the project]

[0007] In one aspect, this application provides a bifunctional protein construct comprising a first binding site and a second binding site, wherein the first binding site specifically binds to a muscle-specific molecule, and the second binding site specifically binds to a Notch receptor and activates the Notch receptor.

[0008] In some embodiments of the bifunctional protein construct described above, the muscle-specific molecule is a target antigen on the sarcoglycan, between the sarcoglycan and the basement membrane, or in the basement membrane. In some embodiments, the target antigen is selected from the group consisting of laminin, agrin, nidogen, perlecan, and M-cadherin (CDH15). In some embodiments, the target antigen is a component of the dystrophin-associated glycoprotein complex (DGC). In some embodiments, the target antigen is selected from the group consisting of α-dystroglycan (α-DG), β-DG, laminin-211, perlecan, collagen, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, ε-sarcoglycan, ζ-sarcoglycan, biglycan, sarcospan, and matriglycan on α-DG. In some embodiments, the target antigen is laminin subunit alpha-2 (LAMA2), CDH15, α-DG, or a matriglycan on α-DG.

[0009] In some embodiments of any of the bifunctional protein constructs described above, the first binding site includes an antibody site that specifically binds to a muscle-specific molecule. In some embodiments, the antibody site is selected from the group consisting of full-length antibody, Fab, Fab', F(ab')2, scFv, and sdAb.

[0010] In some embodiments of any of the bifunctional protein constructs described above, the antibody site specifically binds to LAMA2 (anti-LAMA2 antibody site). In some embodiments, the anti-LAMA2 antibody site comprises (i) HC-CDR1 containing the amino acid sequence of SEQ ID NO: 1, HC-CDR2 containing the amino acid sequence of SEQ ID NO: 2, HC-CDR3 containing the amino acid sequence of SEQ ID NO: 3, LC-CDR1 containing the amino acid sequence of SEQ ID NO: 4, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 6; (ii) HC-CDR1 containing the amino acid sequence of SEQ ID NO: 7, HC-CDR2 containing the amino acid sequence of SEQ ID NO: 8, and SEQ ID NO: 9 (iii) HC-CDR3 containing the amino acid sequence of SEQ ID NO: 10, LC-CDR1 containing the amino acid sequence of SEQ ID NO: 11, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 12; (iii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 54, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 66; (iv) VH HC-CDR1 and HC-C VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequences of DR2, HC-CDR3, and SEQ ID NO: 67; (v) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 54, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 68; (vi) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 54, and SEQ ID NO: 69 (vii) LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 54, and HC-CDR1, LC-CDR2, and LC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 70; (viii) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 71;(ix) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 72; (x) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 73; (xi) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 55, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 65; (xii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 55, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 66; (xiii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 55, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 67; (x iv) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 68; (xv) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 69; (xvi) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 55, and (xvii) LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 70; (xviii) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 71; (xviii) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 72;(xix) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 73; (xx) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 65; (xxi) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 56, and (xxii) LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 66; (xxii) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 67; (xxiii) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 68; (x xiv) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 69; (xxv) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 70; (xxvi) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 56, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 71; (xxvii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 56, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 72; (xxviii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 56, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 73;(xxix) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 65; (xxx) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 66; (xxxi) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 57, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 67; (xxxii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 57, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 68; (xxxiii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 57, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 69; (x xxiv) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 70; (xxxv) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 71; (xxxvi) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 57, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 72; (xxxvii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 57, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 73; (xxxviii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 58, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 65;(xxxix) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 58, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 66; (xl) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 58, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 67; (xli) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 58, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 68; (xlii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 58, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 69; (xliii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 58, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 70; ( (xliv) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 58, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 71; (xlv) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 58, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 72; (xlvi) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 58, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 73; (xlvii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 59, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 65; (xlviii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 59, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 66;(xlix) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 67; (l) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 68; (li) HC-CDR1, HC-CDR2, and HC of VH containing the amino acid sequence of SEQ ID NO: 59; -CDR3, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 69; (lii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 59, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 70; (liii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 59, and VL LC-CDR1, LC -CDR2 and LC-CDR3;(liv) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 72;(lv) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 73;(lvi) VH containing the amino acid sequence of SEQ ID NO: 60 VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequences of HC-CDR1, HC-CDR2, and HC-CDR3 of SEQ ID NO: 65; (lvii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 60; VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 66; (lviii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 60; SEQ ID NO: 67 VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of (lix) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of (lix) SEQ ID NO: 60, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 68; (lx) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 60, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 69;(lxi) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 70; (lxii) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 71; (lxiii) HC-CDR1, HC-CDR2, and HC-CDR of VH containing the amino acid sequence of SEQ ID NO: 60 3, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 72; (lxiv) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 60, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 73; (lxv) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 61, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 65; ( (lxvi) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 66; (lxvii) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 67; (lxviii) HC-CDR1, HC-CDR2, and HC-C of VH containing the amino acid sequence of SEQ ID NO: 61 VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of DR3 and SEQ ID NO: 68; (lxix) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 61, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 69; (lxx) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 61, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 70;(lxxi) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 71; (lxxii) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 72; (lxxiii) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 61 VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of R3 and SEQ ID NO: 73; (lxxiv) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 62, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 65; (lxxv) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 62, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 66; ( (lxxvi) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 62, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 67; (lxxvii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 62, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 68; (lxxviii) VH HC-CDR1, HC-CDR2, and HC-C VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of DR3 and SEQ ID NO: 69; (lxxix) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 62, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 70; (lxxx) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 62, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 71;(lxxxi) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 62, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 72; (lxxxii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 62, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 73; (lxxxiii) VH HC-CDR1, HC-CDR2, and HC-C VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of DR3 and SEQ ID NO: 65; (lxxxiv) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 63, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 66; (lxxxv) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 63, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 67; (lxxxvi) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 68; (lxxxvii) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 69; (lxxxviii) HC-CDR1, HC-CDR2, and H of VH containing the amino acid sequence of SEQ ID NO: 63 VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of C-CDR3 and SEQ ID NO: 70; (lxxxix) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 63, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 71; (xc) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 63, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 72;(xci) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 73; (xcii) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 65; (xciii) VH containing the amino acid sequence of SEQ ID NO: 64 VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of HC-CDR1, HC-CDR2, and HC-CDR3 of SEQ ID NO: 66; (xciv) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 64; VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 67; (xcv) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 64, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 68; (xcvi) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 64, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 69; (xcvii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 64, and VL LC- CDR1, LC-CDR2, and LC-CDR3; (xcviii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 64, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 71; (xcix) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 64, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 72;Alternatively, (c) the anti-LAMA2 antibody site includes VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 64, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-LAMA2 antibody site includes HC-CDR1 containing the amino acid sequence of SEQ ID NO: 1, HC-CDR2 containing the amino acid sequence of SEQ ID NO: 2, HC-CDR3 containing the amino acid sequence of SEQ ID NO: 3, LC-CDR1 containing the amino acid sequence of SEQ ID NO: 4, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-LAMA2 antibody site includes VH containing the amino acid sequence of SEQ ID NO: 49, and VL containing the amino acid sequence of SEQ ID NO: 50.

[0011] In some embodiments of any of the bifunctional protein constructs described above, the anti-LAMA2 antibody site is anti-LAMA2 scFv. In some embodiments, anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the anti-LAMA2 antibody site is anti-LAMA2 Fab. In some embodiments, anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 52 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 53.

[0012] In some embodiments of any of the bifunctional protein constructs described above, the anti-LAMA2 antibody site includes HC-CDR1 containing the amino acid sequence of SEQ ID NO: 7, HC-CDR2 containing the amino acid sequence of SEQ ID NO: 8, HC-CDR3 containing the amino acid sequence of SEQ ID NO: 9, LC-CDR1 containing the amino acid sequence of SEQ ID NO: 10, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 11, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 12.

[0013] In some embodiments of any of the bifunctional protein constructs described above, the anti-LAMA2 antibody site includes VH, which contains the amino acid sequence of SEQ ID NO: 54, and VL, which contains the amino acid sequence of SEQ ID NO: 65. In some embodiments, the anti-LAMA2 antibody site is: (i) VH containing the amino acid sequence of SEQ ID NO: 54 and VL containing the amino acid sequence of SEQ ID NO: 65; (ii) VH containing the amino acid sequence of SEQ ID NO: 54 and VL containing the amino acid sequence of SEQ ID NO: 66; (iii) VH containing the amino acid sequence of SEQ ID NO: 54 and VL containing the amino acid sequence of SEQ ID NO: 67; (iv) VH containing the amino acid sequence of SEQ ID NO: 54 and VL containing the amino acid sequence of SEQ ID NO: 68; (v) VH containing the amino acid sequence of SEQ ID NO: 54 and VL containing the amino acid sequence of SEQ ID NO: 69; (vi) VH containing the amino acid sequence of SEQ ID NO: 54 and VL containing the amino acid sequence of SEQ ID NO: 70; (vii) VH containing the amino acid sequence of SEQ ID NO: 54 and VL containing the amino acid sequence of SEQ ID NO: 71; (viii) VH containing the amino acid sequence of SEQ ID NO: 54 and VL containing the amino acid sequence of SEQ ID NO: 72; (ix) VH containing the amino acid sequence of SEQ ID NO: 54 and VL containing the amino acid sequence of SEQ ID NO: 73; (x) amino acid sequence of SEQ ID NO: 55 VH containing the sequence, and VL containing the amino acid sequence of SEQ ID NO: 65; (xi) VH containing the amino acid sequence of SEQ ID NO: 55, and VL containing the amino acid sequence of SEQ ID NO: 66; (xii) VH containing the amino acid sequence of SEQ ID NO: 55, and VL containing the amino acid sequence of SEQ ID NO: 67; (xiii) VH containing the amino acid sequence of SEQ ID NO: 55, and VL containing the amino acid sequence of SEQ ID NO: 68; (xiv) VH containing the amino acid sequence of SEQ ID NO: 55, and VL containing the amino acid sequence of SEQ ID NO: 69; (xv) VH containing the amino acid sequence of SEQ ID NO: 55, and VL containing the amino acid sequence of SEQ ID NO: 70; (xvi) VH containing the amino acid sequence of SEQ ID NO: 55, and VL containing the amino acid sequence of SEQ ID NO: 71; (xvii) VH containing the amino acid sequence of SEQ ID NO: 55, and VL containing the amino acid sequence of SEQ ID NO: 72; (xviii) VH containing the amino acid sequence of SEQ ID NO: 55, and VL containing the amino acid sequence of SEQ ID NO: 73; (xix) VH containing the amino acid sequence of SEQ ID NO: 56, and VL containing the amino acid sequence of SEQ ID NO: 65;(xx) VH containing the amino acid sequence of SEQ ID NO: 56, and VL containing the amino acid sequence of SEQ ID NO: 66; (xxi) VH containing the amino acid sequence of SEQ ID NO: 56, and VL containing the amino acid sequence of SEQ ID NO: 67; (xxii) VH containing the amino acid sequence of SEQ ID NO: 56, and VL containing the amino acid sequence of SEQ ID NO: 68; (xxiii) VH containing the amino acid sequence of SEQ ID NO: 56, and VL containing the amino acid sequence of SEQ ID NO: 69; (xxiv) VH containing the amino acid sequence of SEQ ID NO: 56, and VL containing the amino acid sequence of SEQ ID NO: 70; (xxv) amino VH containing the amino acid sequence, and VL containing the amino acid sequence of SEQ ID NO: 71; (xxvi) VH containing the amino acid sequence of SEQ ID NO: 56, and VL containing the amino acid sequence of SEQ ID NO: 72; (xxvii) VH containing the amino acid sequence of SEQ ID NO: 56, and VL containing the amino acid sequence of SEQ ID NO: 73; (xxviii) VH containing the amino acid sequence of SEQ ID NO: 57, and VL containing the amino acid sequence of SEQ ID NO: 65; (xxix) VH containing the amino acid sequence of SEQ ID NO: 57, and VL containing the amino acid sequence of SEQ ID NO: 66; (xxx) VH containing the amino acid sequence of SEQ ID NO: 57, and VL containing the amino acid sequence of sequence number 67; (xxxi) VH containing the amino acid sequence of sequence number 57, and VL containing the amino acid sequence of sequence number 68; (xxxii) VH containing the amino acid sequence of sequence number 57, and VL containing the amino acid sequence of sequence number 69; (xxxiii) VH containing the amino acid sequence of sequence number 57, and VL containing the amino acid sequence of sequence number 70; (xxxiv) VH containing the amino acid sequence of sequence number 57, and VL containing the amino acid sequence of sequence number 71; (xxxv) VH containing the amino acid sequence of sequence number 57, and the amino acid sequence of sequence number 72 VL containing the amino acid sequence; (xxxvi) VH containing the amino acid sequence of SEQ ID NO: 57, and VL containing the amino acid sequence of SEQ ID NO: 73; (xxxvii) VH containing the amino acid sequence of SEQ ID NO: 58, and VL containing the amino acid sequence of SEQ ID NO: 65; (xxxviii) VH containing the amino acid sequence of SEQ ID NO: 58, and VL containing the amino acid sequence of SEQ ID NO: 66; (xxxix) VH containing the amino acid sequence of SEQ ID NO: 58, and VL containing the amino acid sequence of SEQ ID NO: 67; (xl) VH containing the amino acid sequence of SEQ ID NO: 58, and VL containing the amino acid sequence of SEQ ID NO: 68;(xli) VH containing the amino acid sequence of SEQ ID NO: 58, and VL containing the amino acid sequence of SEQ ID NO: 69; (xlii) VH containing the amino acid sequence of SEQ ID NO: 58, and VL containing the amino acid sequence of SEQ ID NO: 70; (xliii) VH containing the amino acid sequence of SEQ ID NO: 58, and VL containing the amino acid sequence of SEQ ID NO: 71; (xliv) VH containing the amino acid sequence of SEQ ID NO: 58, and VL containing the amino acid sequence of SEQ ID NO: 72; (xlv) VH containing the amino acid sequence of SEQ ID NO: 58, and VL containing the amino acid sequence of SEQ ID NO: 73; (xlvi) containing the amino acid sequence of SEQ ID NO: 59 VH, and VL containing the amino acid sequence of SEQ ID NO: 65; (xlvii) VH containing the amino acid sequence of SEQ ID NO: 59, and VL containing the amino acid sequence of SEQ ID NO: 66; (xlviii) VH containing the amino acid sequence of SEQ ID NO: 59, and VL containing the amino acid sequence of SEQ ID NO: 67; (xlix) VH containing the amino acid sequence of SEQ ID NO: 59, and VL containing the amino acid sequence of SEQ ID NO: 68; (l) VH containing the amino acid sequence of SEQ ID NO: 59, and VL containing the amino acid sequence of SEQ ID NO: 69; (li) VH containing the amino acid sequence of SEQ ID NO: 59, and VL containing the amino acid sequence of SEQ ID NO: 70 VL;(lii) VH containing the amino acid sequence of SEQ ID NO: 59, and VL;(liii) VH containing the amino acid sequence of SEQ ID NO: 59, and VL;(liv) VH containing the amino acid sequence of SEQ ID NO: 59, and VL;(lv) VH containing the amino acid sequence of SEQ ID NO: 60, and VL;(lvi) VH containing the amino acid sequence of SEQ ID NO: 60, and VL;(lvii59, and VL;(lvii) VH containing the amino acid sequence of SEQ ID NO: 71, and VL;(lvii) VH containing the amino acid sequence of SEQ ID NO: 72, and VL;(lvii) VH containing the amino VH, and VL containing the amino acid sequence of SEQ ID NO: 67; (lviii) VH containing the amino acid sequence of SEQ ID NO: 60, and VL containing the amino acid sequence of SEQ ID NO: 68; (lix) VH containing the amino acid sequence of SEQ ID NO: 60, and VL containing the amino acid sequence of SEQ ID NO: 69; (lx) VH containing the amino acid sequence of SEQ ID NO: 60, and VL containing the amino acid sequence of SEQ ID NO: 70; (lxi) VH containing the amino acid sequence of SEQ ID NO: 60, and VL containing the amino acid sequence of SEQ ID NO: 71; (lxii) VH containing the amino acid sequence of SEQ ID NO: 60, and VL containing the amino acid sequence of SEQ ID NO: 72;(lxiii) VH containing the amino acid sequence of SEQ ID NO: 60, and VL containing the amino acid sequence of SEQ ID NO: 73; (lxiv) VH containing the amino acid sequence of SEQ ID NO: 61, and VL containing the amino acid sequence of SEQ ID NO: 65; (lxv) VH containing the amino acid sequence of SEQ ID NO: 61, and VL containing the amino acid sequence of SEQ ID NO: 66; (lxvi) VH containing the amino acid sequence of SEQ ID NO: 61, and VL containing the amino acid sequence of SEQ ID NO: 67; (lxvii) VH containing the amino acid sequence of SEQ ID NO: 61, and VL containing the amino acid sequence of SEQ ID NO: 68; (lxviii) SEQ ID NO: 61 VH containing the amino acid sequence, and VL containing the amino acid sequence of SEQ ID NO: 69; (lxix) VH containing the amino acid sequence of SEQ ID NO: 61, and VL containing the amino acid sequence of SEQ ID NO: 70; (lxx) VH containing the amino acid sequence of SEQ ID NO: 61, and VL containing the amino acid sequence of SEQ ID NO: 71; (lxxi) VH containing the amino acid sequence of SEQ ID NO: 61, and VL containing the amino acid sequence of SEQ ID NO: 72; (lxxii) VH containing the amino acid sequence of SEQ ID NO: 61, and VL containing the amino acid sequence of SEQ ID NO: 73; (lxxiii) VH containing the amino acid sequence of SEQ ID NO: 62, and VL containing the amino acid sequence of SEQ ID NO: 65; (lxxiv) VH containing the amino acid sequence of SEQ ID NO: 62, VL containing the amino acid sequence of SEQ ID NO: 66; (lxxv) VH containing the amino acid sequence of SEQ ID NO: 62, VL containing the amino acid sequence of SEQ ID NO: 67; (lxxvi) VH containing the amino acid sequence of SEQ ID NO: 62, VL containing the amino acid sequence of SEQ ID NO: 68; (lxxvii) VH containing the amino acid sequence of SEQ ID NO: 62, VL containing the amino acid sequence of SEQ ID NO: 69; (lxxviii) VH containing the amino acid sequence of SEQ ID NO: 62, and SEQ ID NO: 70 VL containing the no-acid sequence; (lxxix) VH containing the amino acid sequence of SEQ ID NO 62, and VL containing the amino acid sequence of SEQ ID NO 71; (lxxx) VH containing the amino acid sequence of SEQ ID NO 62, and VL containing the amino acid sequence of SEQ ID NO 72; (lxxxi) VH containing the amino acid sequence of SEQ ID NO 62, and VL containing the amino acid sequence of SEQ ID NO 73; (lxxxii) VH containing the amino acid sequence of SEQ ID NO 63, and VL containing the amino acid sequence of SEQ ID NO 65; (lxxxiii) VH containing the amino acid sequence of SEQ ID NO 63, and VL containing the amino acid sequence of SEQ ID NO 66;(lxxxiv) a VH comprising the amino acid sequence of SEQ ID NO: 63, and a VL comprising the amino acid sequence of SEQ ID NO: 67; (lxxxv) a VH comprising the amino acid sequence of SEQ ID NO: 63, and a VL comprising the amino acid sequence of SEQ ID NO: 68; (lxxxvi) a VH comprising the amino acid sequence of SEQ ID NO: 63, and a VL comprising the amino acid sequence of SEQ ID NO: 69; (lxxxvii) a VH comprising the amino acid sequence of SEQ ID NO: 63, and a VL comprising the amino acid sequence of SEQ ID NO: 70; (lxxxviii) a VH comprising the amino acid sequence of SEQ ID NO: 63, and a VL comprising the amino acid sequence of SEQ ID NO: 71; (lxxxix) a VH comprising the amino acid sequence of SEQ ID NO: 63, and a VL comprising the amino acid sequence of SEQ ID NO: 72; (xc) a VH comprising the amino acid sequence of SEQ ID NO: 63, and a VL comprising the amino acid sequence of SEQ ID NO: 73; (xci) a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL comprising the amino acid sequence of SEQ ID NO: 65; (xcii) a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL comprising the amino acid sequence of SEQ ID NO: 66; (xciii) a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL comprising the amino acid sequence of SEQ ID NO: 67; (xciv) a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL comprising the amino acid sequence of SEQ ID NO: 68; (xcv) a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL comprising the amino acid sequence of SEQ ID NO: 69; (xcvi) a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL comprising the amino acid sequence of SEQ ID NO: 70; (xcvii) a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL comprising the amino acid sequence of SEQ ID NO: 71; (xcviii) a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL comprising the amino acid sequence of SEQ ID NO: 72; or (xcix) a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL comprising the amino acid sequence of SEQ ID NO: 73.;

[0014] In some embodiments according to any of the bifunctional protein constructs described above, the anti-LAMA2 antibody moiety is an anti-LAMA2 scFv. In some embodiments, the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145.

[0015] In some embodiments of any of the bifunctional protein constructs described above, the anti-LAMA2 antibody site is an anti-LAMA2 Fab. In some embodiments, the anti-LAMA2 Fab comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 165 and a second polypeptide having the amino acid sequence of SEQ ID NO: 166.

[0016] In some embodiments of any of the bifunctional protein constructs described above, the antibody site specifically binds to the matriglycan on α-DG (anti-matriglycan antibody site). In some embodiments, the anti-matriglycan antibody site is (i) HC-CDR1 containing the amino acid sequence of SEQ ID NO: 74, HC-CDR2 containing the amino acid sequence of SEQ ID NO: 75, HC-CDR3 containing the amino acid sequence of SEQ ID NO: 76, LC-CDR1 containing the amino acid sequence of SEQ ID NO: 77, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 78, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 79; (ii) HC-CDR1 and HC-CDR of VH containing the amino acid sequence of SEQ ID NO: 90 (iii) LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of 2, HC-CDR3, and SEQ ID NO: 95; (iv) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 90, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 96; (iv) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 90, and SEQ ID NO: 97 VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of (v) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 90, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 98; (vi) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 91, and VL LC-CDR1 containing the amino acid sequence of SEQ ID NO: 95, LC-CDR2 and LC-CDR3; (vii) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 96; (viii) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 97;(ix) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98; (x) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95; (xi) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96; (xii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97; (xiii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98; (xiv) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95; (xv) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96; (xvi) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97; or (xvii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, the anti-matriglycan antibody moiety comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 89 to 93, and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 94 to 98.;

[0017] In some embodiments of any of the bifunctional protein constructs described above, the anti-matriglycan antibody site is: (i) VH containing the amino acid sequence of SEQ ID NO: 89 and VL containing the amino acid sequence of SEQ ID NO: 94; (ii) VH containing the amino acid sequence of SEQ ID NO: 90 and VL containing the amino acid sequence of SEQ ID NO: 95; (iii) VH containing the amino acid sequence of SEQ ID NO: 90 and VL containing the amino acid sequence of SEQ ID NO: 96; (iv) VH containing the amino acid sequence of SEQ ID NO: 90 and VL containing the amino acid sequence of SEQ ID NO: 97; (v) VH containing the amino acid sequence of SEQ ID NO: 90 and VL containing the amino acid sequence of SEQ ID NO: 98; (vi) VH containing the amino acid sequence of SEQ ID NO: 91 and VL containing the amino acid sequence of SEQ ID NO: 95; (vii) VH containing the amino acid sequence of SEQ ID NO: 91 and VL containing the amino acid sequence of SEQ ID NO: 96; (viii) VH containing the amino acid sequence of SEQ ID NO: 91 and VL containing the amino acid sequence of SEQ ID NO: 97; ( ix) VH containing the amino acid sequence of SEQ ID NO: 91, and VL containing the amino acid sequence of SEQ ID NO: 98; (x) VH containing the amino acid sequence of SEQ ID NO: 92, and VL containing the amino acid sequence of SEQ ID NO: 95; (xi) VH containing the amino acid sequence of SEQ ID NO: 92, and VL containing the amino acid sequence of SEQ ID NO: 96; (xii) VH containing the amino acid sequence of SEQ ID NO: 92, and VL containing the amino acid sequence of SEQ ID NO: 97; (xiii) VH containing the amino acid sequence of SEQ ID NO: 92, and VL containing the amino acid sequence of SEQ ID NO: 98; (xiv) VH containing the amino acid sequence of SEQ ID NO: 93, and VL containing the amino acid sequence of SEQ ID NO: 95; (xv) VH containing the amino acid sequence of SEQ ID NO: 93, and VL containing the amino acid sequence of SEQ ID NO: 96; (xxvi) VH containing the amino acid sequence of SEQ ID NO: 93, and VL containing the amino acid sequence of SEQ ID NO: 97; or (xxvii) VH containing the amino acid sequence of SEQ ID NO: 93, and VL containing the amino acid sequence of SEQ ID NO: 98.

[0018] In some embodiments of any of the bifunctional protein constructs described above, the anti-matriglycan antibody site is an anti-matriglycan scFv. In some embodiments, the anti-matriglycan scFv contains the amino acid sequence of SEQ ID NO: 99.

[0019] In some embodiments of any of the bifunctional protein constructs described above, the anti-matriglycan antibody site is an anti-matriglycan Fab. In some embodiments, the anti-matriglycan Fab comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 100 and a second polypeptide having the amino acid sequence of SEQ ID NO: 101.

