Bifunctional proteins and uses thereof

Abstract

Provided herein are compositions and methods of treating muscular dystrophies by administering an effective amount of a composition comprising a bifunctional protein comprising a first binding moiety that binds to muscle-specific molecules and a second functional domain that activates Notch signaling, thereby spatiotemporally activating Notch signaling in muscle tissues to boost satellite cell renewal in situ, and thus enhancing muscle repair / regeneration to improve muscle function as a treatment of various muscular dystrophies and sarcopenia. Also provided are engineered DLL4 extracellular domains and protein constructs comprising thereof.

Description

BIFUNCTIONAL PROTEINS AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority benefits of U.S. Provisional Patent Application No. 63 / 639,356 filed on April 26, 2024, and U.S. Provisional Patent Application No. 63 / 537,382 filed on September 8, 2023, the contents of each of which are incorporated herein by reference in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (340742000140SEQLIST.xml; Size: 457,221 bytes; and Date of Creation: September 5, 2024) is herein incorporated by reference in its entirety. FIELD OF THE INVENTION

[0003] The present invention relates to bifunctional protein constructs comprising a first binding moiety that specifically binds to a muscle-specific molecule and a second binding moiety that specifically binds to a Notch receptor and activates the Notch receptor. The present invention also relates to methods for making such bifunctional protein constructs and the use of such bifunctional protein constructs for treating muscle-related diseases (e.g., muscular dystrophies and sarcopenia). Also provided are engineered DLL4 extracellular domains (ECDs) and protein constructs comprising thereof. BACKGROUND

[0004] Muscular dystrophy (MD) is a diverse group of muscle diseases of many subtypes that present with muscle weakness and degeneration at different levels of severity. An MD is commonly linked to a defect(s) in a gene encoding a protein that is important for muscle integrity or function. Degenerative MDs are typically caused by defects in structural proteins that are directly or indirectly associated with the dystrophin-associated glycoprotein complex (DGC), which is essential to maintaining muscle cell membrane integrity.

[0005] A direct link between MD and Notch signaling in human was established with limb-girdle muscular dystrophy (LGMD R21), which is caused by Protein O- glucosyltransferase 1 (POGLUT1) biallelic mutations, reduced Notch signaling, and satellite cell loss (Servián-Morilla et al., 2016, EMBO Mol Med. 8(11):1289-1309; Servián-Morilla et al., 2020, Acta Neuropathol. 139(3):565-582).

[0006] All references cited herein, including patent applications, patent publications, and Genbank Accession numbers are herein incorporated by reference, as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety. BRIEF SUMMARY OF THE INVENTION

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

[0008] In some embodiments according to the bifunctional protein construct described above, the muscle-specific molecule is a target antigen on the sarcolemma, between the sarcolemma and the basal lamina, or in the basal lamina. 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 a laminin subunit alpha-2 (LAMA2), CDH15, α-DG, or matriglycan on α-DG.

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

[0010] In some embodiments according to any of the bifunctional protein constructs described above, the antibody moiety specifically binds to LAMA2 (anti-LAMA2 antibody moiety). In some embodiments, the anti-LAMA2 antibody moiety comprises: (i) an HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC- CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9, an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 10, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 12; (iii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (iv) comprises an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (v) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (vi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (vii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (viii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (ix) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72; (x) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (xi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (xii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (xiii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (xiv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (xv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VLcomprising the amino acid sequence of SEQ ID NO: 69; (xvi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (xvii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (xviii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72; (xix) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (xx) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (xxi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (xxii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (xxiii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (xxiv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (xxv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (xxvi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (xxvii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72; (xxviii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (xxix) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acidsequence of SEQ ID NO: 57, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (xxx) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (xxxi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (xxxii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (xxxiii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (xxxiv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (xxxv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (xxxvi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72; (xxxvii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (xxxviii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (xxxix) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (xl) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (xli) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (xlii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ IDNO: 69; (xliii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (xliv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (xlv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72; (xlvi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (xlvii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (xlviii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (xlix) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (l) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (li) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (lii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (liii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (liv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72; (lv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (lvi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (lvii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (lviii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (lix) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (lx) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (lxi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (lxii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (lxiii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72; (lxiv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (lxv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (lxvi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (lxvii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (lxviii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (lxix) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (lxx) an HC-CDR1, HC-CDR2, andHC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (lxxi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (lxxii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72; (lxxiii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (lxxiv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (lxxv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (lxxvi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (lxxvii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (lxxviii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (lxxix) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (lxxx) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (lxxxi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72; (lxxxii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (lxxxiii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VLcomprising the amino acid sequence of SEQ ID NO: 65; (lxxxiv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (lxxxv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (lxxxvi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (lxxxvii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (lxxxviii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (lxxxix) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (xc) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72; (xci) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (xcii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (xciii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (xciv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (xcv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (xcvi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (xcvii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acidsequence of SEQ ID NO: 64, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (xcviii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (xcix) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72; or (c) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-LAMA2 antibody moiety comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-LAMA2 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 49, and a VL comprising the amino acid sequence of SEQ ID NO: 50.