[0020] In some embodiments of any of the bifunctional protein constructs described above, the antibody site specifically binds to CDH15 (anti-CDH15 antibody site). In some embodiments, the anti-CDH15 antibody site includes HC-CDR1 containing the amino acid sequence of SEQ ID NO: 102, HC-CDR2 containing the amino acid sequence of SEQ ID NO: 103, HC-CDR3 containing the amino acid sequence of SEQ ID NO: 104, LC-CDR1 containing the amino acid sequence of SEQ ID NO: 105, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 106, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 107.

[0021] In some embodiments of any of the bifunctional protein constructs described above, the anti-CDH15 antibody site comprises VH, which contains the amino acid sequence of SEQ ID NO: 108, and VL, which contains the amino acid sequence of SEQ ID NO: 109.

[0022] In some embodiments of any of the bifunctional protein constructs described above, the anti-CDH15 antibody site is anti-CDH15 scFv. In some embodiments, anti-CDH15 scFv contains the amino acid sequence of SEQ ID NO: 110.

[0023] In some embodiments of any of the bifunctional protein constructs described above, the anti-CDH15 antibody site is anti-CDH15 Fab. In some embodiments, anti-CDH15 Fab comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 111 and a second polypeptide having the amino acid sequence of SEQ ID NO: 112.

[0024] In some embodiments of any of the bifunctional protein constructs described above, the first binding site includes a non-antibody site that specifically binds to muscle-specific molecules. In some embodiments, the non-antibody site includes a protein domain selected from the group consisting of the laminin G-like domain (LG domain) of laminin, the LG domain of agryn, the LG domain of nidogen, the LG domain of perlecan, the laminin coiled-coil binding domain of agryn, and the laminin γ-binding domain of nidogen. In some embodiments, the non-antibody site includes the LG domain of laminin. In some embodiments, the non-antibody site includes the LG domain of laminin subunit α-1 (LAMA1), LAMA2, laminin subunit α-3 (LAMA3), laminin subunit α-4 (LAMA4), or laminin subunit α-5 (LAMA5). In some embodiments, the non-antibody site includes any of the amino acid sequences of SEQ ID NOs: 113-117.

[0025] In some embodiments of any of the bifunctional protein constructs described above, the non-antibody site includes the LG domain of LAMA2. In some embodiments, the non-antibody site includes the LG4-5 domain of LAMA2 (LAMA2 LG4-5). In some embodiments, LAMA2 LG4-5 includes the amino acid sequence of SEQ ID NO: 118 or 119.

[0026] In some embodiments of any of the bifunctional protein constructs described above, the non-antibody site comprises the LG domain of perlecan. In some embodiments, the non-antibody site comprises the amino acid sequence of SEQ ID NO: 120.

[0027] In some embodiments of any of the bifunctional protein constructs described above, the non-antibody site includes the LG domain of agryn. In some embodiments, the non-antibody site includes the amino acid sequence of SEQ ID NO: 121.

[0028] In some embodiments of any of the bifunctional protein constructs described above, the non-antibody site includes the laminin coiled-coil binding domain of agryn. In some embodiments, the laminin coiled-coil binding domain of agryn includes the amino acid sequence of SEQ ID NO: 122.

[0029] In some embodiments of any of the bifunctional protein constructs described above, the non-antibody site includes the laminin-γ binding domain of nidogen. In some embodiments, the laminin-γ binding domain of nidogen includes the amino acid sequence of SEQ ID NO: 123 or 124.

[0030] In some embodiments of any of the bifunctional protein constructs described above, the second binding site includes the extracellular domain (ECD) or a variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jagged 1 (Jag1), and Jag2.

[0031] In some embodiments of any of the bifunctional protein constructs described above, the second binding site includes DLL4 ECD or a variant thereof. In some embodiments, the second binding site includes DLL4 ECD, which includes the amino acid sequence of SEQ ID NO: 125. In some embodiments, the second binding site includes a variant DLL4 ECD, which includes the MNNL domain, DSL domain, and EGF1-5 domain of DLL4, or a sequence having at least about 90% sequence identity with the sequences of the MNNL domain, DSL domain, and EGF1-5 domain of DLL4. In some embodiments, the variant DLL4 ECD includes the amino acid sequence of SEQ ID NOs: 126-129 and 260-267.

[0032] In some embodiments of any of the bifunctional protein constructs described above, the second binding site comprises DLL1 ECD or a variant thereof. In some embodiments, the second binding site comprises DLL1 ECD, which comprises the amino acid sequence of SEQ ID NO: 130.

[0033] In some embodiments of any of the bifunctional protein constructs described above, the second binding site comprises DLL3 ECD or a variant thereof. In some embodiments, the second binding site comprises DLL3 ECD, which comprises the amino acid sequence of SEQ ID NO: 131.

[0034] In some embodiments of any of the bifunctional protein constructs described above, the second binding site includes Jag1 ECD or a variant thereof. In some embodiments, the second binding site includes Jag1 ECD, which includes the amino acid sequence of SEQ ID NO: 132. In some embodiments, the second binding site includes a variant Jag1 ECD, which includes the MNNL domain, DSL domain, and EGF1-6 domain of Jag1, or a sequence having at least about 90% sequence identity with the sequences of the MNNL domain, DSL domain, and EGF1-6 domain of Jag1. In some embodiments, the variant Jag1 ECD includes the amino acid sequence of SEQ ID NO: 133 or 134.

[0035] In some embodiments of any of the bifunctional protein constructs described above, the second binding site comprises Jag2 ECD or a variant thereof. In some embodiments, the second binding site comprises Jag2 ECD, which comprises the amino acid sequence of SEQ ID NO: 135.

[0036] In some embodiments of any of the bifunctional protein constructs described above, the second binding site is an anti-Notch antibody site that activates the Notch receptor. In some embodiments, the anti-Notch agonist antibody site is selected from the group consisting of full-length antibody, Fab, Fab', F(ab')2, scFv, and sdAb.

[0037] In some embodiments of any of the difunctional protein constructs described above, the first binding site is fused to the second binding site via an optional linker. In some embodiments, the first binding site is fused to the N-terminus of the second binding site. In some embodiments, the first binding site is fused to the C-terminus of the second binding site. In some embodiments, the difunctional protein construct includes a first binding site comprising (i) a second binding site comprising the ECD or a variant of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2; and (ii) (a) an antibody site, wherein the antibody site is scFv, Fab, or sdAb that specifically binds to muscle-specific molecules; or (b) a non-antibody site, wherein the non-antibody site comprises a protein domain selected from the group consisting of the LG domain of laminin, the LG domain of agryn, the LG domain of nidogen, and the LG domain of perlecan. In some embodiments, the first binding site includes two or more non-antibody sites connected in tandem.

[0038] In some embodiments of any of the difunctional protein constructs described above, the first binding site includes LAMA2 LG4-5 containing the amino acid sequence of SEQ ID NO: 118 or 119. In some embodiments, the second binding site includes variant DLL4 ECD, which contains the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267. In some embodiments, the difunctional protein construct includes the amino acid sequence of any of SEQ ID NOs: 136-139. In some embodiments, the second binding site includes DLL1 ECD, which contains the amino acid sequence of SEQ ID NO: 130. In some embodiments, the difunctional protein construct includes the amino acid sequence of SEQ ID NO: 140. In some embodiments, the second binding site includes DLL3 ECD, which contains the amino acid sequence of SEQ ID NO: 131. In some embodiments, the difunctional protein construct includes the amino acid sequence of SEQ ID NO: 141. In some embodiments, the second binding site includes a variant Jag1 ECD, which contains the amino acid sequence of SEQ ID NO: 133 or 134. In some embodiments, the bifunctional protein construct contains the amino acid sequence of SEQ ID NO: 142 or 143. In some embodiments, the second binding site includes a Jag2 ECD, which contains the amino acid sequence of SEQ ID NO: 135. In some embodiments, the bifunctional protein construct contains the amino acid sequence of SEQ ID NO: 144.

[0039] In some embodiments of any of the bifunctional protein constructs described above, the first binding site includes anti-LAMA2 scFv. In some embodiments, anti-LAMA2 scFv includes the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct includes the amino acid sequence of any of SEQ ID NOs: 146-154.

[0040] In some embodiments of any of the difunctional protein constructs described above, the first binding site includes an anti-matriglycan scFv. In some embodiments, the anti-matriglycan scFv includes the amino acid sequence of SEQ ID NO: 155. In some embodiments, the difunctional protein construct includes the amino acid sequence of any of SEQ ID NOs: 156-164.

[0041] In some embodiments of any of the bifunctional protein constructs described above, the first binding site includes Fab, which specifically binds to muscle-specific molecules. In some embodiments, the second binding site is fused to the N-terminus of the VL of Fab via an optional linker.

[0042] In some embodiments of any of the difunctional protein constructs described above, the first binding site comprises an anti-LAMA2 Fab. In some embodiments, the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the difunctional protein construct comprises a fusion polypeptide comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second binding site and the anti-LAMA2 Fab second polypeptide, the fusion polypeptide comprising any of the amino acid sequences of SEQ ID NOs: 167-175.

[0043] In some embodiments of any of the difunctional protein constructs described above, the first binding site comprises an anti-matriglycan Fab. In some embodiments, the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the difunctional protein construct comprises a fusion polypeptide comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second binding site and the second polypeptide of anti-matriglycerin Fab, wherein the fusion polypeptide comprises any of the amino acid sequences of SEQ ID NOs: 178-186.

[0044] In some embodiments of any of the bifunctional protein constructs described above, the first binding site is fused to the second binding site via a carrier protein. In some embodiments, the carrier protein is selected from the group consisting of human serum albumin (HSA), an anti-HSA antibody site, and a subunit of the Fc domain. In some embodiments, the first binding site is fused to the N-terminus of the carrier protein via an optional first linker, and the second binding site is fused to the C-terminus of the carrier protein via an optional second linker. In some embodiments, the first binding site is fused to the C-terminus of the carrier protein via an optional first linker, and the second binding site is fused to the N-terminus of the carrier protein via an optional second linker. In some embodiments, the bifunctional protein construct includes a first binding site comprising (i) a second binding site comprising an ECD or variant of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2; and (ii)(a) an antibody site, the antibody site being scFv, Fab, or sdAb that specifically binds to muscle-specific molecules; or (b) a non-antibody site, the non-antibody site comprising a protein domain selected from the group consisting of the LG domain of laminin, the LG domain of agryn, the LG domain of nidogen, and the LG domain of perlecan. In some embodiments, the first binding site comprises two or more non-antibody sites connected in tandem.

[0045] In some embodiments of any of the difunctional protein constructs described above, the difunctional protein construct further comprises an Fc domain containing a first subunit and a second subunit. In some embodiments, the Fc domain is derived from human IgG1, human IgG2, or human IgG4. In some embodiments, the Fc domain is derived from human IgG1 containing the amino acid sequence of Sequence ID No. 187. In some embodiments, each subunit of the Fc domain contains a mutation that reduces or disables effector function. In some embodiments, each subunit of the Fc domain contains an L234A / L235A mutation (EU numbering).

[0046] In some embodiments of any of the bifunctional protein constructs described above, either the first subunit of the Fc domain or the second subunit of the Fc domain contains the H435R / Y436F mutation (EU numbering). In some embodiments, the Fc domain contains a knob-into-hole mutation, i) the first subunit of the Fc domain contains the knob mutation and the second subunit of the Fc domain contains the hole mutation; or ii) the second subunit of the Fc domain contains the knob mutation and the first subunit of the Fc domain contains the hole mutation. In some embodiments, the knob mutation is T366W (EU numbering) and the hole mutation is T366S / L368A / Y407V (EU numbering). In some embodiments, the Fc domain comprises a charge pair mutation such that i) an amino acid residue in the first subunit of the Fc domain is replaced with a positively charged residue and an amino acid residue in the second subunit of the Fc domain is replaced with a negatively charged residue; or ii) an amino acid residue in the first subunit of the Fc domain is replaced with a negatively charged residue and an amino acid residue in the second subunit of the Fc domain is replaced with a positively charged residue. In some embodiments, the Fc domain is modified in the following ways: i) the amino acid residue at D399 (EU numbering) in the first subunit of the Fc domain is replaced with a positively charged residue, and the amino acid residue at K409 (EU numbering) in the second subunit of the Fc domain is replaced with a negatively charged residue; or ii) the amino acid residue at K409 (EU numbering) in the first subunit of the Fc domain is replaced with a negatively charged residue, and the amino acid residue at D399 (EU numbering) in the second subunit of the Fc domain is replaced with a positively charged residue.

[0047] In some embodiments of any of the bifunctional protein constructs described above, each subunit of the Fc domain contains a mutation that reduces the half-life. In some embodiments, each subunit of the Fc domain contains the H435A mutation (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 188.

[0048] In some embodiments of any of the difunctional protein constructs described above, the difunctional protein construct includes i) a first unit of a second binding site that specifically binds to a first Notch receptor, and ii) a second unit of a second binding site that specifically binds to a second Notch receptor, wherein the first unit of the second binding site and the second unit of the second binding site each independently include an ECD or variant of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2. In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site include a DLL4 ECD, wherein the DLL4 ECD includes one amino acid sequence of sequence numbers 125-129. In some embodiments, the first unit of the second binding site and the second unit of the second binding site are the same. In some embodiments, the first unit of the second binding site and the second unit of the second binding site are different. In some embodiments, the bifunctional protein construct includes i) a first unit of a first binding site that specifically binds to a first muscle-specific molecule, and ii) a second unit of the first binding site that specifically binds to a second muscle-specific molecule. In some embodiments, the first unit of the first binding site and the second unit of the first binding site are the same. In some embodiments, the first unit of the first binding site and the second unit of the first binding site are different. In some embodiments, the first unit of the first binding site includes a first antibody site that specifically binds to a first muscle-specific molecule, and the second unit of the first binding site includes a first antibody site that specifically binds to a second muscle-specific molecule, with the first antibody site and the second antibody site being independently selected from the group consisting of Fab, scFv, and sdAb.

[0049] In some embodiments of any of the bifunctional protein constructs described above, the first antibody site is the first Fab (Fab1), and the second antibody site is the second Fab (Fab2); the bifunctional protein construct comprises: i) a first polypeptide comprising: N' to C': a first unit of a second binding site that specifically binds to the first Notch receptor - an optional linker - a first subunit of an Fc domain - an optional linker - VH1-(H1-CH1); and ii) N' to C': the second Notch receptor The polypeptide comprises: iii) a second unit of a second binding site that specifically binds to the body - an optional linker - a second subunit of the Fc domain - an optional linker - VH2-(H2-CH1); iii) a third polypeptide comprising N' to C': VL1-(L1-CL); and iv) a fourth polypeptide comprising N' to C': VL2-(L2-CL), wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, the first antibody site is the first Fab (Fab1), and the second antibody site is the second Fab (Fab2); the bifunctional protein construct comprises: i) a first polypeptide comprising: a first unit of the second binding site that binds specifically to the first Notch receptor from N' to C' - an optional linker - a first subunit of the Fc domain - an optional linker - VL1-(L1-CL); and ii) a second binding site that binds specifically to the second Notch receptor from N' to C' The polypeptide comprises: a second unit of the site - an arbitrary linker - a second subunit of the Fc domain - an arbitrary linker - a second polypeptide containing VL2-(L2-CL); iii) N' to C': a third polypeptide containing VH1-(H1-CH1); and iv) N' to C': a fourth polypeptide containing VH2-(H2-CH1), where VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.

[0050] In some embodiments of any of the difunctional protein constructs described above, the difunctional protein construct comprises Fab1 and Fab2, both of which specifically bind to LAMA2. In some embodiments, anti-LAMA2 Fab1 and / or anti-LAMA2 Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site comprises a variant DLL4 ECD, wherein the variant DLL4 ECD comprises any of the amino acid sequences of SEQ ID NOs: 126-129 and 260-267. In some embodiments, the third and fourth polypeptides of the difunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 165, and the first and second polypeptides of the difunctional protein construct each comprise any of the amino acid sequences of SEQ ID NOs: 189-192. In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site comprises a DLL1 ECD, the DLL1 ECD comprising the amino acid sequence of SEQ ID NO: 130. In some embodiments, the third and fourth polypeptides of the difunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 165, and the first and second polypeptides of the difunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 193. In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site comprises a DLL3 ECD, the DLL3 ECD comprising the amino acid sequence of SEQ ID NO: 131. In some embodiments, the third and fourth polypeptides of the difunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 165, and the first and second polypeptides of the difunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 194. In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site comprises a variant Jag1 ECD, the variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133 or 134.In some embodiments, the third and fourth polypeptides of the difunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 165, and the first and second polypeptides of the difunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 195 or 196. In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site comprises a Jag2 ECD, and the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the third and fourth polypeptides of the difunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 165, and the first and second polypeptides of the difunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 197.

[0051] In some embodiments of any of the difunctional protein constructs described above, the difunctional protein construct comprises Fab1 and Fab2, both of which specifically bind to matriglycan. In some embodiments, anti-matriglycan Fab1 and / or anti-matriglycan Fab2 comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 176 and a second polypeptide having the amino acid sequence of SEQ ID NO: 177. In some embodiments, the third and fourth polypeptides of the difunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 176, wherein (i) the first and second polypeptides of the difunctional protein construct each comprise the amino acid sequence of any of SEQ ID NOs: 198 to 201; (ii) the first and second polypeptides of the difunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 202; (iii) the first and second polypeptides of the difunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 203; (iv) the first and second polypeptides of the difunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 204 or 205; or (v) the first and second polypeptides of the difunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 206.

[0052] In some embodiments of any of the difunctional protein constructs described above, the first antibody site is a first sdAb (sdAb1), and the second antibody site is a second sdAb (sdAb2); the difunctional protein construct comprises i) a first polypeptide comprising a first unit of a second binding site that specifically binds to a first Notch receptor from N' to C' - an optional linker - a first subunit of an Fc domain - an optional linker - sdAb1; and ii) a second polypeptide comprising a second unit of a second binding site that specifically binds to a second Notch receptor from N' to C' - an optional linker - a second subunit of an Fc domain - an optional linker - sdAb2. In some embodiments, the first antibody site is a first scFv(scFv1), and the second antibody site is a second scFv(scFv2); the bifunctional protein construct comprises i) a first polypeptide comprising a first unit of a second binding site that specifically binds to a first Notch receptor from N' to C' - an optional linker - a first subunit of an Fc domain - an optional linker - scFv1; and ii) a second polypeptide comprising a second unit of a second binding site that specifically binds to a second Notch receptor from N' to C' - an optional linker - a second subunit of an Fc domain - an optional linker - scFv2.

[0053] In some embodiments of any of the difunctional protein constructs described above, the difunctional protein construct includes scFv1 and scFv2, both of which specifically bind to LAMA2. In some embodiments, anti-LAMA2 scFv1 and / or anti-LAMA2 scFv2 include the amino acid sequence of SEQ ID NO: 145 or 268. In some embodiments, the linker includes the amino acid sequence of any one of SEQ ID NOs: 207-222. In some embodiments, the first subunit of the Fc domain and the second subunit of the Fc domain each include the amino acid sequence of SEQ ID NO: 188. In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site includes a variant DLL4 ECD, and the variant DLL4 ECD includes the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267. In some embodiments, the difunctional protein construct includes a first polypeptide and a second polypeptide each containing the amino acid sequence of any one of SEQ ID NOs: 223-226. In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site comprises a DLL1 ECD, the DLL1 ECD comprising the amino acid sequence of SEQ ID NO: 130. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 227. In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site comprises a DLL3 ECD, the DLL3 ECD comprising the amino acid sequence of SEQ ID NO: 131. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 228. In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site comprises a variant Jag1 ECD, the variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133 or 134. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each having the amino acid sequence of SEQ ID NO: 229 or 230, respectively.In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site comprises a Jag2 ECD, the Jag2 ECD comprising the amino acid sequence of SEQ ID NO: 135. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 231.

[0054] In some embodiments of any of the difunctional protein constructs described above, the difunctional protein construct comprises scFv1 and scFv2, both of which bind specifically to matriglycan. In some embodiments, the difunctional protein construct comprises a first polypeptide and a second polypeptide, each comprising (i) any one amino acid sequence of SEQ ID NOs. 232-235; (ii) the amino acid sequence of SEQ ID NOs. 236; (iii) the amino acid sequence of SEQ ID NOs. 237; (iv) the amino acid sequence of SEQ ID NOs. 238 or 239; or (v) the amino acid sequence of SEQ ID NOs. 240, respectively.

[0055] In some embodiments of any of the difunctional protein constructs described above, the first antibody site is a first Fab (Fab1), and the second antibody site is a second Fab (Fab2); the difunctional protein construct comprises: i) a first polypeptide comprising a first unit of a second binding site that specifically binds to a first Notch receptor, with the following characteristics: N' to C':VH1-(H1-CH1)-optional linker-first subunit of an Fc domain-optional linker; ii) N' to C':VH2-(H2-CH1) 1) A second polypeptide comprising a second unit of a second binding site that specifically binds to an arbitrary linker-Fc domain-any linker-second Notch receptor; iii) a third polypeptide comprising N' to C':VL1-(L1-CL); and iv) a fourth polypeptide comprising N' to C':VL2-(L2-CL); VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.

[0056] In some embodiments of any of the difunctional protein constructs described above, the first antibody site is a first sdAb (sdAb1), and the second antibody site is a second sdAb (sdAb2); the difunctional protein construct comprises i) a first polypeptide comprising a first unit of N' to C':sdAb1-any linker-first subunit of an Fc domain-any linker-second binding site that specifically binds to a first Notch receptor; and ii) a second polypeptide comprising a second unit of N' to C':sdAb2-any linker-second subunit of an Fc domain-any linker-second binding site that specifically binds to a second Notch receptor.

[0057] In some embodiments of any of the difunctional protein constructs described above, the first antibody site is a first scFv(scFv1), and the second antibody site is a second scFv(scFv2); the difunctional protein construct comprises i) a first polypeptide comprising a first unit of N' to C':scFv1 - any linker - a first subunit of the Fc domain - any linker - a second binding site that specifically binds to a first Notch receptor; and ii) a second polypeptide comprising a second unit of N' to C':scFv2 - any linker - a second subunit of the Fc domain - any linker - a second binding site that specifically binds to a second Notch receptor.

[0058] In some embodiments of any of the difunctional protein constructs described above, the first antibody site is a first Fab (Fab1), and the second antibody site is a second Fab (Fab2); the difunctional protein construct comprises: i) a first polypeptide comprising a first subunit of an N'-to-C':VH1-(H1-CH1)-any linker-Fc domain; ii) a second polypeptide comprising a second subunit of an N'-to-C':VH2-(H2-CH1)-any linker-Fc domain; ii i) From N' to C': VL1-(L1-CL)-any linker-a third polypeptide comprising a first unit of a second binding site that specifically binds to a first Notch receptor; and iv) From N' to C': VL2-(L2-CL)-any linker-a fourth polypeptide comprising a second unit of a second binding site that specifically binds to a second Notch receptor; VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.

[0059] In some embodiments of any of the difunctional protein constructs described above, the first antibody site is a first Fab (Fab1), and the second antibody site is a second Fab (Fab2); the difunctional protein construct comprises: i) a first polypeptide comprising a first subunit of an N'-to-C':VH1-(H1-CH1)-any linker-Fc domain; ii) a second polypeptide comprising a second subunit of an N'-to-C':VH2-(H2-CH1)-any linker-Fc domain; ii i) From N' to C': A third polypeptide comprising a first unit of a second binding site that specifically binds to a first Notch receptor - an optional linker - VL1-(L1-CL); and iv) From N' to C': A fourth polypeptide comprising a second unit of a second binding site that specifically binds to a second Notch receptor - an optional linker - VL2-(L2-CL); VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.

[0060] In some embodiments of any of the bifunctional protein constructs described above, the first antibody site is the first Fab (Fab1), and the second antibody site is the second Fab (Fab2); the bifunctional protein construct comprises: i) a first polypeptide comprising a first unit of a second binding site that specifically binds to the first Notch receptor (N' to C' - any linker - VH1 - (H1-CH1) - any linker - Fc domain); and ii) a second Notch receptor (N' to C' - any linker) A second polypeptide comprising a second unit of a second binding site that specifically binds to the body - an arbitrary linker - VH2-(H2-CH1) - an arbitrary linker - a second subunit of the Fc domain; iii) a third polypeptide comprising N' to C': VL1-(L1-CL); and iv) a fourth polypeptide comprising N' to C': VL2-(L2-CL); VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.

[0061] In some embodiments of any of the difunctional protein constructs described above, the difunctional protein construct further comprises a third unit of a second binding site that specifically binds to a third Notch receptor, and a fourth unit of a second binding site that specifically binds to a fourth Notch receptor, wherein the third unit of the second binding site and the fourth unit of the second binding site each independently comprise an ECD or variant of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2. In some embodiments, the third unit of the second binding site and the fourth unit of the second binding site each independently comprise an ECD or variant of DLL4. In some embodiments, the third unit of the second binding site and / or the fourth unit of the second binding site comprise a DLL4 ECD, wherein the DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 125. In some embodiments, the third unit and / or fourth unit of the second binding site comprises a variant DLL4 ECD, which comprises the MNNL domain, DSL domain, and EGF1-5 domain of DLL4. In some embodiments, the variant DLL4 ECD comprises one of the amino acid sequences of SEQ ID NOs. 126-129 and 260-267. In some embodiments, all four units of the second binding site are the same. In some embodiments, at least one of the four units of the second binding site is different from the others.

[0062] In some embodiments of any of the difunctional protein constructs described above, the first antibody site is the first Fab (Fab1), and the second antibody site is the second Fab (Fab2); the difunctional protein construct comprises: i) a first polypeptide comprising: N' to C': a first unit of a second binding site that specifically binds to the first Notch receptor - an arbitrary linker - VH1-(H1-CH1)-an arbitrary linker - a first subunit of an Fc domain; ii) N' to C': a second unit of a second binding site that specifically binds to the second Notch receptor - an arbitrary linker - VH2-(H2-CH1) 1) A second polypeptide comprising a second subunit of an arbitrary linker-Fc domain; iii) A third polypeptide comprising a third unit of a second binding site that specifically binds to a third Notch receptor from N' to C' - an arbitrary linker-VL1-(L1-CL); and iv) A fourth polypeptide comprising a fourth unit of a second binding site that specifically binds to a fourth Notch receptor from N' to C' - an arbitrary linker-VL2-(L2-CL); VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.

[0063] In some embodiments of any of the bifunctional protein constructs described above, the first unit of the first binding site includes a first non-antibody site that specifically binds to a first muscle-specific molecule, and the second unit of the first binding site includes a second non-antibody site that specifically binds to a second muscle-specific molecule, with the first and second non-antibody sites being independently selected from the group consisting of the LG domains of laminin, agryn, nidogen, and perlecan. In some embodiments, the first unit of the first binding site includes two or more first non-antibody sites connected in tandem, and the second unit of the first binding site includes two or more second non-antibody sites connected in tandem. In some embodiments, the first and / or second non-antibody sites include LAMA2 LG4-5 containing the amino acid sequence of SEQ ID NO: 118 or 119.

[0064] In some embodiments of any of the difunctional protein constructs described above, the difunctional protein construct comprises: i) a first polypeptide comprising: N' to C': a first unit of a first binding site - an optional linker - a first subunit of an Fc domain - an optional linker - a first unit of a second binding site that specifically binds to a first Notch receptor; and ii) a second polypeptide comprising: N' to C': a second unit of a first binding site - an optional linker - a second subunit of an Fc domain - an optional linker - a second unit of a second binding site that specifically binds to a second Notch receptor.

[0065] In some embodiments of any of the difunctional protein constructs described above, the difunctional protein construct comprises: i) a first polypeptide comprising: N' to C': a first unit of a second binding site that specifically binds to a first Notch receptor - an optional linker - a first subunit of an Fc domain - an optional linker - a first unit of the first binding site; and ii) N' to C': a second polypeptide comprising: a second unit of a second binding site that specifically binds to a second Notch receptor - an optional linker - a second subunit of an Fc domain - an optional linker - a second unit of the first binding site.

[0066] In some embodiments of any of the difunctional protein constructs described above, the first unit of the first binding site comprises two LAMA2 LG4-5 molecules linked in tandem, and the second unit of the first binding site comprises two LAMA2 LG4-5 molecules linked in tandem. In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site comprises a variant DLL4 ECD, the variant DLL4 ECD comprising any of the amino acid sequences of SEQ ID NOs. 126-129 and 260-267. In some embodiments, the difunctional protein construct comprises a first polypeptide and a second polypeptide, each comprising any one of the amino acid sequences of SEQ ID NOs. 241-244. In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site comprises a DLL1 ECD, the DLL1 ECD comprising the amino acid sequence of SEQ ID NO. 130. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each containing the amino acid sequence of SEQ ID NO: 245. In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site comprises a DLL3 ECD, the DLL3 ECD containing the amino acid sequence of SEQ ID NO: 131. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each containing the amino acid sequence of SEQ ID NO: 246. In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site comprises a variant Jag1 ECD, the variant Jag1 ECD containing the amino acid sequence of SEQ ID NO: 133 or 134. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each containing the amino acid sequence of SEQ ID NO: 247 or 248. In some embodiments, the first unit of the second binding site and / or the second unit of the second binding site comprises a Jag2 ECD, the Jag2 ECD comprising the amino acid sequence of SEQ ID NO: 135. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 249.

[0067] In some embodiments of any of the bifunctional protein constructs described above, i) a first binding site is fused to the N-terminus of a first subunit of an Fc domain via an optional first linker, and a second binding site is fused to the N-terminus of a second subunit of an Fc domain via an optional second linker; or ii) a first binding site is fused to the N-terminus of a second subunit of an Fc domain via an optional first linker, and a second binding site is fused to the N-terminus of a first subunit of an Fc domain via an optional second linker. In some embodiments, the first binding site is a Fab that specifically binds to a muscle-specific molecule, and the C-terminus of CH1 of the Fab is fused to the N-terminus of a first subunit of an Fc domain or a second subunit of an Fc domain via an optional first linker. In some embodiments, the second binding site includes an ECD of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2, or a variant thereof.

[0068] In some embodiments of any of the difunctional protein constructs described above, the difunctional protein construct comprises i) a first unit of a second binding site that specifically binds to a first Notch receptor, and ii) a second unit of a second binding site that specifically binds to a second Notch receptor; the first unit of the second binding site is fused to the N-terminus of a first subunit of an Fc domain via an optional first linker, and the second unit of the second binding site is fused to the N-terminus of a second subunit of an Fc domain via an optional second linker; and the first binding site is fused to the C-terminus of either the first subunit of an Fc domain or a second subunit of an Fc domain via an optional third linker. In some embodiments, the bifunctional protein construct comprises i) a first unit of a first binding site that specifically binds to a first muscle-specific molecule, and ii) a second unit of the first binding site that specifically binds to a second muscle-specific molecule; the first unit of the first binding site is fused to the C-terminus of a first subunit of an Fc domain via an optional third linker, and the second unit of the first binding site is fused to the C-terminus of a second subunit of an Fc domain via an optional fourth linker.

[0069] In some embodiments of any of the difunctional protein constructs described above, the second binding site is fused to the N-terminus of the first subunit of the Fc domain via an optional linker; the difunctional protein construct comprises one or more units of the first binding site that specifically bind to one or more muscle-specific molecules; each of the one or more units of the first binding site is independently fused via an optional linker to one of i) the C-terminus of the first subunit of the Fc domain; ii) the N-terminus of the second subunit of the Fc domain; and iii) the C-terminus of the second subunit of the Fc domain.

[0070] In some embodiments of any of the difunctional protein constructs described above, the second binding site is fused to the N-terminus of the second subunit of the Fc domain via an optional linker; the difunctional protein construct comprises one or more units of the first binding site that specifically bind to one or more muscle-specific molecules; each of the one or more units of the first binding site is independently fused via an optional linker to one of the following: i) the N-terminus of the first subunit of the Fc domain; ii) the C-terminus of the first subunit of the Fc domain; and iii) the C-terminus of the second subunit of the Fc domain.

[0071] In some embodiments of any of the difunctional protein constructs described above, the difunctional protein construct comprises i) a first unit of a second binding site that specifically binds to a first Notch receptor, and ii) a second unit of a second binding site that specifically binds to a second Notch receptor; the first unit of the second binding site is fused to the C-terminus of a first subunit of an Fc domain via an optional first linker, and the second unit of the second binding site is fused to the C-terminus of a second subunit of an Fc domain via an optional second linker; and the first binding site is fused to the N-terminus of either the first subunit of an Fc domain or a second subunit of an Fc domain via an optional third linker. In some embodiments, the bifunctional protein construct comprises i) a first unit of a first binding site that specifically binds to a first muscle-specific molecule, and ii) a second unit of the first binding site that specifically binds to a second muscle-specific molecule; the first unit of the first binding site is fused to the N-terminus of a first subunit of the Fc domain via an optional third linker, and the second unit of the first binding site is fused to the N-terminus of a second subunit of the Fc domain via an optional fourth linker.

[0072] In some embodiments of any of the difunctional protein constructs described above, the second binding site is fused to the C-terminus of the first subunit of the Fc domain via an optional linker; the difunctional protein construct comprises one or more units of the first binding site that specifically bind to one or more muscle-specific molecules; each of the one or more units of the first binding site is independently fused via an optional linker to one of the following: i) the N-terminus of the first subunit of the Fc domain; ii) the N-terminus of the second subunit of the Fc domain; and iii) the C-terminus of the second subunit of the Fc domain.

[0073] In some embodiments of any of the difunctional protein constructs described above, the second binding site is fused to the C-terminus of the second subunit of the Fc domain via an optional linker; the difunctional protein construct comprises one or more units of the first binding site that specifically bind to one or more muscle-specific molecules; each of the one or more units of the first binding site is independently fused via an optional linker to one of i) the N-terminus of the first subunit of the Fc domain; ii) the C-terminus of the first subunit of the Fc domain; and iii) the N-terminus of the second subunit of the Fc domain.

[0074] In some embodiments of any of the bifunctional protein constructs described above, i) one or more units of the second binding site each independently comprises an ECD or variant of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2; and ii) one or more units of the first binding site each independently comprises an antibody site that specifically binds to a muscle-specific molecule, the antibody site being independently selected from the group consisting of scFv, Fab, and sdAb. In some embodiments, i) two units of the second binding site are the same; and / or ii) two or more units of the first binding site are the same. In some embodiments, i) two units of the second binding site are different; and / or ii) at least one of the two or more units of the first binding site is different from the others. In some embodiments, the Fc domain includes knob-into-hole mutations and / or charge pair mutations. In some embodiments, (i) the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 348, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 255; (ii) the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 255, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 348; (iii) the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 256, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 257 (iv) The first subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 257, and the second subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 256; (v) The first subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 258, and the second subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 259; or (vi) The first subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 259, and the second subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 258.

[0075] In another embodiment, this specification provides one or more isolated nucleic acids encoding any of the bifunctional protein constructs described above.

[0076] In another embodiment, this specification provides one or more vectors comprising one or more isolated nucleic acids as described above. In some embodiments, one or more vectors are viral vectors. In some embodiments, the viral vectors are adeno-associated virus (AAV) vectors or lentiviral vectors.

[0077] In another embodiment, the Specified herein provides host cells expressing any of the bifunctional protein constructs described above, one or more isolated nucleic acids described above, or one or more vectors described above. In some embodiments, the host cells are Chinese hamster ovary (CHO) cells or HEK293 cells.

[0078] Furthermore, a pharmaceutical composition is provided comprising i) any of the bifunctional protein constructs described above, one or more isolated nucleic acids described above, or one or more vectors described above; and ii) a pharmaceutically acceptable excipient.

[0079] In another embodiment, the Specified provides a method for generating a bifunctional protein construct, comprising: i) culturing a host cell containing one or more of the isolated nucleic acids or one or more of the vectors described above, or the host cell described above, under conditions suitable for the expression of the bifunctional protein construct; and ii) recovering the bifunctional protein construct expressed from the cultured host cell. In some embodiments, the method further comprises introducing one or more isolated nucleic acids or one or more vectors into the host cell.

[0080] In another embodiment, this specification provides a method for treating a muscle-related disease in an individual, comprising administering to the individual an effective amount of any of the above-described bifunctional protein constructs or the above-described pharmaceutically acceptable compositions. In some embodiments, the muscle-related disease is selected from the group consisting of Pompe disease, central nucleus myopathy, fibrodysplasia ossificans progressive (FOP), Friedreich's ataxia (FRDA), familial hypertrophic cardiomyopathy, Laing's distal myopathy, myofibrilous myopathy, and muscular dystrophy. In some embodiments, the muscle disease is muscular dystrophy. In some embodiments, the muscular dystrophy includes one or more of the following: Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophy (CMD), facioscapulohumeral muscular dystrophy (FSHD), myotonic dystrophy (DM), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), congenital myopathy, Charcot-Marie-Tooth (CMT) disorder, and Emery-Dreyfus muscular dystrophy (EDMD). In some embodiments, the bifunctional protein construct or pharmaceutical composition is administered intravenously, subcutaneously, or intramuscularly. In some embodiments, the individual is human.

[0081] In another embodiment, the present invention relates to an antibody construct comprising an antibody site (anti-matriglycan antibody site) that specifically recognizes matriglycan on α-DG, wherein the anti-matriglycan antibody site comprises (i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 75, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76, LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 79. -CDR3; (ii) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 90, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 95; (iii) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 90, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 96; (iv) HC-CD of VH containing the amino acid sequence of SEQ ID NO: 90 (v) LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequences of R1, HC-CDR2, and HC-CDR3, and SEQ ID NO: 97; (v) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 90, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 98; (vi) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 91, and the amino acid sequence of SEQ ID NO: 95 (vii) LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 91, and HC-CDR1, LC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 96, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 91, and HC-CDR1, LC-CDR2, and LC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 97;(ix) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 98; (x) HC-CDR1, HC-CDR2, and HC-CDR3 of VH containing the amino acid sequence of SEQ ID NO: 92, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL containing the amino acid sequence of SEQ ID NO: 95; (xi) HC-CDR1 of VH containing the amino acid sequence of SEQ ID NO: 92, VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequences of HC-CDR2, HC-CDR3, and SEQ ID NO: 96; (xii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 92, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 97; (xiii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 92, and SEQ ID NO: 98 VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence; (xiv) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 93, and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 95; (xv) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 93, and VL LC-CDR1, LC-CDR2, and L containing the amino acid sequence of SEQ ID NO: 96 The present invention provides an antibody construct comprising: C-CDR3; (xvi) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 93; and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 97; or (xvii) VH HC-CDR1, HC-CDR2, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 93; and VL LC-CDR1, LC-CDR2, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 98.

[0082] In some embodiments of any of the antibody constructs described above, the anti-matriglycan antibody site includes a VH containing one of the amino acid sequences of SEQ ID NOs. 89 to 93, and a VL containing one of the amino acid sequences of SEQ ID NOs. 94 to 98. In some embodiments, the anti-matriglycan antibody site is: (i) VH containing the amino acid sequence of SEQ ID NO: 89 and VL containing the amino acid sequence of SEQ ID NO: 94; (ii) VH containing the amino acid sequence of SEQ ID NO: 90 and VL containing the amino acid sequence of SEQ ID NO: 95; (iii) VH containing the amino acid sequence of SEQ ID NO: 90 and VL containing the amino acid sequence of SEQ ID NO: 96; (iv) VH containing the amino acid sequence of SEQ ID NO: 90 and VL containing the amino acid sequence of SEQ ID NO: 97; (v) VH containing the amino acid sequence of SEQ ID NO: 90 and VL containing the amino acid sequence of SEQ ID NO: 98; (vi) VH containing the amino acid sequence of SEQ ID NO: 91 and VL containing the amino acid sequence of SEQ ID NO: 95; (vii) VH containing the amino acid sequence of SEQ ID NO: 91 and VL containing the amino acid sequence of SEQ ID NO: 96; (viii) VH containing the amino acid sequence of SEQ ID NO: 91 and VL containing the amino acid sequence of SEQ ID NO: 97; (ix) amino VH containing an acid sequence, and VL containing the amino acid sequence of SEQ ID NO: 98; (x) VH containing the amino acid sequence of SEQ ID NO: 92, and VL containing the amino acid sequence of SEQ ID NO: 95; (xi) VH containing the amino acid sequence of SEQ ID NO: 92, and VL containing the amino acid sequence of SEQ ID NO: 96; (xii) VH containing the amino acid sequence of SEQ ID NO: 92, and VL containing the amino acid sequence of SEQ ID NO: 97; (xiii) VH containing the amino acid sequence of SEQ ID NO: 92, and VL containing the amino acid sequence of SEQ ID NO: 98; (xiv) VH containing the amino acid sequence of SEQ ID NO: 93, and VL containing the amino acid sequence of SEQ ID NO: 95; (xv) VH containing the amino acid sequence of SEQ ID NO: 93, and VL containing the amino acid sequence of SEQ ID NO: 96; (xvi) VH containing the amino acid sequence of SEQ ID NO: 93, and VL containing the amino acid sequence of SEQ ID NO: 97; or (xvii) VH containing the amino acid sequence of SEQ ID NO: 93, and VL containing the amino acid sequence of SEQ ID NO: 98.

[0083] In some embodiments of any of the antibody constructs described above, the anti-matriglycan antibody site is selected from the group consisting of full-length antibody, Fab, Fab', F(ab')2, and scFv. In some embodiments, the anti-matriglycan antibody site is anti-matriglycan scFv. In some embodiments, anti-matriglycan scFv contains the amino acid sequence of SEQ ID NO: 99.

[0084] In some embodiments of any of the antibody constructs described above, the anti-matriglycan antibody site is an anti-matriglycan Fab. In some embodiments, the anti-matriglycan Fab comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 100 and a second polypeptide having the amino acid sequence of SEQ ID NO: 101.

[0085] In some embodiments of any of the antibody constructs described above, the anti-matriglycan antibody site is an anti-matriglycan Fab. In some embodiments, the anti-matriglycan Fab comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 100 and a second polypeptide having the amino acid sequence of SEQ ID NO: 101.

[0086] In some embodiments of any of the antibody constructs described above, the anti-matriglycan antibody site is a full-length anti-matriglycan antibody. In some embodiments, the full-length anti-matriglycan antibody includes an Fc domain derived from human IgG1, human IgG2, or human IgG4. In some embodiments, the Fc domain is derived from human IgG1 containing the amino acid sequence of SEQ ID NO: 187. In some embodiments, each subunit of the Fc domain contains the L234A / L235A mutation (EU numbering). In some embodiments, each subunit of the Fc domain contains the H435A mutation (EU numbering). In some embodiments, each subunit of the Fc domain contains the L234A / L235A / P329G mutation (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 188. In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 277.

[0087] In some embodiments of any of the antibody constructs described above, the antibody construct further includes a second binding site that specifically binds to a second target molecule. In some embodiments, the second binding site specifically binds to and activates the Notch receptor. In some embodiments, the second binding site includes an ECD or variant of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2.

[0088] In another embodiment, the present invention provides an engineered DLL4 extracellular domain (ECD), wherein the engineered DLL4 ECD comprises a mutation selected from the group consisting of T52N and T135N, and the amino acid position is relative to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, the engineered DLL4 ECD further comprises a mutation at one or more amino acid positions selected from the group consisting of G2, E14, R66, P80, F81, H168, Q220, N231, and N260. In some embodiments, the further mutation is selected from the group consisting of G2S, E14H, R66S, R66T, P80L, F81L, H168Y, Q220H, N231D, and N260D. In some embodiments, the engineered DLL4 ECD includes (i) T52N and T135N; (ii) T52N, R66S, and T135N; (iii) E14H, T52N, R66T, P80L, T135N, and N231D; (iv) T52N, R66T, P80L, T135N, Q220H, and N260D; (v) G2S, T52N, F81L, T135N, and H168Y; (vi) G2S (vii) G2S, E14H, T52N, F81L, R66S, T135N, and H168Y; and (viii) G2S, T52N, R66T, P80L, T135N, H168Y, and N231D; and (viii) G2S, T52N, R66T, P80L, F81L, T135N, H168Y, Q220H, and N260D. In some embodiments, the manipulated DLL4 ECD contains an amino acid sequence selected from the group consisting of any one of sequence numbers 261-264.

[0089] In another embodiment of any of the manipulated DLL4 ECDs described above, the present invention provides a protein construct comprising a manipulated DLL4 ECD. In some embodiments, the protein construct further includes a binding site that specifically binds to muscle-specific molecules.

[0090] It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the present invention. [Brief explanation of the drawing]

[0091] [Figure 1A] This shows different configurations of bifunctional proteins containing anti-muscle target proteins and Notch ligands or agonists. It also shows monovalent Notch ligands with "knob-into-hole" anti-muscle protein antibodies (Ab). [Figure 1B] This shows different configurations of a bifunctional protein containing an anti-muscle target protein and a Notch ligand or agonist. Additional forms and configurations of the bifunctional protein are shown, where R1, R2, R3, and R4 may be an anti-muscle target protein or a Notch ligand / agonist. [Figure 1C] This shows different configurations of a bifunctional protein containing an anti-muscle target protein and a Notch ligand or agonist. Additional forms and configurations of the bifunctional protein are shown, where R1, R2, R3, and R4 may be an anti-muscle target protein or a Notch ligand / agonist. [Figure 1D] This shows different configurations of a bifunctional protein containing an anti-muscle target protein and a Notch ligand or agonist. It also shows a bivalent fusion protein with a Notch ligand fused to the N-terminus of Fc and an anti-muscle protein antibody Fab fused to the C-terminus of Fc. [Figure 1E] This shows different configurations of a bifunctional protein containing an anti-muscle target protein and a Notch ligand or agonist. It also shows a bivalent fusion protein with a Notch ligand fused to the N-terminus of Fc and an anti-muscle protein antibody scFv fused to the C-terminus of Fc. [Figure 1F]This shows different configurations of a bifunctional protein containing an anti-muscle target protein and a Notch ligand or agonist. It also shows a bivalent fusion protein having a Notch ligand fused to the N-terminus of Fc and a single-chain anti-muscle target protein antibody (VHH or nanobody) fused to the C-terminus of Fc. [Figure 1G] This shows different configurations of a bifunctional protein containing an anti-muscle target protein and a Notch ligand or agonist. It also shows a bivalent fusion protein with a Notch ligand fused to the C-terminus of Fc and an anti-muscle protein antibody Fab fused to the N-terminus of Fc. [Figure 1H] This shows different configurations of a bifunctional protein containing an anti-muscle target protein and a Notch ligand or agonist. It shows a bivalent fusion protein having a Notch ligand fused to the C-terminus of Fc and an anti-muscle protein scFv fused to the N-terminus of Fc. [Figure 1I] This shows different configurations of a bifunctional protein containing an anti-muscle target protein and a Notch ligand or agonist. It shows a bivalent fusion protein having a Notch ligand fused to the C-terminus of Fc and an anti-muscle protein VHH / nanobody fused to the N-terminus of Fc. [Figure 1J] This shows different configurations of a bifunctional protein containing an anti-muscle target protein and a Notch ligand or agonist. It also shows a bivalent fusion protein with a Notch ligand fused to the C-terminus of the antibody light chain. [Figure 1K] This shows different configurations of a bifunctional protein containing an anti-muscle target protein and a Notch ligand or agonist. It also shows a bivalent fusion protein with a Notch ligand fused to the N-terminus of the antibody light chain. [Figure 1L] This shows different configurations of a bifunctional protein containing an anti-muscle target protein and a Notch ligand or agonist. It also shows a bivalent fusion protein with a Notch ligand fused to the N-terminus of the antibody heavy chain. [Figure 1M]This shows different configurations of bifunctional proteins containing anti-muscle target proteins and Notch ligands or agonists. It also shows a tetravalent fusion protein with a Notch ligand fused to the N-terminus of the antibody light chain and heavy chain.

[0092] [Figure 2A] This shows different configurations of bifunctional proteins containing non-antibody sites (e.g., the LG domain of laminin, the LG domain of agryn, the LG domain of nidogen, and the LG domain of perlecan) and Notch ligands or agonists. It shows a laminin G-like domain (LG domain) monomer, or alternatively, a monovalent protein with a Notch ligand fused to the N-terminus of scFv, sdAb, or Fab for muscle-specific proteins. [Figure 2B] This shows different configurations of bifunctional proteins containing non-antibody sites (e.g., the LG domain of laminin, agryn, nidogen, and perlecan) and a Notch ligand or agonist. It also shows LG domain dimers, or alternatively, monovalent proteins with a Notch ligand fused to the N-terminus of scFv, sdAb, or Fab that specifically recognizes muscle-specific proteins. [Figure 2C] This shows different configurations of bifunctional proteins containing non-antibody sites (e.g., the LG domain of laminin, agryn, nidogen, and perlecan) and Notch ligands or agonists. It shows tandem LG domain trimers or polymers, or alternatively, monovalent proteins with Notch ligands fused to the N-terminus of scFv, sdAb, or Fab for muscle-specific proteins. [Figure 2D]This shows different configurations of bifunctional proteins containing non-antibody sites (e.g., the LG domain of laminin, the LG domain of agryn, the LG domain of nidogen, and the LG domain of perlecan) and Notch ligands or agonists. From the N' to the C' terminus: This shows monovalent proteins of Notch ligand-albumin, anti-albumin scFv or sdAb, or monomeric human Fc-LG domain monomers. [Figure 2E] This shows different configurations of bifunctional proteins containing non-antibody sites (e.g., the LG domain of laminin, agryn, nidogen, and perlecan) and Notch ligands or agonists. From the N' to the C' terminus: This shows a monovalent protein with Notch ligand-albumin, anti-albumin scFv or sdAb, or monomeric human Fc-LG domain dimer. [Figure 2F] This shows different configurations of bifunctional proteins containing non-antibody sites (e.g., the LG domain of laminin, agryn, nidogen, and perlecan) and Notch ligands or agonists. From N' to C' terminus: Notch ligand - albumin, anti-albumin scFv or sdAb, or monomeric human Fc - tandem LG domain - monovalent protein in a trimer or multimer. [Figure 2G] This shows different configurations of bifunctional proteins containing non-antibody sites (e.g., the LG domain of laminin, agryn, nidogen, and perlecan) and a Notch ligand or agonist. It also shows bivalent fusion proteins having a Notch ligand fused to the N-terminus of Fc and an LG domain fused to the C-terminus of Fc as a monomer, dimer, or multimer (tandem), respectively. [Figure 2H]This shows different configurations of bifunctional proteins containing non-antibody sites (e.g., the LG domain of laminin, agryn, nidogen, and perlecan) and a Notch ligand or agonist. It also shows bivalent fusion proteins having a Notch ligand fused to the N-terminus of Fc and an LG domain fused to the C-terminus of Fc as a monomer, dimer, or multimer (tandem), respectively. [Figure 2I] This shows different configurations of bifunctional proteins containing non-antibody sites (e.g., the LG domain of laminin, agryn, nidogen, and perlecan) and a Notch ligand or agonist. It also shows bivalent fusion proteins having a Notch ligand fused to the C-terminus of Fc and an LG domain fused to the N-terminus of Fc as a monomer, dimer, or multimer (tandem), respectively. [Figure 2J] This shows different configurations of bifunctional proteins containing non-antibody sites (e.g., the LG domain of laminin, agryn, nidogen, and perlecan) and a Notch ligand or agonist. It also shows bivalent fusion proteins having a Notch ligand fused to the C-terminus of Fc and an LG domain fused to the N-terminus of Fc as a monomer, dimer, or multimer (tandem), respectively.