[0011] 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: 51. In some embodiments, the anti-LAMA2 antibody moiety is an anti-LAMA2 Fab. In some embodiments, the 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 according to any of the bifunctional protein constructs described above, the anti-LAMA2 antibody moiety comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9, an LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 10, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 12.

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

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

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

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

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

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

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

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

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

[0024] In some embodiments according to any of the bifunctional protein constructs described above, the first binding moiety comprises a non-antibody moiety specifically binding to the muscle-specific molecule. In some embodiments, the non-antibody moiety comprises a protein domain selected from the group consisting of a laminin G-like domain (LG domain) of laminin, an LG domain of agrin, an LG domain of nidogen, an LG domain of perlecan, the laminin coiled-coil binding domain of agrin, and the laminin γ binding domain of nidogen. In some embodiments, wherein the non-antibody moiety comprises an LGdomain of laminin. In some embodiments, the non-antibody moiety comprises an 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 moiety comprises the amino acid sequence of any of SEQ ID NOs: 113-117.

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

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

[0027] In some embodiments according to any of the bifunctional protein constructs described above, the non-antibody moiety comprises an LG domain of agrin. In some embodiments, the non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 121.

[0028] In some embodiments according to any of the bifunctional protein constructs described above, the non-antibody moiety comprises a laminin coiled-coil binding domain of agrin. In some embodiments, the laminin coiled-coil binding domain of agrin comprises the amino acid sequence of SEQ ID NO: 122.

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

[0030] In some embodiments according to any of the bifunctional protein constructs described above, the second binding moiety comprises an extracellular domain (ECD) or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jagged1 (Jag1), and Jag2.

[0031] In some embodiments according to any of the bifunctional protein constructs described above, the second binding moiety comprises a DLL4 ECD or a variant thereof. In some embodiments, the second binding moiety comprises a DLL4 ECD, and wherein the DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 125. In some embodiments, wherein the second binding moiety comprises a variant DLL4 ECD, and wherein the variantDLL4 ECD comprises MNNL domain, DSL domain, and EGF1-5 domains of DLL4, or a sequence having at least about 90% sequence identity to the sequence of MNNL domain, DSL domain, and EGF1-5 domains of DLL4. In some embodiments, the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267.

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

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

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

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

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

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

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

[0039] In some embodiments according to any of the bifunctional protein constructs described above, the first binding moiety comprises an anti-LAMA2 scFv. In some embodiments, 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 any of SEQ ID NOs: 146-154.

[0040] In some embodiments according to any of the bifunctional protein constructs described above, the first binding moiety comprises an anti-matriglycan scFv. In some embodiments, 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 any of SEQ ID NOs: 156-164.

[0041] In some embodiments according to any of the bifunctional protein constructs described above, the first binding moiety comprises a Fab specifically binding to a muscle- specific molecule. In some embodiments, the second binding moiety is fused to the N- terminus of the VL of the Fab via an optional linker.

[0042] In some embodiments according to any of the bifunctional protein constructs described above, the first binding moiety 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 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 moiety and the second polypeptide of the anti- LAMA2 Fab, and wherein the fusion polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 167-175.

[0043] In some embodiments according to any of the bifunctional protein constructs described above, the first binding moiety 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 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 moiety and the second polypeptide of the anti-matriglycan Fab, and wherein the fusion polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 178-186.