[0093] [Figure 3A] The SDS-PAGE for the reduced and non-reduced DLL4v-Fc-LG4-5 constructs is shown. [Figure 3B] The size exclusion chromatography (SEC) profiles for the protein standard (upper profile) and the DLL4v-Fc-LG4-5 construct (lower profile) are shown.

[0094] [Figure 4A] The SDS-PAGE for the reduced and non-reduced DLL4wt-Fc-LG4-5 constructs is shown. [Figure 4B]The size exclusion chromatography (SEC) profiles for the protein standard (upper profile) and the DLL4wt-Fc-LG4-5 construct (lower profile) are shown.

[0095] [Figure 5A] The SDS-PAGE for the reduced and non-reduced DLL4v-Fc-LAMA2scFv constructs is shown. [Figure 5B] The size exclusion chromatography (SEC) profiles for the protein standard (upper profile) and the DLL4v-Fc-LAMA2scFv construct (lower profile) are shown.

[0096] [Figure 6] This shows the binding of the bifunctional proteins DLL4v-Fc-LG4-5, DLL4wt-Fc-LG4-5, and DLL4v-Fc-LAMA2scFv to Notch-1 using sandwich ELISA. The DLL4v-Fc-LG4-5 and DLL4v-Fc-LAMA2scFv constructs have higher affinity (or avidity) to Notch-1 compared to the DLL4wt-Fc-LG4-5 construct.

[0097] [Figure 7A] This graph shows the kinetic analysis of the DLL4v-Fc-LG4-5 construct bound to purified matriglycerides at different concentrations using the Biacore system. The y-axis represents absolute reaction units (RU), and the x-axis represents time (seconds). The arrows indicate the injection points of the purified matriglycerides. [Figure 7B] The Western blot results of a laminin-overlay assay show that DLL4v-Fc-LG4-5 binds to matrixglycan.

[0098] [Figure 8]This image shows the kinetic analysis of the DLL4v-Fc-LAMA2scFv construct bound to recombinant human LG4-5 at different concentrations using the Biacore system, confirming that LAMA2scFv binds to LG4-5. The arrows indicate the injection points of recombinant human LG4-5.

[0099] [Figure 9A] The study schema is shown below. Male D2.mdx mice, approximately 7-9 weeks old, were evaluated for muscle function by forelimb grip strength. Mice were randomized into two groups (n=10 / group). The control group was treated three times a week with 20 mg / kg of an isotype control antibody against the unrelated antigen trinitrophenol (TNP). The experimental group was treated three times a week with 5 mg / kg of DLL4v-Fc-LAMA2scFv. The wild-type (WT) group received no treatment. Forelimb grip strength was evaluated monthly for 7 months. [Figure 9B] This shows the forelimb grip strength of WT, DLL4v-Fc-LAMA2scFv treated, and isotype control treated D2.mdx mice over a 7-month period. [Figure 9C] This shows the body weight of WT, DLL4v-Fc-LAMA2scFv treated, and isotype control treated D2.mdx mice over a 7-month period.

[0100] [Figure 10A] The size exclusion chromatography (SEC) profiles for the protein standard (upper profile), huD4v11_2N-FcAAG-LG21DS, huD4v12_2N_A2-FcAAG-LG21DS, huD4v13_2N_-FcAAG-LG21DS, and huD4v14_2N_H2-FcAAG-LG21DS are shown. [Figure 10B] The table shows the yield and percentage of the major peaks determined by analytical size exclusion chromatography (aSEC).

[0101] [Figure 11A]This shows the binding affinity of huD4v11_2N-FcAAG-LG21DS, huD4v12_2N_A2-FcAAG-LG21DS, huD4v13_2N_-FcAAG-LG21DS, and huD4v14_2N_H2-FcAAG-LG21DS to the mouse Notch1 receptor, compared to huD4v_G2S_F81L_H168Y-Fc(AAG)-LG21scFvDS (parent) and mud4v construct as determined by ELISA assay. [Figure 11B] The table below summarizes the binding affinity (KD) of each construct as measured in nM. [Modes for carrying out the invention]

[0102] This application provides a bifunctional protein construct that specifically targets and delivers to muscle tissue with spatiotemporally controlled Notch activation. Binding of the bifunctional protein construct to muscle tissue, either via proteins on the muscle (cell) sarcodiae or proteins in the extracellular matrix (basement membrane), immobilizes the bifunctional protein construct and its contained Notch activation sites (e.g., Notch ligands or agonist antibodies that specifically recognize Notch 1-4), which is a requirement for Notch signaling. Therefore, the bifunctional protein construct disclosed herein satisfies three requirements for specific Notch signaling in muscle: 1) muscle targeting to ensure tissue specificity; 2) Notch ligand immobilization required for transactivation; and 3) timely control of Notch activation for satellite cell regeneration while enabling myoblast differentiation during muscle repair.

[0103] Therefore, in one embodiment, this application provides a bifunctional protein construct comprising a first binding site and a second binding site, wherein the first binding site specifically binds to a muscle-specific molecule, and the second binding site specifically binds to a Notch receptor and activates the Notch receptor.

[0104] In another embodiment, a method for preparing the difunctional protein constructs described herein is provided. Isolated nucleic acids, vectors, and host cells encoding any of the difunctional protein constructs are also provided. Furthermore, a method for treating muscle-related diseases in an individual (e.g., a human) is provided by administering an effective amount of the difunctional protein construct or its pharmaceutically acceptable composition to the individual.

[0105] In another embodiment, an engineered DLL4 extracellular domain (ECD) is provided, comprising a mutation selected from the group consisting of T52N and T135N, wherein the amino acid position is relative to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126. A protein construct comprising the DLL4 ECD described herein is also provided.

[0106] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which the present invention pertains. Any methods and substances similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but exemplary methods and substances are described below.

[0107] In this specification, the term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and their antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. The term "antibody site" refers to a full-length antibody or its antigen-binding fragment.

[0108] An "antibody" may refer to an immunoglobulin molecule or fragment thereof (including the basic four-chain antibody unit) that can specifically bind to a particular epitope of an antigen. Antibodies may be intact immunoglobulins of natural or recombinant origin, or they may be the immunoreactive portion of an intact immunoglobulin. The antibodies in this invention may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), antigen-binding fragments (e.g., Fv, Fab, Fab', F(ab)2 and F(ab')2), as well as single-chain antibodies (scFv), heavy-chain antibodies, such as camelid antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0109] A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains may be referred to as "VH" and "VL," respectively. The variable regions in both chains typically contain three highly variable loops called complementarity-determining regions (CDRs) (light chain (LC)CDRs containing LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC)CDRs containing HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the rules of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). Three CDRs of the heavy or light chain are inserted between adjacent extensions known as framework regions (FRs), which are more conserved than the CDRs and form a skeleton supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major antibody classes are classified into subclasses, e.g., IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgGA1 (α1 heavy chain), or IgGA2 (α2 heavy chain).

[0110] The term “antigen-binding fragment” as used herein refers to an antibody fragment that includes, for example, a diabody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (dsdiabody), single-chain Fv (scFv), scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody containing one or more CDRs, a single-domain antibody (sdAb) (e.g., a camelid single-domain antibody), a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. An antigen-binding fragment can bind to the same antigen to which a parent antibody or parent antibody fragment (e.g., parent scFv) binds. In some embodiments, an antigen-binding fragment may include one or more CDRs derived from a particular human antibody grafted onto a framework region derived from one or more different human antibodies.

[0111] "Fv" is the minimal antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer in which one heavy chain variable domain and one light chain variable domain are tightly and noncovalently associated. The folding of these two domains creates six hypervariable loops (three from the heavy chain and three from the light chain) that provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to the antigen, albeit with lower affinity than the entire binding site.

[0112] A "single-stranded Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. In some embodiments, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains, enabling the scFv to form a desired structure for antigen binding. For an overview of scFv, see The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0113] A basic four-chain antibody is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units plus an additional polypeptide called a J chain, containing 10 antigen-binding sites, while IgA antibodies contain 2 to 5 basic four-chain units that can polymerize and combine with the J chain to form a multivalent aggregate. In the case of IgG, a four-chain unit is typically around 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide crosslinks. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for the α and γ chains respectively, and four CH domains for the μ and ε isotypes. Each light chain (L) has a variable domain (VL) at its N-terminus, followed by a constant domain at the other end. The VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain. Certain amino acid residues are thought to form an interface between the variable domains of the light and heavy chains. The pairing of VH and VL together forms a single antigen-binding site. For the structures and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8. thSee Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. Light chains from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, immunoglobulins can be assigned to different classes or isotypes. Five classes of immunoglobulins exist: IgA, IgD, IgE, IgG, and IgM, each having heavy chains denoted as α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0114] The Fc fragment contains the carboxyl-terminal portions of both H chains, held together by disulfide bonds. The effector function of an antibody is determined by the sequence of the Fc region, which is also recognized by an Fc receptor (FcR) found in a given type of cell.

[0115] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the antibody's heavy or light chain. The variable domains of the heavy and light chains may be referred to as "VH" and "VL," respectively. These domains are typically the most variable parts of the antibody (compared to other antibodies of the same class) and contain the antigen-binding site. Antibodies derived solely from camelid species have a single heavy chain variable region, which is referred to as "VHH." Therefore, VHH is a special type of VH.

[0116] The term "variable" refers to the fact that a given segment of the variable domain exhibits extensive sequence differences between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, variability is not uniformly distributed across the entire variable domain. Rather, it is concentrated in three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). The native heavy and light chain variable domains each primarily adopt a beta-sheet structure and contain four FR regions connected by three HVRs, where the three HVRs connect the beta-sheet structure and, in some cases, form loops that form part of it. The HVRs in each chain are held in close proximity to each other by the FR regions and, together with the HVRs from the other chain, contribute to the formation of the antibody's antigen-binding site (see abat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domain does not directly participate in antibody binding to antigens, but it exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cytotoxicity.

[0117] When used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Typically, in contrast to polyclonal antibody preparations, which contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier “monoclonal” describes the characteristic of an antibody as being obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with this application are, for example, those produced by the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd(ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA method (see, e.g., U.S. Patent Nos. 4,816, 567), phage display technology (e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA See 101(34):12467-12472(2004); and Lee et al., J.Immunol.Methods 284(1-2):119-132(2004), and techniques for generating human or human-like antibodies in animals having a human immunoglobulin locus or part or all of a human immunoglobulin gene encoding a human immunoglobulin sequence (e.g., WO1998 / 24893; WO1996 / 34096; WO1996 / 33735; WO1991 / 10741; Jakobovits et al., Proc.Natl.Acad.Sci.USA 90:2551(1993); Jakobovits et al., Nature 362:255-258(1993); Bruggemann et al., Year in Immunol. 7:33 (1993); US Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol.It can be produced using a variety of techniques, including (see 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0118] The terms "full-length antibody," "intact antibody," or "complete antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, full-length four-chain antibodies include those having heavy and light chains containing the Fc region. The constant domain may be the native sequence constant domain (e.g., the human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

[0119] The term "diabody" refers to the pairing of VH and V domains, where pairing is achieved between the V domains rather than within the V domain, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. L This refers to a small antibody fragment prepared by constructing an sFv fragment (see previous paragraph) using short linkers (approximately 5-10 residues) between domains. A bispecific diabody is a combination of the VH and VH of two antibodies. L The domain is a heterodimer of two "crossover" sFv fragments located on different polypeptide chains. The diabody is described in more detail, for example, EP404,097;WO93 / 11161;Hollinger et al.,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993).

[0120] The term "monoclonal antibody" as used herein specifically includes "chimeric" antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies (to the extent that they exhibit the desired biological activity) (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The chimeric antibodies covered herein include PRIMATTZFACTOR D® antibodies in which the antigen-binding region of the antibody is derived, for example, from an antibody produced by immunizing a macaque monkey with the target antigen. As used herein, "humanized antibody" is used as a subset of "chimeric antibody".

[0121] As used herein, the terms “CDR” or “complementarity-determining region” are intended to mean discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions were identified by Kabat et al., J.Biol.Chem.252:6609-6616(1977);Kabat et al.,USDept.of Health and Human Services,“Sequences of proteins of immunological interest”(1991);Chothia et al.,J.Mol.Biol.196:901-917(1987);Al-Lazikani B.et al.,J.Mol.Biol.,273:927-948(1997);MacCallum et al.,J.Mol.Biol.262:732-745(1996);Abhinandan and Martin,Mol.Immunol.,45:3832-3839(2008);Lefranc MPet The definitions, as described in al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plueckthun, J. Mol. Biol., 309:657-670 (2001), include duplicate or subset amino acid residues when compared to one another. However, the application of any definition to refer to a CDR of an antibody or grafted antibody or its variant is intended to be within the scope of the terms defined and used herein. For comparison, the amino acid residues encompassing a CDR as defined by each of the references cited above are shown in Table 1 below. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol.Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015).The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and for possible inclusion in one or more claims herein. [Table 1]

[0122] The expressions "Kabat variable domain residue numbering" or "Kabat amino acid position numbering," and their variations, refer to the numbering system used for the heavy chain or light chain variable domains of antibody synthesis in Kabat et al. (cited above). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortenings or insertions into the FR or hypervariable region (HVR) of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after H2 residue 52 (residue 52a according to Kabat) and an inserted residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody sequence with a homologous region of a "standard" Kabat numbered sequence.

[0123] Unless otherwise specified herein, the numbering of residues in immunoglobulin heavy chains is that of the EU index by Kabat et al. (see above), with minor modifications. "Kabat EU index" refers to the residue numbering of human IgG1 EU antibodies.

[0124] "Framework" or "FR" residues are variable domain residues other than the CDR residues as defined herein.

[0125] As used herein, “immunoassay” refers to any binding assay that uses an antibody capable of specifically binding to a target molecule and detecting and quantifying that target molecule.

[0126] The "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody containing the minimal sequence derived from the non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues derived from the recipient's hypervariable region (HVR) are replaced with residues derived from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, possessing the desired antibody specificity, affinity, and capability. In some cases, framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the recipient or donor antibody. These modifications are made to further improve antibody performance. Typically, a humanized antibody contains substantially all of at least one, usually two, variable domains, all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FRs are from the human immunoglobulin sequence. Humanized antibodies also optionally include at least a portion of the immunoglobulin constant region (Fc), typically that of human immunoglobulin. Suitable human acceptor antibodies may be selected from conventional databases, e.g., the KABAT database, the Los Alamos database, AbM, and the Swiss Protein database, based on homology to the nucleotide and amino acid sequences of the donor antibody. Human antibodies characterized by homology (based on amino acids) to the framework region of the donor antibody may be suitable for providing a heavy chain constant region and / or a heavy chain variable framework region for insertion of the donor CDR. Suitable acceptor antibodies capable of providing a light chain constant region or a variable framework region may be selected in a similar manner. It should be noted that the heavy and light chains of the acceptor antibody do not need to originate from the same acceptor antibody. Several methods for producing such humanized antibodies are described in the prior art (see, e.g., EP-A-0239400 and EP-A-054951).For further details, see, for example, Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0127] A “human antibody” is an antibody that possesses an amino acid sequence corresponding to that of an antibody produced by a human, and / or an antibody produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J.Mol.Biol., 227:381 (1991); Marks et al., J.Mol.Biol., 222:581 (1991). The method described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991) is also available for the preparation of human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies are modified to produce such antibodies in response to antigen loading, but can be prepared by administering antigens to transgenic animals in which the endogenous loci have been deactivated, e.g., immunized xenomouses (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 relating to XENOMOUSE® technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) relating to human antibodies produced via human B-cell hybridoma technology.

[0128] The term "donor antibody" refers to an antibody (monoclonal and / or recombinant) that provides the amino acid sequence of its variable region, CDR, or other functional fragment or analogue thereof to a first immunoglobulin partner in order to provide the modified immunoglobulin coding region and the resulting expressed modified antibody with the antigen specificity and neutralizing activity characteristics of the donor antibody.

[0129] The term "acceptor antibody" refers to a heterologous antibody (monoclonal and / or recombinant) that provides all (or any, but in some embodiments, all) of the amino acid sequences encoding its heavy chain and / or light chain framework region and / or its heavy chain and / or light chain constant region to a first immunoglobulin partner. In a given embodiment, a human antibody is the acceptor antibody.

[0130] The terms “to bind,” “bound,” “fused,” or “fused,” as used herein, mean to connect or integrate two or more components by binding, linking, force or linkage in order to maintain them together, and include both direct and indirect binding, such as when a first polypeptide is directly bound to a second polypeptide or substance, and when one or more intermediate compounds (e.g., amino acids, peptides, polypeptides, etc.) are positioned between a first polypeptide and a second polypeptide or substance.

[0131] With respect to polypeptides and antibody sequences identified herein, “amino acid sequence identity percentage (%)” or “homology” is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the polypeptide being compared, after the sequences have been aligned, taking into account any conservative substitutions as part of sequence identity. Alignment for the purpose of determining amino acid sequence identity percentage can be achieved in various ways within the art, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the greatest possible alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the % amino acid sequence identity value is generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0132] "Homologie" refers to the sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If the positions in both compared sequences are occupied by the same base or amino acid monomer subunit—for example, if the positions in each of two DNA molecules are occupied by adenine—then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences, divided by the number of positions compared, and multiplied by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Typically, comparisons are made when the two sequences are aligned to give maximum homology.

[0133] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the variable domain, which is the other portion of an immunoglobulin that contains the antigen-binding site. The constant domain is the C of the heavy chain H 1, C H 2 and C H 3 domains (collectively, C H ) and the CHL (or C L ) domain of the light chain.

[0134] The "light chains" of antibodies (immunoglobulins) derived from any mammalian species can be assigned to one of two distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of their constant domains.

[0135] A "CH1 domain" (also referred to as "C1" of the "H1" domain) normally extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0136] The "hinge region" is generally defined as the region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds in the same positions.

[0137] The "CH2 domain" (also referred to as the "C2" domain) of a human IgG Fc domain normally extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide an alternative to domain-domain pairing and assist in stabilizing the CH2 domain. Burton, Molec Immunol. 22:161-206 (1985).

[0138] The "CH3 domain" (also called the "C3" domain) includes the extension from the C-terminal residue in the Fc domain to the CH2 domain (i.e., from approximately amino acid residue 341 to the C-terminus in the antibody sequence, typically to amino acid residue 446 or 447 in IgG).

[0139] In this specification, the terms “Fc domain” or “fragment crystallizable region” are used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc domains and variant Fc domains. While the boundaries of the Fc domain of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc domain is typically defined as extending from the amino acid residue position Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc domain (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombination of the nucleic acid encoding the antibody heavy chain. Thus, compositions of intact antibodies may include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0140] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc domain of an antibody. The preferred FcR is the native human FcR sequence. Furthermore, preferred FcRs are those that bind to IgG antibodies (gamma receptors) and include the receptor subclasses FcγRI, FcγRII, and FcγRIII (including allele variants and alternative splicing forms of these receptors). FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activating motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See M. Daeeron, Annu. Rev. Immunol. 15:203-234 (1997). FcR is outlined in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein.)

[0141] The term “epitope,” as used herein, refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody site binds. Two antibodies or antibody sites may bind to the same epitope within an antigen if they exhibit competitive binding to the antigen.

[0142] As used herein, the terms “specifically bind,” “specifically recognize,” and “specific to” refer to measurable and reproducible interactions, such as binding, between a target and an antibody or antibody moiety, which determine the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody or antibody moiety that specifically recognizes a target (which may be an epitope) is an antibody or antibody moiety that binds to this target with greater affinity, avidity, more readily, and / or for a longer duration than its binding to other targets. In some embodiments, the degree of antibody binding to an unrelated target is less than 10% of the antibody binding to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target is ≤10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, ≤10 -12 , ≤10 -13 , or ≤10 -14 The dissociation constant of M (K D ) are present. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved between proteins of different species. In some embodiments, specific binding may include, but is not required, exclusive binding. The binding specificity of the antibody or antigen-binding domain can be experimentally determined by methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, EIA, BIACORE® tests, and peptide scans.

[0143] The term "specificity" refers to the selective recognition of an antigen-binding protein or antibody against a specific epitope of an antigen. Natural antibodies are, for example, monospecific. The term "multispecific," as used herein, indicates that an antigen-binding protein or antibody has two or more antigen-binding sites, at least two of which bind to different antigens or different epitopes of the same antigen. "Dual-specific," as used herein, indicates that an antigen-binding protein or antibody has two different antigen-binding specificities. The term "monospecific" antibody, as used herein, refers to an antibody that has one or more binding sites (each of which binds to the same epitope of the same antigen).

[0144] Effector cells are leukocytes that express one or more FcRs and exhibit effector function. In one embodiment, effector cells express at least FcγRII and exhibit ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from native sources, such as blood. Effector cells are typically lymphocytes associated with the effector phase that function to produce cytokines (helper T cells), kill pathogen-infected cells (cytotoxic T cells), or secrete antibodies (differentiated B cells).

[0145] Complement-dependent cell injury, or CDC, refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) (bound to their corresponding antigens). To assess complement activation, a CDC assay may be performed, for example, as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996). Antibody variants having modified Fc region amino acid sequences and increased or decreased C1q binding capacity are described in U.S. Patent Nos. 6,194,551B1 and WO99 / 51642. The contents of those patent publications are specifically incorporated herein by reference. See also Idusogie et al., J.Immunol. 164:4178-4184 (2000).

[0146] "Binding affinity" typically refers to the strength of the combined non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can typically be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies typically tend to bind gradually to antigens and dissociate easily, while high-affinity antibodies typically tend to bind more quickly to antigens and remain bound for longer. A variety of methods for measuring binding affinity are known in the art, and any of these may be used for the purposes of this application. Specific descriptive and exemplary embodiments for measuring binding affinity are described below.

[0147] "On speed", "meeting speed", "meeting speed", or "k on When used herein, the values ​​may also be determined as described above using methods such as biolayer interferometry and surface plasmon resonance (SPR).

[0148] "Isolated" polypeptides are those identified, separated, and / or recovered from components of their production environment (e.g., natural or recombinant). Preferably, isolated polypeptides are not associated with all other components from their production environment. Contaminations of the production environment, e.g., those resulting from recombinant transfection cells, are typically substances that would interfere with the study, diagnosis, or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteolytic or non-proteolytic solutes. In preferred embodiments, polypeptides are purified to (1) up to more than 95% by weight of antibody, for example, as determined by the Lowry method, and in some embodiments up to more than 99% by weight; (1) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer; or (3) to homogeneity by SDS-PAGE under non-reducible or reducing conditions using Coomassie blue or, preferably, silver staining. Isolated polypeptides contain polypeptides in situ within recombinant cells, because at least one component of the polypeptide's natural environment is absent. However, isolated polypeptides are typically prepared through at least one purification step.

[0149] The “isolated” nucleic acid molecules encoding constructs, antibodies, or their antigen-binding fragments described herein are nucleic acid molecules identified and isolated from at least one contaminating nucleic acid molecule that is normally associated with the environment in which they are produced. Preferably, the isolated nucleic acid does not associate with any components that are associated with the production environment. The isolated nucleic acid molecules encoding polypeptides and antibodies described herein are in a form or context different from that which is found in nature. Therefore, isolated nucleic acid molecules are distinguished from nucleic acids encoding polypeptides and antibodies described herein that are naturally present in cells. Isolated nucleic acids include nucleic acid molecules that are normally present in cells that contain nucleic acid molecules, but whose nucleic acid molecules are located outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0150] Nucleic acids are "functionally ligated" when they are in a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretion leader is functionally ligated to DNA for a polypeptide if it is expressed as a preprotein involved in polypeptide secretion; a promoter or enhancer is functionally ligated to a coding sequence if it affects the transcription of that sequence; or a ribosome binding site is functionally ligated to a coding sequence if it is positioned to facilitate translation. Typically, "functionally ligated" means that the ligated DNA sequences are contiguous, and in the case of a secretion leader, contiguous and within the reading frame. Enhancers, however, do not need to be contiguous. Ligation is achieved by ligation at a convenient restriction site. If such a site does not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0151] "Isolated" means modified or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in the normal environment of a living organism is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from coexisting substances in its natural environment is "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-native environment, such as a host cell.

[0152] The term "hybridoma," as used herein, refers to a cell resulting from the fusion of a B lymphocyte and a fusion partner, such as a myeloma cell. Hybridomas can be cloned and maintained indefinitely in cell culture and can generate monoclonal antibodies. Hybridomas can also be considered hybrid cells.

[0153] The terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” are used interchangeably and may refer to polymers of nucleotides. Such polymers of nucleotides may contain native and / or non-native nucleotides, including but not limited to DNA, RNA, and PNA. “Nucleic acid sequence” refers to a linear sequence of nucleotides containing a nucleic acid molecule or polynucleotide. “Isolated nucleic acid” refers to a nucleic acid segment or fragment separated from adjacent sequences in its naturally occurring state, i.e., a DNA fragment removed from sequences normally adjacent to that fragment in a naturally occurring genome. The term also applies to other components naturally associated with nucleic acids, i.e., nucleic acids that are substantially purified from RNA or DNA or proteins naturally associated with them in cells. Thus, the term includes, for example, recombinant DNA incorporated into vectors, autonomously replicating plasmids or viruses, or the genomic DNA of prokaryotes or eukaryotes, or recombinant DNA existing as a distinct molecule independent of other sequences (i.e., cDNA or as a genomic or cDNA fragment produced by PCR or restriction enzyme digestion). It also includes recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0154] When used herein to refer to nucleic acids, “complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid chains or between two regions of the same nucleic acid chain. It is known that an adenine residue in a first nucleic acid region can form a specific hydrogen bond ("base pairing") with a residue in a second nucleic acid region that is antiparallel to the first region, if the residue is thymine or uracil. Similarly, a cytosine residue in a first nucleic acid chain can base pair with a residue in a second nucleic acid chain that is antiparallel to the first chain, if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel configuration, at least one nucleotide residue in the first region can base pair with a residue in the second region. In some embodiments, the first region includes a first portion, and the second region includes a second portion, so that when the first and second portions are arranged in an antiparallel configuration, at least about 50%, and / or at least about 75%, or at least about 90%, or at least about 95% of the nucleotide residues in the first portion can be base-paired with nucleotide residues in the second portion. In some embodiments, all nucleotide residues in the first portion can be base-paired with nucleotide residues in the second portion.

[0155] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is ligated. The term includes vectors as self-replicating nucleic acid structures, as well as vectors integrated into the genome of a host cell into which they are introduced. A vector may be a viral vector, plasmid, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. A vector may be an adeno-associated virus (AAV) vector or a lentiviral vector. A vector may be a DNA or RNA vector. A vector may be either a self-replicating extrachromosomal vector or a vector integrated into a host genome. A given vector is capable of inducing the expression of a nucleic acid to which it is functionally ligated. Such a vector is referred to herein as an “expression vector.”

[0156] "Code" refers to the inherent properties and resulting biological properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, that serve as a template for the synthesis of other polymers and macromolecules in a biological process having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing) and the non-coding strand used as a template for the transcription of a gene or cDNA can be said to code for a protein or other product of that gene or cDNA.

[0157] The terms “polypeptide” and “peptide” are used interchangeably to refer to polymers of amino acid residues and are not limited to the minimum length. Such polymers of amino acid residues may contain native or non-native amino acid residues. Both full-length proteins and fragments thereof are included in the definition. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, and phosphorylation. Furthermore, “polypeptide” includes modifications to the native sequence, such as deletions, additions, and substitutions (generally inherently conserved), as long as the polypeptide maintains the desired activity. These modifications may be planned (e.g., via site-directed mutagenesis) or accidental (e.g., via host mutations resulting in a protein or error due to PCR amplification).

[0158] As used herein, “conjugated” refers to a covalent bond between one molecule and a second molecule.

[0159] Where used herein, a “variant” is a nucleic acid or peptide sequence that differs in sequence from a reference nucleic acid or peptide sequence but retains the essential biological properties of the reference molecule. Variations in the nucleic acid variant sequence may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or they may result in amino acid substitutions, additions, deletions, fusions, and cleavages. Variations in the peptide variant sequence are typically limited or conserved, so the sequences of the reference peptide and the variant are very similar overall and identical in many regions. Variants and reference peptides may differ in their amino acid sequences by any combination of one or more substitutions, additions, or deletions. Nucleic acid or peptide variants may be naturally occurring, such as allelic variants, or they may be variants not known to exist naturally. Variants of nucleic acids and peptides that do not exist naturally may be produced by mutagenesis or direct synthesis. In various embodiments, the variant sequence is at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, and at least 85% identical to the reference sequence.

[0160] The term “control” as used herein may mean any method of altering the level or activity of a substrate. Non-limiting examples of controlling a protein include affecting its expression (including transcription and / or translation), its folding, its degradation or protein turnover, and its localization. Non-limiting examples of controlling an enzyme further include affecting its enzyme activity. “Regulator” refers to a molecule whose activity affects the level or activity of a substrate. Regulators may be direct or indirect. Regulators may function to activate, inhibit, or otherwise modulate their substrate.

[0161] The term "control sequence" refers to a DNA sequence necessary for the expression of a functionally linked coding sequence in a particular host organism. For example, a suitable control sequence for prokaryotes includes a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0162] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulant, formulation aid, or carrier commonly used in the art for use with a therapeutic agent containing a "pharmaceutically acceptable composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration used and is compatible with the other components of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation used.

[0163] The terms "pharmaceutical preparation" and "pharmaceutical composition" refer to preparations that are in a form that allows the biological activity of the active ingredient(s) to be effective, and that do not contain additional ingredients that are unacceptably toxic to the target population to which the preparation will be administered. Such preparations may be sterile.

[0164] "Sterile" preparations are sterile or essentially free of live microorganisms and their spores.

[0165] A "reconstituted" formulation is prepared by dissolving a lyophilized protein or antibody formulation in a diluent so that the protein is evenly dispersed. Reconstituted formulations are suitable for administration to patients treated with the target protein (e.g., subcutaneous administration) and, in certain embodiments, may be suitable for parenteral or intravenous administration.

[0166] An "isotonic" preparation is one that has essentially the same osmotic pressure as human blood. Isotonic preparations typically have an osmotic pressure of about 250–350 mOsm. The term "hypotonic" refers to a preparation that has an osmotic pressure lower than that of human blood. Correspondingly, the term "hypertonic" is used to refer to a preparation that has an osmotic pressure higher than that of human blood. Isotonicity can be measured, for example, using vapor pressure or an ice-type osmometer. The preparations of this application may be hypertonic as a result of the addition of salts and / or buffers.

[0167] The terms “transfected,” “transformed,” or “transduced,” as used herein, refer to the process by which an exogenous nucleic acid is transferred to or introduced into a host cell. A “transfected,” “transformed,” or “transduced” cell is one which has been transfected, transformed, or transduced with an exogenous nucleic acid. Cells include primary target cells and their offspring.

[0168] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced (including the offspring of such cells). Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring (regardless of the number of passages). Offspring may not be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring having the same function or biological activity as those screened or selected in the original transformed cells are included herein.

[0169] As used herein, “treatment” or “to treat” is an approach to obtain beneficial or desired outcomes, including clinical outcomes. For the purposes of this application, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: reducing one or more symptoms caused by the disease; reducing the severity of the disease; stabilizing the disease (e.g., preventing or delaying disease exacerbation); preventing or delaying the spread of the disease (e.g., metastasis); preventing or delaying disease recurrence; delaying or slowing disease progression; improving the disease state; providing remission (partial or total) of the disease; reducing the dose of one or more other medicines required to treat the disease; delaying disease progression; improving or enhancing quality of life; improving weight gain; and / or extending survival. A method of application is intended to achieve any one or more of these aspects of treatment.

[0170] The terms “effective dose” and “pharmaceutically effective dose,” as used herein, refer to an amount of drug sufficient to deliver a desired biological effect. This effect may be a reduction (e.g., a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and / or mitigation of a sign, symptom, or cause of a disease or disorder, or any other desired modification of a biological system.

[0171] The term “inhibit” or “inhibit” refers to a reduction or absence of any phenotypic feature, or a reduction or absence of the occurrence, degree, or possibility of such feature. To “reduce” or “inhibit” is to reduce, reduce, or prevent activity, function, and / or quantity compared to the reference. In a given embodiment, “reduce” or “inhibit” means the ability to cause an overall reduction of 20% or more (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%). In another embodiment, “reduce” or “inhibit” means the ability to cause an overall reduction of 50% or more. In yet another embodiment, “reduce” or “inhibit” means the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more.

[0172] As used herein, “suppressing” function or activity means reducing function or activity compared to conditions that are identical except for the condition or parameter being studied, or alternatively, compared to another condition. For example, an antibody that suppresses tumor growth reduces the rate of tumor growth compared to the rate of tumor growth in the absence of the antibody.

[0173] As used herein, the terms “patient,” “subject,” “individual,” etc., are used interchangeably herein and refer to any animal having a complement system, mammals in some embodiments, and humans (including humans who require or are susceptible to therapy for a disease or its sequelae). Individuals may include, for example, dogs, cats, pigs, cattle, sheep, goats, horses, rats, monkeys, mice, and humans. In some embodiments, the individual is human.

[0174] Affinity-matured antibodies are those having one or more modifications in one or more CDRs, resulting in improved antibody affinity to an antigen compared to parent antibodies without those modifications. In some embodiments, affinity-matured antibodies have nanomolar or even picomolar affinity to the target antigen. Affinity-matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation by VH- and VL-domain shuffling. Random mutagenesis of CDRs and / or framework residues has been described, for example, by Barbas et al. Proc Nat.Acad.Sci.USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J.Immunol.155:1994-2004 (1995); Jackson et al., J.Immunol.154(7):3310-9 (1995); and Hawkins et al., J.Mal.Biol.226:889-896 (1992).

[0175] It is understood that embodiments of this application described herein include embodiments "consisting of" and / or "essentially consisting of".

[0176] References to values ​​or parameters "about" in this specification include (and shall be described) variations in the value or parameter itself. For example, a statement referring to "about X" includes a statement of "X".

[0177] Where used herein, a reference to "not" a certain value or parameter usually means and describes "other than" a certain value or parameter. For example, "a method is not used to treat disease of type X" means "a method is used to treat disease of any other type than X."

[0178] As used herein, the term "approximately XY" has the same meaning as "approximately X to approximately Y".

[0179] As used herein and in the appended claims, the singular forms "a," "or," and "the" refer to multiple subjects unless the context otherwise clearly indicates.

[0180] II.2 Functional Protein Constructs This application provides, in one embodiment, a bifunctional protein construct comprising a first binding site and a second binding site, wherein the first binding site specifically binds to a muscle-specific molecule, and the second binding site specifically binds to and activates a Notch receptor. The term “bifunctional protein construct” as used herein refers to a protein construct that includes binding function to both a muscle-specific molecule and a Notch receptor, and may encompass constructs with additional functionality. For example, in some embodiments, the bifunctional protein construct is multispecific. In some embodiments, the bifunctional protein construct comprises a first binding site and a second binding site, wherein the first binding site includes means for binding to a muscle-specific molecule, and the second binding site includes means for binding to and activating a Notch receptor.

[0181] In some embodiments, the first binding site includes an antibody site, including but not limited to full-length antibodies, scFv, Fab, or sdAb. In some embodiments, the first binding site includes a non-antibody site. Exemplary first binding sites include, but are not limited to, anti-LAMA2 antibody sites (e.g., any of the anti-human LAMA2 antibody sites described herein), anti-matriglycan antibody sites (e.g., any of the anti-human ADG41 antibody sites described herein), and anti-CDH15 antibodies (e.g., any of the anti-human CDH15 antibody sites described herein). These antibody sites are described in more detail in the following sections. Means for binding to muscle-specific molecules as described herein may be any of the antibody sites and their functional equivalents described herein.

[0182] In some embodiments, the first binding site includes a non-antibody binding site that specifically binds to muscle-specific molecules, such as a protein domain of an extracellular matrix (ECM) protein capable of binding to matriglycan (e.g., a laminin G-like domain (LG domain)). In some embodiments, the non-antibody site includes a protein domain selected from the group consisting of the LG domain of laminin, the LG domain of agryn, the LG domain of nidogen, and the LG domain of perlecan. In some embodiments, the first binding site includes two or more non-antibody sites connected in tandem. The means for binding to muscle-specific molecules described herein may be any of the non-antibody binding sites and their functional equivalents described herein.

[0183] The second binding site described herein specifically binds to and activates the Notch receptor. In some embodiments, the second binding site includes the extracellular domain (ECD) or variant thereof of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jagged 1 (Jag1), and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs. 126-129 and 260-267). In some embodiments, the second binding site includes a DLL1 ECD containing the amino acid sequence of SEQ ID NO: 130, or a variant thereof containing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 130. In some embodiments, the second binding site includes a DLL3 ECD containing the amino acid sequence of SEQ ID NO: 131, or a variant thereof containing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 131. In some embodiments, the second binding site includes a DLL4 ECD containing the amino acid sequence of SEQ ID NO: 125, or a variant thereof containing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 125. In some embodiments, the second binding site includes a variant DLL4 ECD containing the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267, or a variant thereof containing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs: 126-129 and 260-267. In some embodiments, the second binding site includes a Jag1 ECD containing the amino acid sequence of SEQ ID NO: 132, or a variant thereof containing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 132.In some embodiments, the second binding site includes a variant Jag1 ECD containing the amino acid sequence of SEQ ID NO: 133 or 134, or a variant thereof containing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 133 or 134. In some embodiments, the second binding site includes a Jag2 ECD containing the amino acid sequence of SEQ ID NO: 135, or a variant thereof containing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 135. The means for binding to (and optionally activating) the Notch receptor as described herein may be any of the non-antibody sites and their functional equivalents as described herein. In some embodiments, the second binding site is an anti-Notch antibody site that activates the Notch receptor. The means for binding to (and optionally activating) the Notch receptor as described herein may be any of the anti-Notch antibody sites and their functional equivalents as described herein.

[0184] The first and second binding sites described herein may be fused (i.e., covalently linked) to each other directly or indirectly. In some embodiments, the first binding site is fused to the second binding site via a carrier protein (e.g., albumin, an anti-human serum albumin antibody, or a monomeric Fc domain). In some embodiments, the first binding site is fused to the second binding site via any peptide linker (e.g., any of the peptide linkers described under the “Linkers” subsection), for example, a peptide linker having an amino acid length of about 30 or fewer (e.g., one or fewer of about 25, 20, or 15). In some embodiments, the bifunctional protein construct provided herein comprises an Fc domain comprising a first subunit and a second subunit.

[0185] The bifunctional protein constructs provided herein may be used in any one of the various bispecific or multispecific antibody construct forms known in the art. Numerous forms have been developed in the art to address the therapeutic opportunities presented by molecules with multiple binding specificities. Several approaches have been described for preparing bispecific antibody constructs in which a specific antibody light chain or fragment pairs with a specific antibody heavy chain or fragment.

[0186] For example, international patent application number PCT / EP2011 / 056388 (WO2011 / 131746) describes an in vitro method for generating a heterodimer protein in which asymmetric mutations are introduced into the CH3 region of two monospecific starting proteins to induce directional "Fab arm" or "half-body" exchange between two monospecific IgG4 or IgG4-like antibodies by incubation under reducing conditions.

[0187] Schaefer et al. (Roche Diagnostics GmbH) describe a method for assembling two heavy chains and two light chains derived from two existing antibodies into a human bivalent bispecific IgG antibody without the use of an artificial linker (PNAS (2011) 108(27):11187-11192 and US2009 / 0232811). The method involves exchanging one or more heavy chain and light chain domains within one half of the antigen-binding fragment (Fab) of the bispecific antibody (CrossMab). Based on a knob-into-hole technique that enables heavy chain heterodimerization, the correct association of the light chains and their corresponding heavy chains is achieved by exchanging the heavy chain and light chain domains within one half of the antigen-binding fragment (Fab) of the bispecific antibody. This "crossover" preserves antigen-binding affinity but makes the two arms very different, so light chain mispairing can no longer occur. See WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, and WO2009 / 080254 (each incorporated herein by reference in its entirety).

[0188] 1. Direct fusion or fusion via carrier proteins The first and second binding sites may be linked to each other directly or indirectly (i.e., via a peptide linker or carrier protein, e.g., a monomeric carrier protein). The first binding site may be fused to the second binding site via any linker (e.g., any of SEQ ID NOs. 211, 212, 337, and 338). The first and second binding sites may also be fused to each other directly. The first binding site may be fused to the N-terminus or C-terminus of the second binding site, e.g., to the N-terminus or C-terminus of any one of the polypeptides of the second binding site.

[0189] In some embodiments, the first and second binding sites are linked to each other via a carrier protein. In some embodiments, the carrier protein is selected from the group consisting of human serum albumin (HSA), anti-HSA antibody sites, and Fc domain subunits. Anti-HSA antibody sites are known in the art, for example, Mandrup et al., Commun Biol., 4(1):310 (2021) and Benjamin et al., Hybridoma, 6(2):183-90 (1987). In some embodiments, the first binding site is fused to the N-terminus of the carrier protein via an optional first linker, and the second binding site is fused to the C-terminus of the carrier protein via an optional second linker. In some embodiments, the first binding site is fused to the C-terminus of the carrier protein via an optional first linker, and the second binding site is fused to the N-terminus of the carrier protein via an optional second linker. In some embodiments, the carrier protein (e.g., HSA) extends the half-life of the bifunctional protein construct (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more).

[0190] In some embodiments, the first binding site comprises an scFv containing VH and VL that specifically bind to muscle-specific molecules, and the scFv is fused to a second binding site that specifically binds to the Notch receptor. The second binding site may be fused to the N-terminus or C-terminus of the scFv that specifically recognizes muscle-specific molecules via an optional linker. In some embodiments, the optional linker between the second binding site and the scFv comprises a carrier protein, e.g., HSA, an anti-HSA antibody site, and a subunit of the Fc domain. In some embodiments, the optional linker between the second binding site and the scFv is a peptide linker. In some embodiments, the bifunctional protein construct comprises a second binding site that specifically binds to the Notch receptor, forming the scFv from N' to C': [VH - optional first linker (e.g., peptide linker) - VL] - optional second linker (e.g., peptide linker or carrier protein). In some embodiments, the bifunctional protein construct includes a second binding site that specifically binds to the Notch receptor, forming N' to C':scFv [VL-a first arbitrary linker (e.g., peptide linker)-VH]-a second arbitrary linker (e.g., peptide linker or carrier protein). In some embodiments, the bifunctional protein construct includes a second binding site that specifically binds to the Notch receptor, forming N' to C':scFv [VH-a second arbitrary linker (e.g., peptide linker)-VL]. In some embodiments, the bifunctional protein construct includes a second binding site that specifically binds to the Notch receptor, forming N' to C':scFv [VL-a second arbitrary linker (e.g., peptide linker)-VH]. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. In some embodiments, the first binding site includes an anti-matriglycan scFv (e.g., SEQ ID NO: 155).In some embodiments, the first binding site includes an anti-LAMA2 scFv (e.g., SEQ ID NO: 145 or 268). In some embodiments, the second binding site includes an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding site includes any of the amino acid sequences of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the first binding site optionally includes a plurality (e.g., two or three) of scFv linked in tandem via a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).

[0191] In some embodiments, the first binding site comprises a Fab comprising a first polypeptide chain containing VH and CH1 and a second polypeptide chain containing VL and CL, wherein the Fab is fused to a second binding site that specifically binds to muscle-specific molecules and specifically to Notch receptors via an optional linker. In some embodiments, the second binding site is fused to the N-terminus of the first polypeptide chain of the Fab. In some embodiments, the second binding site is fused to the C-terminus of the first polypeptide chain of the Fab. In some embodiments, the second binding site is fused to the N-terminus of the second polypeptide chain of the Fab. In some embodiments, the second binding site is fused to the C-terminus of the second polypeptide chain of the Fab. In some embodiments, the bifunctional protein construct comprises i) a fusion polypeptide comprising a second binding site that specifically binds to a Notch receptor via N' to C':VH-CH1-any linker (e.g., a peptide linker or carrier protein); and ii) a second polypeptide comprising N' to C':VL-CL; VH-CH1 and VL-CL form a Fab that specifically binds to muscle-specific molecules. In some embodiments, the bifunctional protein construct comprises i) a fusion polypeptide comprising N'-C': a second binding site that specifically binds to the Notch receptor - an arbitrary linker (e.g., a peptide linker or carrier protein) - VH-CH1; and ii) a second polypeptide comprising N'-C': VL-CL; VH-CH1 and VL-CL form a Fab that specifically binds to muscle-specific molecules.In some embodiments, the bifunctional protein construct comprises i) a first polypeptide comprising N' to C':VH-CH1; and ii) a fusion polypeptide comprising N' to C':a second binding site that specifically binds to the Notch receptor—an optional linker (e.g., a peptide linker or carrier protein)—VL-CL; VH-CH1 and VL-CL form a Fab that specifically binds to a muscle-specific molecule. In some embodiments, the optional linker between the second binding site and the Fab comprises a carrier protein, e.g., HSA, an anti-HSA antibody site, and a subunit of the Fc domain. In some embodiments, the optional linker between the second binding site and the Fab is a peptide linker. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. In some embodiments, the first binding site includes an anti-matriglycan Fab, e.g., an anti-matriglycan Fab comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 176 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 177. In some embodiments, the first binding site includes an anti-LAMA2 Fab, e.g., an anti-LAMA2 Fab comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 165 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 166. In some embodiments, the second binding site includes an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, e.g., any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding site includes any of the amino acid sequences of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the first binding site optionally includes multiple (e.g., two or three) Fabs linked in tandem via a peptide linker (e.g., any of SEQ ID NOs. 211, 212, 337, and 338).

[0192] In some embodiments, the first binding site comprises an sdAb containing a VHH domain that specifically binds to muscle-specific molecules, and the sdAb is fused to a second binding site that specifically binds to the Notch receptor. The second binding site may be fused to the N-terminus or C-terminus of the sdAb. In some embodiments, the bifunctional protein construct comprises an sdAb (e.g., VHH) that specifically binds to muscle-specific molecules from N' to C' - an optional linker (e.g., a peptide linker or carrier protein) - a second binding site that specifically binds to the Notch receptor. In some embodiments, the bifunctional protein construct comprises an sdAb (e.g., VHH) that specifically binds to muscle-specific molecules from N' to C' - an optional linker (e.g., a peptide linker or carrier protein) - an sdAb (e.g., VHH) that specifically binds to muscle-specific molecules. In some embodiments, the optional linker between the second binding site and the sdAb comprises a carrier protein, e.g., HSA, an anti-HSA antibody site, and a subunit of the Fc domain. In some embodiments, the optional linker between the second binding site and the sdAb is a peptide linker. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. In some embodiments, the second binding site includes an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs. 126-129 and 260-267). In some embodiments, the second binding site includes any of the amino acid sequences of SEQ ID NOs. 126-135 and 260-267. In some embodiments, the first binding site optionally includes a plurality (e.g., two or three) of sdAbs linked in tandem via a peptide linker (e.g., any of SEQ ID NOs. 211, 212, 337, and 338).

[0193] In some embodiments, the first binding site includes a non-antibody site that specifically binds to a muscle-specific molecule, and the non-antibody site includes an LG domain selected from the group consisting of, for example, the LG domain of laminin, the LG domain of agryn, the LG domain of nidogen, and the LG domain of perlecan. In some embodiments, the bifunctional protein construct includes a second binding site that specifically binds to the Notch receptor, N' to C': a non-antibody site that specifically binds to a muscle-specific molecule - an optional linker (e.g., a peptide linker or carrier protein). In some embodiments, the bifunctional protein construct includes a second binding site that specifically binds to the Notch receptor, N' to C': an optional linker (e.g., a peptide linker or carrier protein) - a non-antibody site that specifically binds to a muscle-specific molecule. In some embodiments, the optional linker between the second binding site and the non-antibody site includes a carrier protein, e.g., HSA, an anti-HSA antibody site, and a subunit of the Fc domain. In some embodiments, the optional linker between the second binding site and the non-antibody site is a peptide linker. In some embodiments, the first binding site includes LAMA2 LG4-5 containing the amino acid sequence of SEQ ID NO: 118 or 119. In some embodiments, the second binding site includes an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding site includes the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the first binding site optionally includes a plurality of (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) non-antibody sites (e.g., LG domains) linked in tandem via a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).

[0194] Exemplary direct fusion In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising N' to C':DLL4 ECD or a variant thereof (e.g., any of SEQ ID NOs. 126-135 and 260-267); an arbitrary peptide linker (e.g., any of SEQ ID NOs. 211, 212, 337, and 338); and a first binding site comprising the LG domain of LAMA2 (e.g., LAMA2 LG4-5).

[0195] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising variant DLL4 ECD from N' to C', wherein variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 136 (hereinafter referred to as "DLL4wt-LG4-5").

[0196] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising variant DLL4 ECD from N' to C', wherein variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 137 (hereinafter referred to as "DLL4v-LG4-5").

[0197] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising N' to C': DLL4 variant ECD, wherein DLL4 variant ECD comprises the amino acid sequence of SEQ ID NO: 128; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 138 (hereinafter referred to as "DLL4max-LG4-5").

[0198] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising variant DLL4 ECD from N' to C', wherein variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 139 (hereinafter referred to as "DLL4deimmune(DLL4di)-LG4-5").

[0199] In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N' to C':DLL1 ECD or a variant thereof; an optional peptide linker; and a first binding site comprising the LG domain of LAMA2 (e.g., LAMA2 LG4-5). In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N' to C':DLL1 ECD, wherein DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 140 (hereinafter referred to as "DLL1wt-LG4-5").

[0200] In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N' to C':DLL3 ECD or a variant thereof; an optional peptide linker; and a first binding site comprising the LG domain of LAMA2 (e.g., LAMA2 LG4-5). In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N' to C':DLL3 ECD, wherein DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 141 (hereinafter referred to as "DLL3wt-LG4-5").

[0201] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site from N' to C' containing Jag1 ECD or a variant thereof (e.g., any of the Jag1 ECDs described herein, e.g., any of SEQ ID NOs. 132-134); an arbitrary peptide linker (e.g., any of SEQ ID NOs. 211, 212, 337, and 338); and a first binding site containing the LG domain of LAMA2 (e.g., LAMA2 LG4-5) (e.g., SEQ ID NO. 119).

[0202] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising variant Jag1 ECD from N' to C', wherein variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 142 (hereinafter referred to as "Jag1wt-LG4-5").