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

[0045] In some embodiments according to any of the bifunctional protein constructs described above, the bifunctional protein construct further comprises an Fc domain comprising 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 comprising the amino acid sequence of SEQ ID NO: 187. In some embodiments, each subunit of the Fc domain comprises a mutation that reduces or abolishes the effector function. In some embodiments, each subunit of the Fc domain comprises an L234A / L235A mutation (EU numbering).

[0046] In some embodiments according to any of the bifunctional protein constructs described above, either the first subunit of the Fc domain or the second subunit of the Fc domain comprises an H435R / Y436F mutation (EU numbering). In some embodiments, the Fc domain comprises knob-into-hole mutations, and wherein: 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 second subunit of the Fc domain comprises a knob mutation, and the first subunit of the Fc domain comprises a 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 charged-pair mutations, and wherein: 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 comprises i) an amino acid residue at D399 (EU numbering) inthe first subunit of the Fc domain is replaced with a positively charged residue, and an amino acid residue at K409 (EU numbering) in the second subunit of the Fc domain is replaced with a negatively charged residue; or ii) an amino acid residue at K409 (EU numbering) in the first subunit of the Fc domain is replaced with a negatively charged residue, and an 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 according to any of the bifunctional protein constructs described above, each subunit of the Fc domain comprises a mutation that reduces half-life. In some embodiments, each subunit of the Fc domain comprises an H435A mutation (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188.

[0048] In some embodiments according to any of the bifunctional protein constructs described above, the bifunctional protein construct comprises: i) a first unit of a second binding moiety specifically binding to a first Notch receptor, and ii) a second unit of a second binding moiety specifically binding to a second Notch receptor, and wherein the first unit of the second binding moiety and the second unit of the second binding moiety each independently comprises an ECD or a variant thereof 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 moiety and / or the second unit of the second binding moiety comprises a DLL4 ECD, and wherein the DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NO: 125-129. In some embodiments, the first unit of the second binding moiety and the second unit of the second binding moiety are the same. In some embodiments, the first unit of the second binding moiety and the second unit of the second binding moiety are different. In some embodiments, the bifunctional protein construct comprises: i) a first unit of a first binding moiety specifically binding to a first muscle-specific molecule, and ii) a second unit of a first binding moiety specifically binding to a second muscle-specific molecule. In some embodiments, the first unit of the first binding moiety and the second unit of the first binding moiety are the same. In some embodiments, the first unit of the first binding moiety and the second unit of the first binding moiety are different. In some embodiments, the first unit of the first binding moiety comprises a first antibody moiety specifically binding to the first muscle-specific molecule, the second unit of the first binding moiety comprises a second antibody moiety specifically binding to the second muscle- specific molecule, and wherein the first antibody moiety and the second antibody moiety are each independently selected from the group consisting of a Fab, an scFv, and an sdAb.

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

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

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

[0052] In some embodiments according to any of the bifunctional protein constructs described above, the first antibody moiety is a first sdAb (sdAb1), and the second antibody moiety is a second sdAb (sdAb2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – the first subunit of the Fc domain – optional linker – sdAb1; and ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – the second subunit of the Fc domain – optional linker – sdAb2. In some embodiments, the first antibody moiety is a first scFv (scFv1), and the second antibody moiety is a second scFv (scFv2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – the first subunit of the Fc domain – optional linker – scFv1; and ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – the second subunit of the Fc domain – optional linker – scFv2.

[0053] In some embodiments according to any of the bifunctional protein constructs described above, the bifunctional protein construct comprises scFv1 and scFv2 both 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. In some embodiments, the linker comprises an 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 comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a variant DLL4 ECD, and wherein the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide each comprising the amino acid sequence of any of SEQ ID NOs: 223-226. In some embodiments, the first unit of the second binding moiety and / or the second unit of the second bindingmoiety comprises a DLL1 ECD, and wherein the DLL1 ECD comprises 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 moiety and / or the second unit of the second binding moiety comprises a DLL3 ECD, and wherein the DLL3 ECD comprises 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 moiety and / or the second unit of the second binding moiety comprises a variant Jag1 ECD, and wherein the variant Jag1 ECD comprises 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 comprising the amino acid sequence of SEQ ID NO: 229 or 230. In some embodiments, the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a Jag2 ECD, and wherein the Jag2 ECD comprises 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 according to any of the bifunctional protein constructs described above, the bifunctional protein construct comprises scFv1 and scFv2 both specifically bind to matriglycan. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide each comprising: (i) the amino acid sequence of any one of SEQ ID NOs: 232-235; (ii) the amino acid sequence of SEQ ID NO: 236; (iii) the amino acid sequence of SEQ ID NO: 237; (iv) the amino acid sequence of SEQ ID NO: 238 or 239; or (v) the amino acid sequence of SEQ ID NO: 240.