[0203] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising variant Jag1 ECD from N' to C', wherein variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 143 (hereinafter referred to as "Jag1v-LG4-5").

[0204] In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N'-to-C':Jag2 ECD or a variant thereof; an optional peptide linker; and a first binding site comprising the LG domain of LAMA2 (e.g., LAMA2 LG4-5). In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N'-to-C':Jag2 ECD, wherein Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 144 (hereinafter referred to as "Jag2wt-LG4-5").

[0205] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising N' to C': DLL4 ECD or a variant thereof (e.g., either the DLL4 ECD described herein or an engineered DLL4 ECD, e.g., any of SEQ ID NOs: 126-129 and 260-267); an arbitrary peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding site comprising anti-LAMA2 scFv (e.g., SEQ ID NOs: 145 or 268).

[0206] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant DLL4 ECD from N' to C', wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 146 (hereinafter referred to as "DLL4wt-LG21scFv").

[0207] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant DLL4 ECD from N' to C', wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 147 (hereinafter referred to as "DLL4v-LG21scFv").

[0208] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant DLL4 ECD from N' to C', wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 128; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 148 (hereinafter referred to as "DLL4max-LG21scFv").

[0209] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant DLL4 ECD from N' to C', wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 149 (hereinafter referred to as "DLL4di-LG21scFv").

[0210] In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N'-to-C':DLL1 ECD or a variant thereof; an optional peptide linker; and a first binding site comprising anti-LAMA2 scFv. In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N'-to-C':DLL1 ECD, wherein DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising anti-LAMA2 scFv, wherein anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 150 (hereinafter referred to as "DLL1wt-LG21scFv").

[0211] In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N'-to-C':DLL3 ECD or a variant thereof; an optional peptide linker; and a first binding site comprising anti-LAMA2 scFv. In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N'-to-C':DLL3 ECD, wherein DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising anti-LAMA2 scFv, wherein anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 151 (hereinafter referred to as "DLL3wt-LG21scFv").

[0212] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising N' to C': Jag1 ECD or a variant thereof (e.g., any of the Jag1 ECDs described herein, e.g., any of SEQ ID NOs. 132-134); any peptide linker (e.g., any of SEQ ID NOs. 211, 212, 337, and 338); and a first binding site comprising anti-LAMA2 scFv (e.g., SEQ ID NOs. 145 or 268).

[0213] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant Jag1 ECD from N' to C', wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 152 (hereinafter referred to as "Jag1wt-LG21scFv").

[0214] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant Jag1 ECD from N' to C', wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 153 (hereinafter referred to as "Jag1v-LG21scFv").

[0215] In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N'-to-C':Jag2 ECD or a variant thereof; an optional peptide linker; and a first binding site comprising anti-LAMA2 scFv. In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N'-to-C':Jag2 ECD, wherein Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising anti-LAMA2 scFv, wherein anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 154 (hereinafter referred to as "Jag2wt-LG21scFv").

[0216] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site from N' to C' containing a DLL4 ECD or a variant thereof (e.g., either the DLL4 ECD described herein or an engineered DLL4 ECD, e.g., any of SEQ ID NOs. 126-129 and 260-267); an arbitrary peptide linker (e.g., any of SEQ ID NOs. 211, 212, 337, and 338); and a first binding site containing an anti-matriglycan scFv (e.g., SEQ ID NO. 155).

[0217] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant DLL4 ECD from N' to C', wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising an anti-matriglycan scFv, wherein the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 156 (hereinafter referred to as "DLL4wt-ADG41scFv").

[0218] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant DLL4 ECD from N' to C', wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising an anti-matriglycan scFv, wherein the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 157 (hereinafter referred to as "DLL4v-ADG41scFv").

[0219] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant DLL4 ECD from N' to C', wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 128; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising an anti-matriglycan scFv, wherein the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 158 (hereinafter referred to as "DLL4max-ADG41scFv").

[0220] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant DLL4 ECD from N' to C', wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising an anti-matriglycan scFv, wherein the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 159 (hereinafter referred to as "DLL4di-ADG41scFv").

[0221] In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N' to C':DLL1 ECD or a variant thereof; an optional peptide linker; and a first binding site comprising anti-matriglycan scFv. In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N' to C':DLL1 ECD, wherein DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising anti-matriglycan scFv, wherein anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 160 (hereinafter referred to as "DLL1wt-ADG41scFv").

[0222] In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N'-to-C':DLL3 ECD or a variant thereof; an optional peptide linker; and a first binding site comprising an anti-matriglycan scFv. In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N'-to-C':DLL3 ECD, wherein DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising an anti-matriglycan scFv, wherein anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 161 (hereinafter referred to as "DLL3wt-ADG41scFv").

[0223] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising N' to C': Jag1 ECD or a variant thereof (e.g., any of the Jag1 ECDs described herein, e.g., any of SEQ ID NOs. 132-134); any peptide linker (e.g., any of SEQ ID NOs. 211, 212, 337, and 338); and a first binding site comprising anti-matriglycan scFv (e.g., SEQ ID NO. 155).

[0224] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant Jag1 ECD from N' to C', wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising an anti-matriglycan scFv, wherein the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 162 (hereinafter referred to as "Jag1wt-ADG41scFv").

[0225] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant Jag1 ECD from N' to C', wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134; an arbitrary peptide linker (e.g., SEQ ID NO: 211); and a first binding site comprising an anti-matriglycan scFv, wherein the anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 163 (hereinafter referred to as "Jag1v-ADG41scFv").

[0226] In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N' to C':Jag2 ECD or a variant thereof; an arbitrary peptide linker (e.g., any of SEQ ID NOs. 211, 212, 337, and 338); and a first binding site comprising anti-matriglycan scFv. In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N' to C':Jag2 ECD, wherein Jag2 ECD comprises the amino acid sequence of SEQ ID NO. 135; an arbitrary peptide linker (e.g., SEQ ID NO. 211); and a first binding site comprising anti-matriglycan scFv, wherein anti-matriglycan scFv comprises the amino acid sequence of SEQ ID NO. 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO. 164 (hereinafter referred to as "Jag2wt-ADG41scFv").

[0227] In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising anti-LAMA2 Fab (e.g., SEQ ID NO: 165) and a second binding site comprising DLL4 ECD or a variant thereof (e.g., any of the DLL4 ECDs described herein or manipulated DLL4 ECDs, e.g., any of SEQ ID NOs: 126-129 and 260-267), wherein the second binding site is fused to the N-terminus of the VL of anti-LAMA2 Fab (e.g., SEQ ID NO: 165) via an arbitrary peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).

[0228] In some embodiments, a bifunctional protein construct is provided, comprising a first binding site comprising anti-LAMA2 Fab and a second binding site comprising variant DLL4 ECD, wherein the second binding site is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126, and anti-LAMA2 Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a fusion polypeptide comprising the second binding site and the second polypeptide of anti-LAMA2 Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 167 (hereinafter referred to as "DLL4wt-LG21-Fab").

[0229] In some embodiments, a bifunctional protein construct is provided, comprising a first binding site comprising anti-LAMA2 Fab and a second binding site comprising variant DLL4 ECD, wherein the second binding site is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 127, and anti-LAMA2 Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a fusion polypeptide comprising the second binding site and the second polypeptide of anti-LAMA2 Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 168 (hereinafter referred to as "DLL4v-LG21-Fab").

[0230] In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising anti-LAMA2 Fab and a second binding site comprising variant DLL4 ECD, wherein the second binding site is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 128, and anti-LAMA2 Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises a fusion polypeptide comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second binding site and the second polypeptide of anti-LAMA2 Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 169 (hereinafter referred to as "DLL4max-LG21-Fab").

[0231] In some embodiments, a bifunctional protein construct is provided, comprising a first binding site comprising anti-LAMA2 Fab and a second binding site comprising variant DLL4 ECD, wherein the second binding site is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 129, and anti-LAMA2 Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a fusion polypeptide comprising the second binding site and the second polypeptide of anti-LAMA2 Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 170 (hereinafter referred to as "DLL4di-LG21-Fab").

[0232] In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising anti-LAMA2 Fab and a second binding site comprising DLL1 ECD or a variant thereof, wherein the second binding site is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising anti-LAMA2 Fab and a second binding site comprising DLL1 ECD, wherein the second binding site is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130, and the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide having a second binding site and a second polypeptide containing anti-LAMA2 Fab, the fusion polypeptide having the amino acid sequence of SEQ ID NO: 171 (hereinafter referred to as "DLL1wt-LG21-Fab").

[0233] In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising anti-LAMA2 Fab and a second binding site comprising DLL3 ECD or a variant thereof, wherein the second binding site is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising anti-LAMA2 Fab and a second binding site comprising DLL3 ECD, wherein the second binding site is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), the DLL3 ECD comprising the amino acid sequence of SEQ ID NO: 131, and the anti-LAMA2 Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide having a second binding site and a second polypeptide containing anti-LAMA2 Fab, the fusion polypeptide having the amino acid sequence of SEQ ID NO: 172 (hereinafter referred to as "DLL3wt-LG21-Fab").

[0234] In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising anti-LAMA2 Fab (e.g., SEQ ID NO: 165) and a second binding site comprising Jag1 ECD or a variant thereof (e.g., any of the Jag1 ECDs described herein, e.g., any of SEQ ID NOs: 132-134), wherein the second binding site is fused to the N-terminus of the VL of anti-LAMA2 Fab (e.g., SEQ ID NO: 165) via an arbitrary peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).

[0235] In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising anti-LAMA2 Fab and a second binding site comprising variant Jag1 ECD, wherein the second binding site is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133, and anti-LAMA2 Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a fusion polypeptide comprising the second binding site and the second polypeptide of anti-LAMA2 Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 173 (hereinafter referred to as "Jag1wt-LG21-Fab").

[0236] In some embodiments, a bifunctional protein construct is provided, comprising a first binding site comprising anti-LAMA2 Fab and a second binding site comprising variant Jag1 ECD, wherein the second binding site is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 134, and anti-LAMA2 Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a fusion polypeptide comprising the second binding site and the second polypeptide of anti-LAMA2 Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 174 (hereinafter referred to as "Jag1v-LG21-Fab").

[0237] In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising anti-LAMA2 Fab and a second binding site comprising Jag2 ECD or a variant thereof, wherein the second binding site is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising anti-LAMA2 Fab and a second binding site comprising Jag2 ECD, wherein the second binding site is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), the Jag2 ECD comprising the amino acid sequence of SEQ ID NO: 135, and the anti-LAMA2 Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide containing a second binding site and an anti-LAMA2 Fab second polypeptide, the fusion polypeptide containing the amino acid sequence of SEQ ID NO: 175 (hereinafter referred to as "Jag2wt-LG21-Fab").

[0238] In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising an anti-matriglycan Fab (e.g., SEQ ID NO: 176) and a second binding site comprising a DLL4 ECD or a variant thereof (e.g., any of the DLL4 ECDs described herein or manipulated DLL4 ECDs, e.g., any of SEQ ID NOs: 126-129 and 260-267), wherein the second binding site is fused to the N-terminus of the VL of the anti-matriglycan Fab (e.g., SEQ ID NO: 176) via an arbitrary peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).

[0239] In some embodiments, a difunctional protein construct is provided, the difunctional protein construct comprising a first binding site comprising anti-matriglycan Fab and a second binding site comprising variant DLL4 ECD, wherein the second binding site is fused to the N-terminus of the VL of anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126, and anti-matriglycan Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the difunctional protein construct comprises a fusion polypeptide comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and the second binding site and the second polypeptide of anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 178 (hereinafter referred to as "DLL4wt-ADG41-Fab").

[0240] In some embodiments, a difunctional protein construct is provided, the difunctional protein construct comprising a first binding site comprising anti-matriglycan Fab and a second binding site comprising variant DLL4 ECD, wherein the second binding site is fused to the N-terminus of the VL of anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 127, and anti-matriglycan Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the difunctional protein construct comprises a fusion polypeptide comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second binding site and the second polypeptide of anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 179 (hereinafter referred to as "DLL4v-ADG41-Fab").

[0241] In some embodiments, a difunctional protein construct is provided, the difunctional protein construct comprising a first binding site comprising anti-matriglycan Fab and a second binding site comprising variant DLL4 ECD, wherein the second binding site is fused to the N-terminus of the VL of anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 128, and anti-matriglycan Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the difunctional protein construct comprises a fusion polypeptide comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second binding site and the second polypeptide of anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 180 (hereinafter referred to as "DLL4max-ADG41-Fab").

[0242] In some embodiments, a difunctional protein construct is provided, the difunctional protein construct comprising a first binding site comprising anti-matriglycan Fab and a second binding site comprising variant DLL4 ECD, wherein the second binding site is fused to the N-terminus of the VL of anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 129, and anti-matriglycan Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the difunctional protein construct comprises a fusion polypeptide comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second binding site and the second polypeptide of anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 181 (hereinafter referred to as "DLL4di-ADG41-Fab").

[0243] In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising an anti-matriglycan Fab and a second binding site comprising DLL1 ECD or a variant thereof, wherein the second binding site is fused to the N-terminus of the VL of the anti-matriglycan Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising an anti-matriglycan Fab and a second binding site comprising DLL1 ECD, wherein the second binding site is fused to the N-terminus of the VL of the anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130, and the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the bifunctional protein construct comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide having a second binding site and a second polypeptide of anti-matriglycan Fab, the fusion polypeptide having the amino acid sequence of SEQ ID NO: 182 (hereinafter referred to as "DLL1wt-ADG41-Fab").

[0244] In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising an anti-matriglycan Fab and a second binding site comprising a DLL3 ECD or a variant thereof, wherein the second binding site is fused to the N-terminus of the VL of the anti-matriglycan Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising an anti-matriglycan Fab and a second binding site comprising a DLL3 ECD, wherein the second binding site is fused to the N-terminus of the VL of the anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), the DLL3 ECD comprising the amino acid sequence of SEQ ID NO: 131, and the anti-matriglycan Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the bifunctional protein construct comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide having a second binding site and a second polypeptide of anti-matriglycan Fab, the fusion polypeptide having the amino acid sequence of SEQ ID NO: 183 (hereinafter referred to as "DLL3wt-ADG41-Fab").

[0245] In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising an anti-matriglycan Fab (e.g., SEQ ID NO: 176) and a second binding site comprising a Jag1 ECD or a variant thereof (e.g., any of the Jag1 ECDs described herein, e.g., any of SEQ ID NOs: 132-134), wherein the second binding site is fused to the N-terminus of the VL of the anti-matriglycan Fab (e.g., SEQ ID NO: 176) via an arbitrary peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).

[0246] In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising anti-matriglycan Fab and a second binding site comprising variant Jag1 ECD, wherein the second binding site is fused to the N-terminus of the VL of anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133, and anti-matriglycan Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a fusion polypeptide comprising the second binding site and the second polypeptide of anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 184 (hereinafter referred to as "Jag1wt-ADG41-Fab").

[0247] In some embodiments, a difunctional protein construct is provided, the difunctional protein construct comprising a first binding site comprising anti-matriglycan Fab and a second binding site comprising variant Jag1 ECD, wherein the second binding site is fused to the N-terminus of the VL of anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 134, and anti-matriglycan Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the difunctional protein construct comprises a fusion polypeptide comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second binding site and the second polypeptide of anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 185 (hereinafter referred to as "Jag1v-ADG41-Fab").

[0248] In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising an anti-matriglycan Fab and a second binding site comprising a Jag2 ECD or a variant thereof, wherein the second binding site is fused to the N-terminus of the VL of the anti-matriglycan Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, the bifunctional protein construct comprising a first binding site comprising an anti-matriglycan Fab and a second binding site comprising a Jag2 ECD, wherein the second binding site is fused to the N-terminus of the VL of the anti-matriglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), the Jag2 ECD comprising the amino acid sequence of SEQ ID NO: 135, and the anti-matriglycan Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the bifunctional protein construct comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide having a second binding site and a second polypeptide of anti-matriglycan Fab, the fusion polypeptide having the amino acid sequence of SEQ ID NO: 186 (hereinafter referred to as "Jag2wt-ADG41-Fab").

[0249] In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N' to C':DLL4 ECD or a variant thereof (e.g., SEQ ID NO: 339); an arbitrary peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising a laminin-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0250] In some embodiments, from N' to C', there is provided a bifunctional protein construct comprising: a second binding site comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 339; an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising a laminin-α2 LG4-5 domain, wherein the laminin-α2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 319 (hereinafter referred to as "DLL4(EGF1-6)-GS linker-laminin-α2 LG4-5").

[0251] In some embodiments, from N' to C', there is provided a bifunctional protein construct comprising: a second binding site comprising DLL4 ECD or a variant thereof (e.g., SEQ ID NO: 339); an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising a laminin-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0252] In some embodiments, from N' to C', there is provided a bifunctional protein construct comprising: a second binding site comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 339; an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising a laminin-α2 LG4-5 domain, wherein the laminin-α2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 320 (hereinafter referred to as "DLL4(EGF1-6)-HSA-laminin-α2 LG4-5").

[0253] In some embodiments, from N' to C', there is provided a bifunctional protein construct comprising: a second binding site comprising DLL1 ECD or a variant thereof (e.g., SEQ ID NO: 340); an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising laminin-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0254] In some embodiments, from N' to C', there is provided a bifunctional protein construct comprising: a second binding site comprising a variant DLL1 ECD, wherein the variant DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 340; an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising laminin-α2 LG4-5 domain, wherein the laminin-α2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 323 (hereinafter referred to as "DLL1(EGF1-6)-HSA-laminin-α2 LG4-5").

[0255] In some embodiments, from N' to C', there is provided a bifunctional protein construct comprising: a second binding site comprising Jag1 ECD or a variant thereof (e.g., SEQ ID NO: 341); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising laminin-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0256] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant Jag1 ECD from N' to C', wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 341; an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising a laminin-α2 LG4-5 domain, wherein the laminin-α2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 325 (hereinafter referred to as "Jagged1(EGF1-6)-laminin-α2 LG4-5").

[0257] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising N' to C':Jag1 ECD or a variant thereof (e.g., SEQ ID NO: 341); an arbitrary peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an arbitrary peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising a laminin-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0258] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising variant Jag1 ECD from N' to C', wherein variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 341; an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising a laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 326 (hereinafter referred to as "Jagged1(EGF1-6)-HSA-laminin-a2 LG4-5").

[0259] In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N' to C':Jag2 ECD or a variant thereof (e.g., SEQ ID NO: 342); an arbitrary peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising a laminin-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0260] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant Jag2 ECD from N' to C', wherein the variant Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 342; an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising a laminin-α2 LG4-5 domain, wherein the laminin-α2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 328 (hereinafter referred to as "Jagged2(EGF1-6)-laminin-α2 LG4-5").

[0261] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising N' to C':Jag2 ECD or a variant thereof (e.g., SEQ ID NO: 342); an arbitrary peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an arbitrary peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising a laminin-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0262] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant Jag2 ECD from N' to C', wherein the variant Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 342; an optional peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising a laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 329 (hereinafter referred to as "Jagged2(EGF1-6)-HSA-laminin-a2 LG4-5").

[0263] In some embodiments, a bifunctional protein construct is provided comprising a second binding site comprising N' to C':DLL1 ECD or a variant thereof (e.g., SEQ ID NO: 340); an arbitrary peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising a laminin-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0264] In some embodiments, a bifunctional protein construct is provided comprising: a second binding site comprising a variant DLL1 ECD from N' to C', wherein the variant DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 340; an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding site comprising a laminin-α2 LG4-5 domain, wherein the laminin-α2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 322 (hereinafter referred to as "DLL1(EGF1-6)-laminin-α2 LG4-5").

[0265] 2. Fc domain fusion In some embodiments, the bifunctional protein construct includes an Fc domain comprising a first subunit and a second subunit. The Fc domain may be a third site connected to the first and second binding sites. In some embodiments, if the first or second binding site is a full-length antibody, the Fc domain may be part of the full-length antibody.

[0266] In some embodiments, the Fc domain is a variant Fc domain that has minimal effector function or no effector function at all. In some embodiments, the variant Fc domain is derived from IgG1 Fc or IgG4 Fc.

[0267] One or more amino acid modifications may be introduced into the Fc domain, thereby generating an Fc domain variant. The Fc domain variant may include a human Fc domain sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc domain) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0268] In some embodiments, the Fc domain possesses some, but not all, effector functions, making it a desirable candidate for applications where the half-life of the antibody site in vivo is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In some embodiments, the Fc domain contains one or more mutations that increase the half-life of the antibody site in vivo. In some embodiments, the variant Fc domain has increased FcRn binding at pH 6.0. In some embodiments, the variant Fc domain contains the M252Y / S254T / T256E ("YTE") mutation (EU numbering). See, for example, Dall'Acqua et al., J Immunol., 169:5171-5180 (2002) and Wang et al., Protein & Cell, 9(1):63-73 (2018). In some embodiments, the variant Fc domain contains the M428L / N434S mutation. For example, see Zalevsky et al., Nat Biotechnol., 28:157-159 (2010) and Wang et al., Protein & Cell, 9(1):63-73 (2018).

[0269] In vitro and / or in vivo cytotoxicity assays can be performed to analyze the CDC and / or ADCC activity of the Fc domain. For example, Fc receptor (FcR) binding assays can be performed to determine whether an antibody possesses FcgR binding (and thus potential ADCC activity) and / or FcRn binding ability. NK cells, the main cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of target molecules are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); see 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, for example, the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA) and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule may be evaluated in vivo in animal models, such as those disclosed in, for example, Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity.See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0270] Antibodies with reduced effector function include those having one or more substitutions at Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include the so-called "DANA" Fc variant having substitutions to alanine at residues 265 and 297, and variant Fc having substitutions at two or more amino acid positions 265, 269, 270, 297, and 327 (U.S. Patent No. 7,332,581). In some embodiments, the Fc domain of a bifunctional protein construct contains one or more of these mutations. In some embodiments, the variant Fc domain contains the N297A, N297Q, or N297G mutation. See, for example, Bolt et al., Eur J Immunol., 23:403-411 (1993); Leabman et al., Mabs, 5:896-903 (2013); Tao and Morrison, J Immunol., 143:2595-2601 (1989); Walker et al., Biochem J., 259:347-353 (1989); and Wang et al., Protein & Cell, 9(1):63-73 (2018). In some embodiments, the variant Fc domain contains the L235E mutation. See, for example, Alegre et al., J Immunol., 148:3461-3468 (1992) and Wang et al., Protein & Cell, 9(1):63-73 (2018).

[0271] Certain antibody variants having improved or reduced binding to FcR are described. See, for example, U.S. Patent No. 6,737,056;WO2004 / 056312 and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001).

[0272] In some embodiments, the Fc domain is derived from human IgG1, human IgG2, or human IgG4. In some embodiments, the Fc domain is derived from the IgG1 Fc domain (e.g., the human IgG1 Fc domain). In some embodiments, human IgG1 Fc contains the amino acid sequence of Sequence ID No. 187. In some embodiments, each subunit of the Fc domain contains a mutation that reduces or disables effector function. In some embodiments, effector function is eliminated via a mutation in the constant region where glycosylation is eliminated, e.g., an "effectorless mutation". In one embodiment, the effectorless mutation is the N297A or DANA mutation (D265A+N297A) (EU numbering) in the CH2 region. See, for example, Shields et al., J. Biol. Chem. 276(9):6591-6604 (2001). Alternatively or additionally, effector function can be reduced or eliminated through generative techniques such as expression in non-glycosylating host cells (e.g., E. coli) or in host cells that result in modified glycosylation patterns that are ineffective or less effective in promoting effector function (e.g., Shinkawa et al.). al., J. Biol. Chem. 278(5):3466-3473 (2003). In some embodiments, each subunit of the Fc domain contains the L234A / L235 "LALA" mutation (EU numbering). In some embodiments, each subunit of the Fc domain contains the L234A / L235A / P329G "LALALPG" mutation (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 277. In some embodiments, the Fc domain contains a mutation that reduces the half-life of the bifunctional protein construct in the blood circulation, e.g., the H435A mutation (EU numbering). In some embodiments, each subunit of the Fc domain (e.g., human IgG1 Fc) includes L234A / L235A and H435A mutations. In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 188.In some embodiments, the Fc domain is an IgG4 (e.g., human IgG4) Fc domain. In some embodiments, the Fc domain includes a mutation that reduces Fab exchange, e.g., the S228P mutation. In some embodiments, the IgG4 Fc domain includes the F234A / L235A mutation. See, for example, Xu et al., Cell Immunol., 200:16-26 (2000) and Wang et al., Protein & Cell, 9(1):63-73 (2018). For other examples of Fc domain variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351.

[0273] In some embodiments, if the bifunctional protein construct is a heterodimer protein containing two different polypeptide chains, the Fc domain may contain one or more mutations that facilitate the assembly of the heterodimer protein. For example, knob-into-hole is a heterodimerization technique for the CH3 domain of an antibody. To date, the knob-into-hole technique has been applied to the production of human full-length bispecific antibodies having a single common light chain (LC) (Merchant et al. “An efficient route to human bispecific IgG.” Nat Biotechnol. 1998; 16: 677-81; Jackman et al. “Development of a two-part strategy to identify a therapeutic human bispecific antibody that inhibits IgE receptor signaling.” J Biol Chem. 2010; 285: 20850-9). See also WO1996027011 (which is incorporated herein by reference in its entirety for all purposes). All of these mutations may be incorporated into the bifunctional protein constructs described herein.