[0055] In some embodiments according to any of the bifunctional protein constructs described above, the first antibody moiety is a first Fab (Fab1), and the second antibody moiety is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH1-(H1-CH1) – optional linker – the first subunit of the Fc domain – optional linker – a first unit of a second binding moiety specifically binding to a first Notch receptor; ii) a second polypeptide comprising from N’ to C’: VH2-(H2-CH1) – optional linker – the second subunit of the Fc domain – optional linker – a second unit of a second binding moiety specifically binding to a second Notch receptor; iii) a third polypeptide comprising from N’ to C’: VL1-(L1-CL); and iv) a fourth polypeptidecomprising from N’ to C’: VL2-(L2-CL); and wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.

[0056] In some embodiments according to any of the bifunctional protein constructs described above, the first antibody moiety is a first sdAb (sdAb1), and the second antibody moiety is a second sdAb (sdAb2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: sdAb1 – optional linker – the first subunit of the Fc domain – optional linker – a first unit of a second binding moiety specifically binding to a first Notch receptor; and ii) a second polypeptide comprising from N’ to C’: sdAb2– optional linker – the second subunit of the Fc domain – optional linker – a second unit of a second binding moiety specifically binding to a second Notch receptor.

[0057] In some embodiments according to any of the bifunctional protein constructs described above, the first antibody moiety is a first scFv (scFv1), and the second antibody moiety is a second scFv (scFv2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: scFv1– optional linker – the first subunit of the Fc domain – optional linker – a first unit of a second binding moiety specifically binding to a first Notch receptor; and ii) a second polypeptide comprising from N’ to C’: scFv2– optional linker – the second subunit of the Fc domain – optional linker – a second unit of a second binding moiety specifically binding to a second Notch receptor.

[0058] In some embodiments according to any of the bifunctional protein constructs described above, the first antibody moiety is a first Fab (Fab1), and the second antibody moiety is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH1-(H1-CH1) – optional linker – the first subunit of the Fc domain; ii) a second polypeptide comprising from N’ to C’: VH2-(H2-CH1) – optional linker – the second subunit of the Fc domain; iii) a third polypeptide comprising from N’ to C’: VL1-(L1-CL) – optional linker – a first unit of a second binding moiety specifically binding to a first Notch receptor; and iv) a fourth polypeptide comprising from N’ to C’: VL2-(L2-CL) – optional linker – a second unit of a second binding moiety specifically binding to a second Notch receptor; and wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.

[0059] In some embodiments according to any of the bifunctional protein constructs described above, the first antibody moiety is a first Fab (Fab1), and the second antibody moiety is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH1-(H1-CH1) – optional linker – the first subunit of the Fc domain; ii) a second polypeptide comprising from N’ to C’: VH2-(H2-CH1) – optionallinker – the second subunit of the Fc domain; iii) a third polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – VL1-(L1-CL); and iv) a fourth polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – VL2-(L2-CL); and wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.

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

[0061] In some embodiments according to any of the bifunctional protein constructs described above, the bifunctional protein construct further comprises a third unit of a second binding moiety specifically binding to a third Notch receptor, and a fourth unit of a second binding moiety specifically binding to a fourth Notch receptor, and wherein the third unit of the second binding moiety and the fourth unit of the second binding moiety each independently comprises an ECD or a variant thereof 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 moiety and the fourth unit of the second binding moiety each independently comprises an ECD or a variant thereof of DLL4. In some embodiments, the third unit of the second binding moiety and / or the fourth unit of the second binding moiety comprises a DLL4 ECD, and wherein the DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 125. In some embodiments, the third unit of the second binding moiety and / or the fourth unit of the second binding moiety comprises a variant DLL4 ECD, and wherein the variant DLL4 ECD comprises MNNL domain, DSL domain, and EGF1-5 domains of DLL4. In some embodiments, the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NO: 126-129 and 260-267. In some embodiments, all four units of the secondbinding moiety are the same. In some embodiments, at least one of the four units of the second binding moiety is different from the others.

[0062] In some embodiments according to any of the bifunctional protein constructs described above, the first antibody moiety is a first Fab (Fab1), and the second antibody moiety is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – VH1-(H1-CH1) – optional linker – the first subunit of the Fc domain; ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – VH2-(H2-CH1) – optional linker – the second subunit of the Fc domain; iii) a third polypeptide comprising from N’ to C’: a third unit of a second binding moiety specifically binding to a third Notch receptor – optional linker – VL1-(L1-CL); and iv) a fourth polypeptide comprising from N’ to C’: a fourth unit of a second binding moiety specifically binding to a fourth Notch receptor – optional linker – VL2-(L2-CL); and wherein VL1-(L1- CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.

[0063] In some embodiments according to any of the bifunctional protein constructs described above, the first unit of the first binding moiety comprises a first non-antibody moiety specifically binding to the first muscle-specific molecule, the second unit of the first binding moiety comprises a second non-antibody moiety specifically binding to the second muscle-specific molecule, and wherein the first non-antibody moiety and the second non- antibody moiety are each independently selected from the group consisting of an LG domain of laminin, an LG domain of agrin, an LG domain of nidogen, and an LG domain of perlecan. In some embodiments, the first unit of the first binding moiety comprises two or more first non-antibody moieties connected in tandem, and the second unit of the first binding moiety comprises two or more second non-antibody moieties connected in tandem. In some embodiments, the first non-antibody moiety and / or the second non-antibody moiety comprises LAMA2 LG4-5 comprising the amino acid sequence of SEQ ID NO: 118 or 119.

[0064] In some embodiments according to any of the bifunctional protein constructs described above, the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the first binding moiety – optional linker – the first subunit of the Fc domain – optional linker – a first unit of a second binding moiety specifically binding to a first Notch receptor; and ii) a second polypeptide comprising from N’ to C’: the second unit of the first binding moiety – optional linker – the second subunit ofthe Fc domain – optional linker – a second unit of a second binding moiety specifically binding to a second Notch receptor.

[0065] In some embodiments according to any of the bifunctional protein constructs described above, the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – the first subunit of the Fc domain – optional linker – the first unit of the first binding moiety; and ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – the second subunit of the Fc domain – optional linker – the second unit of the first binding moiety.

[0066] In some embodiments according to any of the bifunctional protein constructs described above, the first unit of the first binding moiety comprises two LAMA2 LG4-5 connected in tandem, and the second unit of the first binding moiety comprises two LAMA2 LG4-5 connected in tandem. In some embodiments, the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a variant DLL4 ECD, and wherein the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide each comprising the amino acid sequence of any one of SEQ ID NOs: 241-244. In some embodiments, the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a DLL1 ECD, and wherein the DLL1 ECD comprises 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: 245. In some embodiments, the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a DLL3 ECD, and wherein the DLL3 ECD comprises 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: 246. In some embodiments, the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a variant Jag1 ECD, and wherein the variant Jag1 ECD comprises 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 comprising the amino acid sequence of SEQ ID NO: 247 or 248. In some embodiments, the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a Jag2 ECD, and wherein the Jag2 ECD comprisesthe 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 according to any of the bifunctional protein constructs described above, i) the first binding moiety is fused to the N-terminus of the first subunit of the Fc domain via an optional first linker, and the second binding moiety is fused to the N- terminus of the second subunit of the Fc domain via an optional second linker; or ii) the first binding moiety is fused to the N-terminus of the second subunit of the Fc domain via an optional first linker, and the second binding moiety is fused to the N-terminus of the first subunit of the Fc domain via an optional second linker. In some embodiments, the first binding moiety is a Fab specifically binding to the muscle-specific molecule, and wherein the C-terminus of the CH1 of the Fab is fused to the N-terminus of the first subunit of the Fc domain or the second subunit of the Fc domain via the optional first linker. In some embodiments, the second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2.