[0274] Therefore, for example, in some embodiments, the bifunctional protein construct comprises a first CH3 domain and an Fc domain comprising a second CH3 domain (containing a knob-into-hole (KIH) residue). In some embodiments, the first CH3 domain is modified to generate a protrusion on the surface of the first CH3 domain that interacts with the second CH3 domain by replacing one or more amino acid residues having a larger side chain volume within the CH3 / CH3 interface; the second CH3 domain is modified to generate a depression on the surface of the second CH3 domain that interacts with the first CH3 domain by replacing one or more amino acid residues having a smaller side chain volume within the CH3 / CH3 interface. In some embodiments, the protrusion is a knob. In some embodiments, the modification for generating a knob is T366W. In some embodiments, the depression is a hole. In some embodiments, the modification for generating a hole is T366S / L368A / Y407V. Therefore, in some embodiments, the Fc domain described herein includes a knob-into-hole mutation, i) the first subunit of the Fc domain includes a knob mutation and the second subunit of the Fc domain includes a hole mutation; or ii) the second subunit of the Fc domain includes a knob mutation and the first subunit of the Fc domain includes a hole mutation. In some embodiments, the knob mutation is T366W (EU numbering) and the hole mutation is T366S / L368A / Y407V (EU numbering). Unless otherwise indicated, all amino acid positions within the Fc domain are numbered according to the EU numbering system.

[0275] In some embodiments, the bifunctional protein construct includes an Fc domain containing a mutation that allows for the purification of the asymmetric Fc domain. In some embodiments, either the first subunit or the second subunit of the Fc domain contains the H435R / Y436F mutation (EU numbering). In some embodiments, each subunit of the Fc domain contains the H435R / Y436F mutation (EU numbering).

[0276] Strop et al. (Rinat-Pfizer Inc.) describe a method for generating stable bispecific antibodies by separately expressing and purifying the two target antibodies, then mixing them together under specified redox conditions (J. Mol. Biol. (2012) 420:204-19).

[0277] Other heterodimerization domains that have a strong preference for forming heterodimers over homodimers can be incorporated into the bifunctional protein construct. Illustrative examples include, but are not limited to, for example, WO2007147901 (Kjaergaard et al.-Novo Nordisk: describes ionic interactions); WO2009 / 089004 (Kannan et al.-Amgen: describes electrostatic steering effects); WO2010 / 034605 (Christensen et al.-Genentech; describes coiled-coils). See also, for example, Pack, P. & Plueckthun, A., Biochemistry 31, 1579-1584 (1992) which describes leucine zippers, or Pack et al., Bio / Technology 11, 1271-1277 (1993) which describes helix-turn-helix motifs. The term "heterodimerization domain" does not exclude additional units beyond the two heterodimers in the bifunctional construct. In certain embodiments, the bifunctional protein construct comprises one or more heterodimerization domains.

[0278] In some embodiments, when the bifunctional protein construct is a heterodimer protein comprising two different polypeptide chains, the Fc domain may contain one or more charge pair mutations to facilitate the assembly of the heterodimer protein. In some embodiments, an amino acid residue in the first subunit of the Fc domain is replaced with a positively charged residue (e.g., R, H, or K), and an amino acid residue in the second subunit of the Fc domain is replaced with a negatively charged residue (e.g., D or E). In some embodiments, an amino acid residue in the first subunit of the Fc domain is replaced with a negatively charged residue, and an amino acid residue in the second subunit of the Fc domain is replaced with a positively charged residue. In some embodiments, the amino acid residue at D399 (EU numbering) in the first subunit of the Fc domain is replaced with a positively charged residue, and the amino acid residue at K409 (EU numbering) in the second subunit of the Fc domain is replaced with a negatively charged residue. In some embodiments, the amino acid residue at K409 (EU numbering) in the first subunit of the Fc domain is replaced with a negatively charged residue, and the amino acid residue at D399 (EU numbering) in the second subunit of the Fc domain is replaced with a positively charged residue.

[0279] In some embodiments, the Fc domain used in the bifunctional protein construct described herein comprises one of the amino acid sequences of SEQ ID NOs: 188, 254, and 277.

[0280] In some embodiments, a bifunctional protein construct is provided comprising: i) one or more units of a first binding site that specifically binds to a muscle-specific molecule; ii) a first unit of a second binding site that specifically binds to and activates a first Notch receptor; iii) a second unit of a second binding site that specifically binds to and activates a second Notch receptor; and iv) an Fc domain comprising a first subunit and a second subunit, wherein optionally the first unit of the second binding site and the second unit of the second binding site each independently comprise an ECD or variant of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the first unit of the second binding site and the second unit of the second binding site are the same. In some embodiments, the first unit of the second binding site and the second unit of the second binding site are different. One or more units of the first binding site may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, the bifunctional protein construct further includes a third unit of the second binding site that specifically binds to a third Notch receptor, and a fourth unit of the second binding site that specifically binds to a fourth Notch receptor. In some embodiments, the third unit of the second binding site and the fourth unit of the second binding site each independently include an ECD or variant of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs. 126-129 and 260-267). The first, second, third, and / or fourth units of the second binding site may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, all four units of the second binding site are identical. In some embodiments, at least one of the four units of the second binding site is different from the others.One or more units of the first binding site may include antibody or non-antibody sites. Various binding sites may independently fuse to the N and / or C-terminus of the Fc domain. The bifunctional protein construct may be homodimer or heterodimer.

[0281] In some embodiments, a bifunctional protein construct is provided comprising: i) a first unit of a first binding site that specifically binds to a first muscle-specific molecule; ii) a second unit of the first binding site that specifically binds to a second muscle-specific molecule; iii) one or more units of a second binding site that specifically binds to and activates a Notch receptor; and iv) an Fc domain comprising the first and second subunits. In some embodiments, a bifunctional protein construct is provided comprising: i) a first unit of a first binding site including a first antibody site that specifically binds to a first muscle-specific molecule; ii) a second unit of the first binding site including a first antibody site that specifically binds to a second muscle-specific molecule; iii) one or more units of a second binding site that specifically binds to and activates a Notch receptor; and iv) an Fc domain comprising a first subunit and a second subunit, wherein the first and second antibody sites are independently selected from the group consisting of Fab, scFv, and sdAb. In some embodiments, a bifunctional protein construct is provided comprising: i) a first unit of a first binding site including a first non-antibody site that specifically binds to a first muscle-specific molecule; ii) a second unit of the first binding site including a second non-antibody site that specifically binds to a second muscle-specific molecule; iii) one or more units of a second binding site that specifically binds to and activates the Notch receptor; and iv) an Fc domain comprising a first subunit and a second subunit, wherein the first non-antibody site and the second non-antibody site are independently selected from the group consisting of the LG domain of laminin, the LG domain of agryn, the LG domain of nidogen, and the LG domain of perlecan. In some embodiments, the first non-antibody site and / or the second non-antibody site comprises LAMA2 LG4-5 including the amino acid sequence of SEQ ID NO: 118 or 119. In some embodiments, the first unit of the first binding site comprises two or more first non-antibody sites connected in tandem, and the second unit of the first binding site comprises two or more second non-antibody sites connected in tandem.In some embodiments, the first unit of the first binding site and the second unit of the first binding site are the same. In some embodiments, the first unit of the first binding site and the second unit of the first binding site are different. One or more units of the second binding site may be the same or different; and / or may bind to the same or different Notch receptors. Various binding sites may independently fuse to the N and / or C-terminus of the Fc domain. The bifunctional protein construct may be homodimer or heterodimer.

[0282] In some embodiments, one or more units of the first binding site in a bifunctional protein construct include (or are) a Fab, which may be connected to the C-terminus of a subunit of the Fc domain via a VH or VL, for example, via any linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). For example, in some embodiments, a bifunctional protein construct comprising: a first Fab(Fab1) that specifically binds to a first muscle-specific molecule comprising VH(VH1), CH1(H1-CH1), VL(VL1), and CL(L1-CL); a second Fab(Fab2) that specifically binds to a second muscle-specific molecule comprising VH(VH2), CH1(H2-CH1), VL(VL2), and CL(L2-CL); a first unit of a second binding site that specifically binds to and activates a first Notch receptor; a second unit of a second binding site that specifically binds to and activates a second Notch receptor; and an Fc domain comprising the first and second subunits, wherein the bifunctional protein construct The invention provides a bifunctional protein construct comprising: i) a first polypeptide comprising N' to C': a first unit of a second binding site - an arbitrary linker - a first subunit of an Fc domain - an arbitrary linker - VH1-(H1-CH1); ii) a second polypeptide comprising N' to C': a second unit of a second binding site - an arbitrary linker - a second subunit of an Fc domain - an arbitrary linker - VH2-(H2-CH1); iii) a third polypeptide comprising N' to C': VL1-(L1-CL); and iv) a fourth polypeptide comprising N' to C': VL2-(L2-CL); where VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.In some embodiments, a bifunctional protein construct comprising: a first Fab(Fab1) that specifically binds to a first muscle-specific molecule comprising VH(VH1), CH1(H1-CH1), VL(VL1), and CL(L1-CL); a second Fab(Fab2) that specifically binds to a second muscle-specific molecule comprising VH(VH2), CH1(H2-CH1), VL(VL2), and CL(L2-CL); a first unit of a second binding site that specifically binds to and activates a first Notch receptor; a second unit of a second binding site that specifically binds to and activates a second Notch receptor; and an Fc domain comprising the first and second subunits, wherein the bifunctional protein construct is A bifunctional protein construct is provided comprising: i) a first polypeptide comprising N' to C': a first unit of a second binding site - an arbitrary linker - a first subunit of an Fc domain - an arbitrary linker - VL1-(L1-CL); ii) a second polypeptide comprising N' to C': a second unit of a second binding site - an arbitrary linker - a second subunit of an Fc domain - an arbitrary linker - VL2-(L2-CL); iii) a third polypeptide comprising N' to C': VH1-(H1-CH1); and iv) a fourth polypeptide comprising N' to C': VH2-(H2-CH1); where VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, any linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which may be the same or different within the bifunctional protein construct. In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. Fab1 and Fab2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, both Fab1 and Fab2 bind specifically to matriglycan Fab.In some embodiments, anti-matriglycan Fab1 and / or anti-matriglycan Fab2 comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 176 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 177. In some embodiments, both Fab1 and Fab2 specifically bind to LAMA2. In some embodiments, anti-LAMA2 Fab1 and / or anti-LAMA2 Fab2 comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 165 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 166. The first unit of the second binding site and the second unit of the second binding site may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding site (one or both units) comprises an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding site (one or both units) includes any of the amino acid sequences of SEQ ID NOs: 126-135 and 260-267.

[0283] In some embodiments, one or more units of the first binding site include (or are) scFv, and the scFv may be attached to the C-terminus of a subunit of the Fc domain via VH or VL, for example, via any linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). For example, in some embodiments, a first scFv (scFv1) specifically binds to a first muscle-specific molecule including VH(VH1) and VL(VL1); a second scFv (scFv2) specifically binds to a second muscle-specific molecule including VH(VH2) and VL(VL2); a first unit of the second binding site specifically binds to and activates a first Notch receptor; a second unit of the second binding site specifically binds to and activates a second Notch receptor; and a bifunctional protein construct comprising an Fc domain including the first and second subunits. A difunctional protein construct is provided, comprising: i) a first polypeptide comprising N' to C': a first unit of a second binding site - an arbitrary linker - a first subunit of an Fc domain - an arbitrary linker - scFv1 (VH1 - an arbitrary linker - VL1, or VL1 - an arbitrary linker - VH1); and ii) a second polypeptide comprising N' to C': a second unit of a second binding site - an arbitrary linker - a second subunit of an Fc domain - an arbitrary linker - scFv2 (VH2 - an arbitrary linker - VL2, or VL2 - an arbitrary linker - VH2). In some embodiments, the arbitrary linker is a peptide linker (e.g., any of SEQ ID NOs. 211, 212, 337, and 338), which may be the same or different within the difunctional protein construct. In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. scFv1 and scFv2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, both scFv1 and scFv2 bind specifically to matriglycans.In some embodiments, anti-matriglycan scFv1 and / or anti-matriglycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, both scFv1 and scFv2 specifically bind to LAMA2. In some embodiments, anti-LAMA2 scFv1 and / or anti-LAMA2 scFv2 comprises the amino acid sequence of SEQ ID NO: 145 or 268. The first unit of the second binding site and the second unit of the second binding site may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding site (one or both units) comprises an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding site (one or both units) includes any of the amino acid sequences of SEQ ID NOs: 126-135 and 260-267.

[0284] In some embodiments, one or more units of the first binding site include (or are) an sdAb, which may be linked to the C-terminus of a subunit of the Fc domain via, for example, any linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). For example, in some embodiments, a first sdAb (sdAb1, e.g., VHH1) that specifically binds to a first muscle-specific molecule; a second sdAb (sdAb2, e.g., VHH2) that specifically binds to a second muscle-specific molecule; a first unit of the second binding site that specifically binds to and activates a first Notch receptor; a second unit of the second binding site that specifically binds to and activates a second Notch receptor; and the first and second subunits A difunctional protein construct is provided comprising an Fc domain containing a linker, wherein the difunctional protein construct comprises: i) a first polypeptide comprising N' to C': a first unit of the second binding site - an arbitrary linker - a first subunit of the Fc domain - an arbitrary linker - sdAb1; and ii) a second polypeptide comprising N' to C': a second unit of the second binding site - an arbitrary linker - a second subunit of the Fc domain - an arbitrary linker - sdAb2. In some embodiments, the arbitrary linker is a peptide linker (e.g., any of SEQ ID NOs. 211, 212, 337, and 338), which may be the same or different within the difunctional protein construct. In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. sdAb1 and sdAb2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. The first unit of the second binding site and the second unit of the second binding site may be the same or different; and / or may bind to the same or different Notch receptors.In some embodiments, the second binding site (one or both units) includes an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs. 126-129 and 260-267). In some embodiments, the second binding site (one or both units) includes the amino acid sequence of any of SEQ ID NOs. 126-135 and 260-267.

[0285] In some embodiments, one or more units of the first binding site include (or are) a Fab, which may be connected to the N-terminus of a subunit of the Fc domain via CH1 or CL, for example, via any linker (e.g., any of sequence numbers 211, 212, 337, and 338). In some embodiments, a bifunctional protein construct comprising: a first Fab(Fab1) that specifically binds to a first muscle-specific molecule comprising VH(VH1), CH1(H1-CH1), VL(VL1), and CL(L1-CL); a second Fab(Fab2) that specifically binds to a second muscle-specific molecule comprising VH(VH2), CH1(H2-CH1), VL(VL2), and CL(L2-CL); a first unit of a second binding site that specifically binds to and activates a first Notch receptor; a second unit of a second binding site that specifically binds to and activates a second Notch receptor; and an Fc domain comprising the first and second subunits, wherein the bifunctional protein construct is A bifunctional protein construct is provided comprising: i) a first polypeptide comprising N' to C':VH1-(H1-CH1)-an arbitrary linker-first subunit of an Fc domain-an arbitrary linker-first unit of a second binding site; ii) a second polypeptide comprising N' to C':VH2-(H2-CH1)-an arbitrary linker-second subunit of an Fc domain-an arbitrary linker-second unit of a second binding site; iii) a third polypeptide comprising N' to C':VL1-(L1-CL); and iv) a fourth polypeptide comprising N' to C':VL2-(L2-CL); where VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.In some embodiments, a bifunctional protein construct comprising: a first Fab(Fab1) that specifically binds to a first muscle-specific molecule comprising VH(VH1), CH1(H1-CH1), VL(VL1), and CL(L1-CL); a second Fab(Fab2) that specifically binds to a second muscle-specific molecule comprising VH(VH2), CH1(H2-CH1), VL(VL2), and CL(L2-CL); a first unit of a second binding site that specifically binds to and activates a first Notch receptor; a second unit of a second binding site that specifically binds to and activates a second Notch receptor; and an Fc domain comprising the first and second subunits, wherein the bifunctional protein construct is configured such that i) from N' to C' A bifunctional protein construct is provided comprising: ii) a first polypeptide comprising: VL1-(L1-CL)-any linker-first subunit of an Fc domain-any linker-first unit of a second binding site; ii) a second polypeptide comprising: VL2-(L2-CL)-any linker-second subunit of an Fc domain-any linker-second unit of a second binding site that specifically binds to a second Notch receptor; iii) a third polypeptide comprising: VH1-(H1-CH1)-N'-C'; and iv) a fourth polypeptide comprising: VH2-(H2-CH1)-N'-C'; VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, any linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which may be the same or different within the bifunctional protein construct. In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. Fab1 and Fab2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, both Fab1 and Fab2 bind specifically to matriglycan Fab.In some embodiments, anti-matriglycan Fab1 and / or anti-matriglycan Fab2 comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 176 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 177. In some embodiments, both Fab1 and Fab2 specifically bind to LAMA2. In some embodiments, anti-LAMA2 Fab1 and / or anti-LAMA2 Fab2 comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 165 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 166. The first unit of the second binding site and the second unit of the second binding site may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding site (one or both units) comprises an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding site (one or both units) includes any of the amino acid sequences of SEQ ID NOs: 126-135 and 260-267.

[0286] In some embodiments, a bifunctional protein construct is provided comprising: i) a first binding site comprising a full-length antibody that specifically binds to a muscle-specific molecule, wherein the full-length antibody comprises an Fc domain comprising a first subunit and a second subunit; ii) a first unit of a second binding site fused (directly or via an optional linker) to the C-terminus of the first subunit of the Fc domain; and iii) a second unit of a second binding site fused (directly or via an optional linker) to the C-terminus of the second subunit of the Fc domain, wherein the first unit of the second binding site specifically binds to a first Notch receptor and activates the first Notch receptor, and the second unit of the second binding site specifically binds to a second Notch receptor and activates the second Notch receptor. In some embodiments, any linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which may be the same or different within a bifunctional protein construct. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. The first unit of the second binding site and the second unit of the second binding site may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding site (one or both units) comprises an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, e.g., any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding site (one or both units) includes any of the amino acid sequences of SEQ ID NOs: 126-135 and 260-267.

[0287] In some embodiments, one or more units of the first binding site include (or are) scFv, and the scFv may be attached to the N-terminus of a subunit of the Fc domain via VH or VL, for example, via any linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). In some embodiments, a first scFv (scFv1) specifically binds to a first muscle-specific molecule including VH (VH1) and VL (VL1); a second scFv (scFv2) specifically binds to a second muscle-specific molecule including VH (VH2) and VL (VL2); a first unit of the second binding site specifically binds to and activates a first Notch receptor; a second unit of the second binding site specifically binds to and activates a second Notch receptor; and a bifunctional protein structure including an Fc domain comprising the first and second subunits. A difunctional protein construct is provided, comprising: i) a first polypeptide comprising N' to C':scFv1(VH1-any linker-VL1 or VL1-any linker-VH1)-any linker-first subunit of an Fc domain-any linker-first unit of a second binding site; and ii) a second polypeptide comprising N' to C':scFv2(VH2-any linker-VL2 or VL2-any linker-VH2)-any linker-second subunit of an Fc domain-any linker-second unit of a second binding site. In some embodiments, the arbitrary linker is a peptide linker (e.g., any of SEQ ID NOs. 211, 212, 337, and 338), which may be the same or different within the difunctional protein construct. In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. scFv1 and scFv2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, both scFv1 and scFv2 bind specifically to matriglycans.In some embodiments, anti-matriglycan scFv1 and / or anti-matriglycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, both scFv1 and scFv2 specifically bind to LAMA2. In some embodiments, anti-LAMA2 scFv1 and / or anti-LAMA2 scFv2 comprises the amino acid sequence of SEQ ID NO: 145 or 268. The first unit of the second binding site and the second unit of the second binding site may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding site (one or both units) comprises an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding site (one or both units) includes any of the amino acid sequences of SEQ ID NOs: 126-135 and 260-267.

[0288] In some embodiments, one or more units of the first binding site include (or are) an sdAb, which may be linked to the N-terminus of a subunit of the Fc domain via, for example, any linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). In some embodiments, the first sdAb (sdAb1, e.g., VHH1) specifically binds to a first muscle-specific molecule; the second sdAb (sdAb2, e.g., VHH2) specifically binds to a second muscle-specific molecule; the first unit of the second binding site specifically binds to and activates a first Notch receptor; the second unit of the second binding site specifically binds to and activates a second Notch receptor; and the first and second subunits. A difunctional protein construct is provided comprising a Fc domain, the difunctional protein construct comprising: i) a first polypeptide comprising a first unit of a second binding site, i) N' to C':sdAb1 - any linker - a first subunit of the Fc domain - any linker - and ii) a second polypeptide comprising a second unit of a second binding site, i) N' to C':sdAb2 - any linker - a second subunit of the Fc domain - any linker - a second binding site. In some embodiments, the arbitrary linker is a peptide linker (e.g., any of SEQ ID NOs. 211, 212, 337, and 338), which may be the same or different within the difunctional protein construct. In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. sdAb1 and sdAb2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. The first unit of the second binding site and the second unit of the second binding site may be the same or different; and / or may bind to the same or different Notch receptors.In some embodiments, the second binding site (one or both units) includes an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs. 126-129 and 260-267). In some embodiments, the second binding site (one or both units) includes the amino acid sequence of any of SEQ ID NOs. 126-135 and 260-267.

[0289] In some embodiments, a bifunctional protein construct comprising: a first Fab(Fab1) that specifically binds to a first muscle-specific molecule comprising VH(VH1), CH1(H1-CH1), VL(VL1), and CL(L1-CL); a second Fab(Fab2) that specifically binds to a second muscle-specific molecule comprising VH(VH2), CH1(H2-CH1), VL(VL2), and CL(L2-CL); a first unit of a second binding site that specifically binds to and activates a first Notch receptor; a second unit of a second binding site that specifically binds to and activates a second Notch receptor; and an Fc domain comprising the first and second subunits, wherein the bifunctional protein construct is A bifunctional protein construct is provided comprising: i) a first polypeptide comprising a first subunit of an arbitrary linker-Fc domain from N' to C':VH1-(H1-CH1); ii) a second polypeptide comprising a second subunit of an arbitrary linker-Fc domain from N' to C':VH2-(H2-CH1); iii) a third polypeptide comprising a first unit of an arbitrary linker-second binding site from N' to C':VL1-(L1-CL); and iv) a fourth polypeptide comprising a second unit of an arbitrary linker-second binding site from N' to C':VL2-(L2-CL); where VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, any linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which may be the same or different within the bifunctional protein construct. In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. Fab1 and Fab2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, both Fab1 and Fab2 bind specifically to matriglycan Fab.In some embodiments, anti-matriglycan Fab1 and / or anti-matriglycan Fab2 comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 176 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 177. In some embodiments, both Fab1 and Fab2 specifically bind to LAMA2. In some embodiments, anti-LAMA2 Fab1 and / or anti-LAMA2 Fab2 comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 165 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 166. The first unit of the second binding site and the second unit of the second binding site may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding site (one or both units) comprises an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding site (one or both units) includes any of the amino acid sequences of SEQ ID NOs: 126-135 and 260-267.

[0290] In some embodiments, a bifunctional protein construct is provided comprising: i) a first binding site comprising a full-length antibody that specifically binds to a muscle-specific molecule; ii) a first unit of a second binding site fused (directly or via an optional linker) to the C-terminus of a first light chain of the full-length antibody; and iii) a second unit of a second binding site fused (directly or via an optional linker) to the C-terminus of a second light chain of the full-length antibody, wherein the first unit of the second binding site specifically binds to a first Notch receptor and activates the first Notch receptor, and the second unit of the second binding site specifically binds to a second Notch receptor and activates the second Notch receptor. In some embodiments, the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which may be the same or different within the bifunctional protein construct. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. The first unit of the second binding site and the second unit of the second binding site may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding site (one or both units) includes an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding site (one or both units) includes the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

[0291] In some embodiments, a bifunctional protein construct comprising: a first Fab(Fab1) that specifically binds to a first muscle-specific molecule comprising VH(VH1), CH1(H1-CH1), VL(VL1), and CL(L1-CL); a second Fab(Fab2) that specifically binds to a second muscle-specific molecule comprising VH(VH2), CH1(H2-CH1), VL(VL2), and CL(L2-CL); a first unit of a second binding site that specifically binds to and activates a first Notch receptor; a second unit of a second binding site that specifically binds to and activates a second Notch receptor; and an Fc domain comprising the first and second subunits, wherein the bifunctional protein construct is A bifunctional protein construct is provided comprising: i) a first polypeptide comprising a first subunit of the Fc domain from N' to C': VH1-(H1-CH1)-any linker; ii) a second polypeptide comprising a second subunit of the Fc domain from N' to C': VH2-(H2-CH1)-any linker; iii) a third polypeptide comprising a first unit of the second binding site-any linker-VL1-(L1-CL) from N' to C'; and iv) a fourth polypeptide comprising a second unit of the second binding site-any linker-VL2-(L2-CL) from N' to C'; VL1-(L1-CL) and VH1-(H1-CH1) forming Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) forming Fab2. In some embodiments, any linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which may be the same or different within the bifunctional protein construct. In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. Fab1 and Fab2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, both Fab1 and Fab2 bind specifically to matriglycan Fab.In some embodiments, anti-matriglycan Fab1 and / or anti-matriglycan Fab2 comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 176 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 177. In some embodiments, both Fab1 and Fab2 specifically bind to LAMA2. In some embodiments, anti-LAMA2 Fab1 and / or anti-LAMA2 Fab2 comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 165 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 166. The first unit of the second binding site and the second unit of the second binding site may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding site (one or both units) comprises an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding site (one or both units) includes any of the amino acid sequences of SEQ ID NOs: 126-135 and 260-267.

[0292] In some embodiments, a bifunctional protein construct is provided comprising a first binding site containing a full-length antibody that specifically binds to a muscle-specific molecule, a first unit of a second binding site fused (directly or via an optional linker) to the N-terminus of a first light chain of the full-length antibody, and a second unit of a second binding site fused (directly or via an optional linker) to the N-terminus of a second light chain of the full-length antibody, wherein the first unit of the second binding site specifically binds to a first Notch receptor and activates the first Notch receptor, and the second unit of the second binding site specifically binds to a second Notch receptor and activates the second Notch receptor. In some embodiments, the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which may be the same or different within the bifunctional protein construct. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. The first unit of the second binding site and the second unit of the second binding site may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding site (one or both units) includes an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding site (one or both units) includes the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

[0293] In some embodiments, a bifunctional protein construct comprising: a first Fab(Fab1) that specifically binds to a first muscle-specific molecule comprising VH(VH1), CH1(H1-CH1), VL(VL1), and CL(L1-CL); a second Fab(Fab2) that specifically binds to a second muscle-specific molecule comprising VH(VH2), CH1(H2-CH1), VL(VL2), and CL(L2-CL); a first unit of a second binding site that specifically binds to and activates a first Notch receptor; a second unit of a second binding site that specifically binds to and activates a second Notch receptor; and an Fc domain comprising the first and second subunits, wherein the bifunctional protein construct is A bifunctional protein construct is provided comprising: i) a first polypeptide comprising a first unit of a second binding site from N' to C' - an arbitrary linker - VH1-(H1-CH1) - an arbitrary linker - a first subunit of an Fc domain; ii) a second polypeptide comprising a second unit of a second binding site from N' to C' - an arbitrary linker - VH2-(H2-CH1) - an arbitrary linker - a second subunit of an Fc domain; iii) a third polypeptide comprising VL1-(L1-CL) from N' to C'; and iv) a fourth polypeptide comprising VL2-(L2-CL) from N' to C'; where VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, any linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which may be the same or different within the bifunctional protein construct. In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. Fab1 and Fab2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, both Fab1 and Fab2 bind specifically to matriglycan Fab.In some embodiments, anti-matriglycan Fab1 and / or anti-matriglycan Fab2 comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 176 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 177. In some embodiments, both Fab1 and Fab2 specifically bind to LAMA2. In some embodiments, anti-LAMA2 Fab1 and / or anti-LAMA2 Fab2 comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 165 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 166. The first unit of the second binding site and the second unit of the second binding site may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding site (one or both units) comprises an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding site (one or both units) includes any of the amino acid sequences of SEQ ID NOs: 126-135 and 260-267.

[0294] In some embodiments, a bifunctional protein construct is provided comprising a first binding site containing a full-length antibody that specifically binds to a muscle-specific molecule, a first unit of a second binding site fused (directly or via an optional linker) to the N-terminus of a first heavy chain of the full-length antibody, and a second unit of a second binding site fused (directly or via an optional linker) to the N-terminus of a second heavy chain of the full-length antibody, wherein the first unit of the second binding site specifically binds to a first Notch receptor and activates the first Notch receptor, and the second unit of the second binding site specifically binds to a second Notch receptor and activates the second Notch receptor. In some embodiments, the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which may be the same or different within the bifunctional protein construct. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycans on α-DG. The first unit of the second binding site and the second unit of the second binding site may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding site (one or both units) includes an ECD or variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, for example, any of the manipulated DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding site (one or both units) includes the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

[0295] In some embodiments, a first Fab (Fab1) specifically binds to a first muscle-specific molecule comprising VH(VH1), CH1(H1-CH1), VL(VL1), and CL(L1-CL); a second Fab (Fab2) specifically binds to a second muscle-specific molecule comprising VH(VH2), CH1(H2-...

Claims

1. A bifunctional protein construct comprising a first binding site and a second binding site, wherein the first binding site specifically binds to a muscle-specific molecule, and the second binding site specifically binds to a Notch receptor and activates the Notch receptor.

2. The bifunctional protein construct according to claim 1, wherein the muscle-specific molecule is a target antigen on the sarcoplasmic sheath, between the sarcoplasmic sheath and the basement membrane, or in the basement membrane.

3. The bifunctional protein construct according to claim 2, wherein the target antigen is selected from the group consisting of laminin, agrin, nidogen, perlecan, and M-cadherin (CDH15).

4. The bifunctional protein construct according to claim 2 or 3, wherein the target antigen is a component of a dystrophin-related glycoprotein complex (DGC).

5. The bifunctional protein construct according to claim 4, wherein the target antigen is selected from the group consisting of α-dystroglycan (α-DG), β-DG, laminin-211, perlecan, collagen, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, ε-sarcoglycan, ζ-sarcoglycan, biglycan, sarcospan, and matriglycan on α-DG.

6. The bifunctional protein construct according to any one of claims 3 to 5, wherein the target antigen is the laminin subunit alpha-2 (LAMA2) of laminin-211, CDH15, α-DG, or a matrixglycan on α-DG.

7. The bifunctional protein construct according to any one of claims 1 to 6, wherein the first binding site includes an antibody site that specifically binds to the muscle-specific molecule.

8. The antibody sites are full-length antibody, Fab, Fab', and F(ab'). ２ A bifunctional protein construct according to claim 7, selected from the group consisting of scFv and sdAb.

9. The antibody site specifically binds to LAMA2 (anti-LAMA2 antibody site), the bifunctional protein construct according to claim 7 or 8.

10. The bifunctional protein construct according to claim 9, wherein the anti-LAMA2 antibody site comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO: 1, HC-CDR2 containing the amino acid sequence of SEQ ID NO: 2, HC-CDR3 containing the amino acid sequence of SEQ ID NO: 3, LC-CDR1 containing the amino acid sequence of SEQ ID NO: 4, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:

6.

11. The bifunctional protein construct according to claim 10, wherein the anti-LAMA2 antibody site comprises VH containing the amino acid sequence of SEQ ID NO: 49 and VL containing the amino acid sequence of SEQ ID NO:

50.

12. The bifunctional protein construct according to claim 9, wherein the anti-LAMA2 antibody site comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO: 7, HC-CDR2 containing the amino acid sequence of SEQ ID NO: 8, HC-CDR3 containing the amino acid sequence of SEQ ID NO: 9, LC-CDR1 containing the amino acid sequence of SEQ ID NO: 10, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 11, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:

12.

13. The bifunctional protein construct according to claim 12, wherein the anti-LAMA2 antibody site comprises VH containing the amino acid sequence of SEQ ID NO: 54 and VL containing the amino acid sequence of SEQ ID NO:

65.

14. The antibody site specifically binds to the matrix on α-DG (anti-matriglycan antibody site), the bifunctional protein construct according to claim 7 or 8.

15. The bifunctional protein construct according to claim 14, wherein the anti-matriglycan antibody site comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO: 74, HC-CDR2 containing the amino acid sequence of SEQ ID NO: 75, HC-CDR3 containing the amino acid sequence of SEQ ID NO: 76, LC-CDR1 containing the amino acid sequence of SEQ ID NO: 83, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 84, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:

85.

16. The bifunctional protein construct according to claim 15, wherein the anti-matriglycan antibody site comprises VH containing the amino acid sequence of SEQ ID NO: 90 and VL containing the amino acid sequence of SEQ ID NO:

95.

17. The antibody site specifically binds to CDH15 (anti-CDH15 antibody site), the bifunctional protein construct according to claim 7 or 8.

18. The bifunctional protein construct according to claim 17, wherein the anti-CDH15 antibody site comprises HC-CDR1 containing the amino acid sequence of SEQ ID NO: 102, HC-CDR2 containing the amino acid sequence of SEQ ID NO: 103, HC-CDR3 containing the amino acid sequence of SEQ ID NO: 104, LC-CDR1 containing the amino acid sequence of SEQ ID NO: 105, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 106, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:

107.

19. The bifunctional protein construct according to claim 18, wherein the anti-CDH15 antibody site comprises VH containing the amino acid sequence of SEQ ID NO: 108 and VL containing the amino acid sequence of SEQ ID NO:

109.

20. The bifunctional protein construct according to any one of claims 1 to 6, wherein the first binding site includes a non-antibody site that specifically binds to the muscle-specific molecule.

21. The bifunctional protein construct according to claim 20, wherein the non-antibody site comprises a protein domain selected from the group consisting of the laminin G-like domain (LG domain) of laminin, the LG domain of agryn, the LG domain of nidogen, the LG domain of perlecan, the laminin coiled-coil binding domain of agryn, and the laminin γ-binding domain of nidogen.

22. The non-antibody site comprises the LG domain of laminin, as described in claim 21, for the bifunctional protein construct.

23. The bifunctional protein construct according to claim 22, wherein the non-antibody site comprises the LG4-5 domain of LAMA2 (LAMA2 LG4-5).

24. The LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 118 or 119, as described in claim 23.

25. The bifunctional protein construct according to any one of claims 1 to 24, wherein the second binding site comprises the extracellular domain (ECD) or a variant of a Notch ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jagged 1 (Jag1), and Jag2.

26. The second bonding site is, a) DLL4 ECD or a variant thereof, wherein the DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 125, and the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267; b) DLL1 ECD or a variant thereof, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; c) DLL3 ECD or a variant thereof, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; d) Jag1 ECD or a variant thereof, wherein the Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 132, and the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134; or e) Jag2 ECD or a variant thereof, wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135 A bifunctional protein construct according to claim 25, comprising:

27. The bifunctional protein construct according to any one of claims 1 to 26, wherein the first binding site is fused to the second binding site via an arbitrary linker.

28. a) The second binding site comprises a variant DLL4 ECD containing any of the amino acid sequences of SEQ ID NOs: 126-129 and 260-267; b) The second binding site comprises a DLL1 ECD containing the amino acid sequence of SEQ ID NO: 130; c) The second binding site comprises a DLL3 containing the amino acid sequence of SEQ ID NO: 131; d) The second binding site comprises a variant Jag1 ECD containing the amino acid sequence of SEQ ID NO: 133 or 134; or e) The second binding site includes Jag2 ECD containing the amino acid sequence of SEQ ID NO: 135, The bifunctional protein construct according to claim 27.

29. The difunctional protein construct according to claim 28, wherein the difunctional protein construct comprises any of the amino acid sequences of SEQ ID NOs: 136 to 144.

30. The bifunctional protein construct according to claim 28, wherein the first binding site comprises Fab, which specifically binds to muscle-specific molecules.

31. The bifunctional protein construct according to claim 30, wherein the second binding site is fused to the N-terminus of the VL of Fab via an arbitrary linker.

32. The difunctional protein construct according to any one of claims 1 to 26, further comprising an Fc domain containing a first subunit and a second subunit.

33. a) The Fc domain is derived from human IgG1 containing the amino acid sequence of SEQ ID NO: 187; b) Each subunit of the Fc domain contains the L234A / L235A mutation (EU numbering); c) Each subunit of the Fc domain contains the H435A mutation (EU numbering); d) Each subunit of the Fc domain contains the P329G mutation (EU numbering); e) Each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188 or 277; f) Either the first subunit of the Fc domain or the second subunit of the Fc domain contains the H435R / Y436F mutation (EU numbering); and / or g) The Fc domain contains a knob-into-hole mutation, i) The first subunit of the Fc domain comprises a knob mutation, and the second subunit of the Fc domain comprises a hole mutation; or ii) The bifunctional protein construct according to claim 32, wherein the second subunit of the Fc domain comprises a knob mutation, and the first subunit of the Fc domain comprises a hole mutation.

34. The bifunctional protein construct according to claim 33, wherein the knob mutation comprises T366W (EU numbering) and the hole mutation comprises T366S / L368A / Y407V (EU numbering).

35. a) The bifunctional protein construct comprises i) a first unit of a second binding site that specifically binds to a first Notch receptor, and ii) a second unit of a second binding site that specifically binds to a second Notch receptor, wherein the first unit of the second binding site and the second unit of the second binding site each independently comprise an ECD or variant of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2; and / or b) The bifunctional protein construct includes i) a first unit of a first binding site that specifically binds to a first muscle-specific molecule, and ii) a second unit of a first binding site that specifically binds to a second muscle-specific molecule. A bifunctional protein construct according to any one of claims 32 to 34.

36. The bifunctional protein construct according to any one of claims 32 to 35, wherein the first unit of the first binding site includes a first antibody site that specifically binds to the first muscle-specific molecule, the second unit of the first binding site includes a first antibody site that specifically binds to the second muscle-specific molecule, and the first antibody site and the second antibody site are independently selected from the group consisting of Fab, scFv, and sdAb.

37. The first antibody site is the first Fab (Fab1), and the second antibody site is the second Fab (Fab2); The aforementioned bifunctional protein construct is i) N' to C': A first polypeptide comprising a first unit of a second binding site that specifically binds to a first Notch receptor—an optional linker—the first subunit of the Fc domain—an optional linker—VH1-(H1-CH1); ii) N' to C': A second polypeptide comprising a second binding site that specifically binds to a second Notch receptor—an optional linker—the second subunit of the Fc domain—an optional linker—VH2-(H2-CH1); iii) From N' to C': A third polypeptide containing VL1-(L1-CL); and iv) From N' to C': A fourth polypeptide containing VL2-(L2-CL) The material includes VL1-(L1-CL) and VH1-(H1-CH1) which form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) which form Fab2. The bifunctional protein construct according to claim 36.

38. The first antibody site is the first Fab (Fab1), and the second antibody site is the second Fab (Fab2); The aforementioned bifunctional protein construct is i) N' to C': A first polypeptide comprising a first unit of a second binding site that specifically binds to a first Notch receptor—an arbitrary linker—the first subunit of the Fc domain—an arbitrary linker—VL1—(L1-CL); ii) N' to C': A second polypeptide comprising a second unit of a second binding site that specifically binds to a second Notch receptor—an optional linker—the second subunit of the Fc domain—an optional linker—VL2-(L2-CL); iii) From N' to C': A third polypeptide containing VH1-(H1-CH1); and iv) From N' to C': A fourth polypeptide containing VH2-(H2-CH1) The material includes VL1-(L1-CL) and VH1-(H1-CH1) which form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) which form Fab2. The bifunctional protein construct according to claim 36.

39. The bifunctional protein construct according to claim 37 or 38, wherein both Fab1 and Fab2 specifically bind to LAMA2, and anti-LAMA2 Fab1 and / or anti-LAMA2 Fab2 comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 165 and a second polypeptide having the amino acid sequence of SEQ ID NO:

166.

40. a) The first unit of the second binding site and / or the second unit of the second binding site comprises variant DLL4 ECD, wherein variant DLL4 ECD comprises any of the amino acid sequences of SEQ ID NOs: 126-129 and 260-267; b) The first unit of the second binding site and / or the second unit of the second binding site comprises a DLL1 ECD, the DLL1 ECD comprising the amino acid sequence of SEQ ID NO: 130; c) The first unit of the second binding site and / or the second unit of the second binding site comprises a DLL3 ECD, the DLL3 ECD comprising the amino acid sequence of SEQ ID NO: 131; d) The first unit of the second binding site and / or the second unit of the second binding site comprises the variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134; or e) The first unit of the second binding site and / or the second unit of the second binding site comprises Jag2 ECD, wherein Jag2 ECD comprises the amino acid sequence of SEQ ID NO:

135. A bifunctional protein construct according to any one of claims 35 to 39.

41. The first antibody site is the first sdAb (sdAb1), and the second antibody site is the second sdAb (sdAb2); The aforementioned bifunctional protein construct is i) N' to C': a first polypeptide comprising a first unit of a second binding site that specifically binds to a first Notch receptor—an arbitrary linker—the first subunit of the Fc domain—an arbitrary linker—sdAb1; and ii) N' to C': A second unit of a second binding site that specifically binds to a second Notch receptor - an optional linker - the second subunit of the Fc domain - an optional linker - a second polypeptide comprising sdAb2 A bifunctional protein construct according to claim 36, comprising:

42. The first antibody site is the first scFv (scFv1), and the second antibody site is the second scFv (scFv2); The aforementioned bifunctional protein construct is i) N' to C': a first polypeptide comprising a first unit of a second binding site that specifically binds to a first Notch receptor—an arbitrary linker—the first subunit of the Fc domain—an arbitrary linker—scFv1; and ii) N' to C': A second polypeptide comprising a second binding site that specifically binds to a second Notch receptor, a second linker, the second subunit of the Fc domain, a second linker, and scFv2. A bifunctional protein construct according to claim 36, comprising:

43. The bifunctional protein construct according to claim 42, wherein both scFv1 and scFv2 specifically bind to LAMA2, and anti-LAMA2 scFv1 and / or anti-LAMA2 scFv2 comprises the amino acid sequence of SEQ ID NO: 145 or 268.

44. a) The first unit of the second binding site and / or the second unit of the second binding site comprises a variant DLL4 ECD containing any of the amino acid sequences of SEQ ID NOs: 126-129 and 260-267; b) The first unit of the second binding site and / or the second unit of the second binding site comprises a DLL1 ECD containing the amino acid sequence of SEQ ID NO: 130; c) The first unit of the second binding site and / or the second unit of the second binding site comprises a DLL3 ECD containing the amino acid sequence of SEQ ID NO: 131; d) The first unit of the second binding site and / or the second unit of the second binding site comprises the variant Jag1 ECD having the amino acid sequence of SEQ ID NO: 133 or 134; or e) The first unit of the second binding site and / or the second unit of the second binding site comprises Jag2 ECD containing the amino acid sequence of SEQ ID NO:

135. A bifunctional protein construct according to any one of claims 41 to 43.

45. The difunctional protein construct according to claim 44, wherein the difunctional protein construct comprises a first polypeptide and a second polypeptide, each having one of the amino acid sequences of SEQ ID NOs. 223-231 and 269-276, respectively.

46. The first antibody site is the first Fab (Fab1), and the second antibody site is the second Fab (Fab2); The aforementioned bifunctional protein construct is i) N' to C': a first unit of a second binding site that specifically binds to a first Notch receptor – an arbitrary linker – VH1 – (H1 – CH1) – an arbitrary linker – a first polypeptide comprising the first subunit of the Fc domain; ii) N' to C': a second unit of a second binding site that specifically binds to a second Notch receptor - an arbitrary linker - VH2 - (H2-CH1) - an arbitrary linker - a second polypeptide comprising the second subunit of the Fc domain; iii) N' to C': A third polypeptide comprising a third unit of a second binding site that specifically binds to a third Notch receptor—an arbitrary linker—VL1-(L1-CL); and iv) N' to C': A fourth polypeptide containing a fourth unit of the second binding site that specifically binds to the fourth Notch receptor—an arbitrary linker—VL2-(L2-CL). The material includes VL1-(L1-CL) and VH1-(H1-CH1) which form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) which form Fab2. The bifunctional protein construct according to claim 35 or 36.

47. The bifunctional protein construct according to any one of claims 32 to 35, wherein the first unit of the first binding site includes a first non-antibody site that specifically binds to the first muscle-specific molecule, the second unit of the first binding site includes a second non-antibody site that specifically binds to the second muscle-specific molecule, and the first non-antibody site and the second non-antibody site are independently selected from the group consisting of the LG domain of laminin, the LG domain of agryn, the LG domain of nidogen, and the LG domain of perlecan.

48. The bifunctional protein construct according to claim 47, wherein the first non-antibody site and / or the second non-antibody site comprises LAMA2 LG4-5 having the amino acid sequence of SEQ ID NO: 118 or 119.

49. The aforementioned bifunctional protein construct is i) N' to C': a first polypeptide comprising the first unit of the first binding site—an arbitrary linker—the first subunit of the Fc domain—an arbitrary linker—a first unit of the second binding site that specifically binds to the first Notch receptor; and ii) N' to C': The second unit of the first binding site - an arbitrary linker - the second subunit of the Fc domain - an arbitrary linker - a second polypeptide comprising the second unit of the second binding site that specifically binds to the second Notch receptor. A bifunctional protein construct according to claim 47 or 48, comprising:

50. The aforementioned bifunctional protein construct is i) N' to C': a first polypeptide comprising a first unit of a second binding site that specifically binds to a first Notch receptor—an arbitrary linker—the first subunit of the Fc domain—an arbitrary linker—the first unit of the first binding site; and ii) N' to C': A second polypeptide comprising a second unit of a second binding site that specifically binds to a second Notch receptor - an optional linker - the second subunit of the Fc domain - an optional linker - the second unit of the first binding site. A bifunctional protein construct according to claim 47 or 48, comprising:

51. a) The first unit of the second binding site and / or the second unit of the second binding site comprises variant DLL4 ECD, wherein variant DLL4 ECD comprises any of the amino acid sequences of SEQ ID NOs: 126-129 and 260-267; b) The first unit of the second binding site and / or the second unit of the second binding site comprises a DLL1 ECD, the DLL1 ECD comprising the amino acid sequence of SEQ ID NO: 130; c) The first unit of the second binding site and / or the second unit of the second binding site comprises a DLL3 ECD, the DLL3 ECD comprising the amino acid sequence of SEQ ID NO: 131; d) The first unit of the second binding site and / or the second unit of the second binding site comprises the variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134; or e) The first unit of the second binding site and / or the second unit of the second binding site comprises Jag2 ECD, wherein Jag2 ECD comprises the amino acid sequence of SEQ ID NO:

135. A bifunctional protein construct according to any one of claims 47 to 50.

52. i) The first binding site is fused to the N-terminus of the first subunit of the Fc domain via an optional first linker, and the second binding site is fused to the N-terminus of the second subunit of the Fc domain via an optional second linker; or ii) The first binding site is fused to the N-terminus of the second subunit of the Fc domain via an arbitrary first linker, and the second binding site is fused to the N-terminus of the first subunit of the Fc domain via an arbitrary second linker. The bifunctional protein construct according to claim 32.

53. One or more isolated nucleic acids encoding a bifunctional protein construct according to any one of claims 1 to 52.

54. One or more vectors comprising one or more isolated nucleic acids as described in claim 53.

55. One or more vectors according to claim 54, which are viral vectors.

56. A host cell expressing a bifunctional protein construct according to any one of claims 1 to 52, wherein the host cell comprises one or more isolated nucleic acids according to claim 53, or one or more vectors according to claim 54 or 55.

57. i) a bifunctional protein construct according to any one of claims 1 to 52, one or more isolated nucleic acids according to claim 53, or one or more vectors according to claim 54 or 55; and ii) a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

58. A method for generating a bifunctional protein construct, i) culturing a host cell containing one or more isolated nucleic acids according to claim 53 or one or more vectors according to claim 54 or 55, or a host cell according to claim 56, under conditions suitable for the expression of the bifunctional protein construct; and ii) Recovering the expressed bifunctional protein construct from the cultured host cells. The method, including the method described above.

59. A method for treating a muscle-related disease in an individual, comprising administering to the individual an effective amount of a bifunctional protein construct according to any one of claims 1 to 52 or a pharmaceutical composition according to claim 57.

60. The method according to claim 59, wherein the muscle-related disease is selected from the group consisting of Pompe disease, central nucleus myopathy, fibrodysplasia ossificans progressive (FOP), Friedreich's ataxia (FRDA), familial hypertrophic cardiomyopathy, Laing type distal myopathy, myofibrilary myopathy, and muscular dystrophy.

61. The method according to claim 60, wherein the muscle disease is muscular dystrophy.

62. The method according to claim 61, wherein the muscular dystrophy includes one or more of the following: Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophy (CMD), facioscapulohumeral muscular dystrophy (FSHD), myotonic dystrophy (DM), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), congenital myopathy, Charcot-Marie-Tooth (CMT) disorder, and Emery-Dreyfuss muscular dystrophy (EDMD).

63. The method according to any one of claims 59 to 62, wherein the bifunctional protein construct or the pharmaceutical composition is administered intravenously, subcutaneously, or intramuscularly.

64. An engineered DLL4 extracellular domain (ECD), wherein the engineered DLL4 ECD contains a mutation selected from the group consisting of T52N and T135N, and the amino acid position is relative to a reference DLL4 ECD containing the amino acid sequence of SEQ ID NO:

126.

65. The manipulated DLL4 ECD according to claim 64, further comprising mutations at one or more amino acid positions selected from the group consisting of G2, E14, R66, P80, F81, H168, Q220, N231, and N260.

66. The manipulated DLL4 ECD according to claim 65, wherein the further mutation is selected from the group consisting of G2S, E14H, R66S, R66T, P80L, F81L, H168Y, Q220H, N231D, and N260D.

67. The aforementioned operated DLL4 ECD is (i) T52N and T135N; (ii) T52N, R66S, and T135N; (iii) E14H, T52N, R66T, P80L, T135N, and N231D; (iv) T52N, R66T, P80L, T135N, Q220H, and N260D; (v) G2S, T52N, F81L, T135N, and H168Y; (vi) G2S, T52N, F81L, R66S, T135N, and H168Y; (vii) G2S, E14H, T52N, F81L, R66T, P80L, T135N, H168Y, and N231D; and (viiii) G2S, T52N, R66T, P80L, F81L, T135N, H168Y, Q220H, and N260D An operated DLL4 ECD according to any one of claims 64 to 66, comprising a mutation selected from the group consisting of the following.

68. The manipulated DLL4 ECD according to claim 67, wherein the manipulated DLL4 ECD comprises an amino acid sequence selected from the group consisting of any one of sequence numbers 261 to 264.

69. A protein construct comprising the manipulated DLL4 ECD according to any one of claims 64 to 68.

70. The protein construct according to claim 69, further comprising a binding site that specifically binds to muscle-specific molecules.

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