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

[0069] In some embodiments according to any of the bifunctional protein constructs described above, the second binding moiety is fused to the N-terminus of the first subunit of the Fc domain via an optional linker; wherein the bifunctional protein construct comprises one or more units of the first binding moiety specifically binding to one or more muscle-specific molecules; and wherein each of the one or more units of the first binding moiety 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 according to any of the bifunctional protein constructs described above, the second binding moiety is fused to the N-terminus of the second subunit of the Fc domain via an optional linker; wherein the bifunctional protein construct comprises one or more units of the first binding moiety specifically binding to one or more muscle- specific molecules; and wherein each of the one or more units of the first binding moiety 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 C- terminus of the second subunit of the Fc domain.

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

[0072] In some embodiments according to any of the bifunctional protein constructs described above, the second binding moiety is fused to the C-terminus of the first subunit of the Fc domain via an optional linker; wherein the bifunctional protein construct comprises one or more units of the first binding moiety specifically binding to one or more muscle- specific molecules; and wherein each of the one or more units of the first binding moiety is independently fused via an optional linker to one of: i) the N-terminus of the first subunit ofthe 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 according to any of the bifunctional protein constructs described above, the second binding moiety is fused to the C-terminus of the second subunit of the Fc domain via an optional linker; wherein the bifunctional protein construct comprises one or more units of the first binding moiety specifically binding to one or more muscle- specific molecules; and wherein each of the one or more units of the first binding moiety 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 according to any of the bifunctional protein constructs described above, i) the one or more units of the second binding moiety each independently comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2; and ii) the one or more units of the first binding moiety each independently comprises an antibody moiety specifically binding to a muscle- specific molecule, and wherein the antibody moiety is independently selected from the group consisting of an scFv, a Fab, and an sdAb. In some embodiments, i) the two units of the second binding moiety are the same; and / or ii) the two or more units of the first binding moiety are the same. In some embodiments, i) the two units of the second binding moiety are different; and / or ii) at least one of the two or more units of the first binding moiety is different from the other(s). In some embodiments, the Fc domain comprises knob-into-hole mutations and / or charged-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 comprises the amino acid sequence of SEQ ID NO: 257, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 256; (v) the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 258, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 259; or (vi) the first subunit of the Fc domain comprises the amino acidsequence of SEQ ID NO: 259, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 258.

[0075] In another aspect, provided herein is one or more isolated nucleic acids encoding any of the bifunctional protein construct described above.

[0076] In another aspect, provided herein is one or more vectors comprising one or more isolated nucleic acids described above. In some embodiments, the one or more vectors is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector.

[0077] In another aspect, provided herein is a host cell expressing any of the bifunctional protein constructs described above, the one or more isolated nucleic acids described above, or the one or more vectors described above. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell or an HEK293 cell.

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

[0079] In another aspect, provided herein is a method of producing a bifunctional protein construct, comprising: i) culturing a host cell comprising the one or more isolated nucleic acids described above or the one or more vectors described above, or the host cell described above, under a condition suitable for the expression of the bifunctional protein construct; and ii) recovering the expressed bifunctional protein construct from the cultured host cell. In some embodiments, the method further comprises introducing the one or more isolated nucleic acids or the one or more vectors into the host cell.

[0080] In another aspect, provided herein is a method of treating a muscle-related disease in an individual, comprising administering to the individual an effective amount of any of the bifunctional protein constructs described above or the pharmaceutical composition described above. In some embodiments, the muscle-related disease is selected from the group consisting of Pompe disease, centronuclear myopathy, fibrodysplasia ossificans progressive (FOP), Friedreich’s ataxia (FRDA), familial hypertrophic cardiomyopathy, Laing distal myopathy, myofibrillar myopathy, and muscular dystrophy. In some embodiments, the muscle disease is muscular dystrophy. In some embodiments, the muscular dystrophy comprises one or more of: 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-Dreifuss muscular dystrophy (EDMD). In some embodiments, the bifunctional protein construct or the pharmaceutical composition is administered intravenously, subcutaneously, or intramuscularly. In some embodiments, the individual is a human.

[0081] In another aspect, the present inventions provides an antibody construct comprising an antibody moiety specifically recognizing matriglycan on α-DG (anti- matriglycan antibody moiety), wherein the anti-matriglycan antibody moiety comprises: (i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 75, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 79; (ii) an HC-CDR1, HC- CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95; (iii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96; (iv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97; (v) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98; (vi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95; (vii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96; (viii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97; (ix) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98; (x) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95; (xi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96; (xii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97; (xiii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98; (xiv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95; (xv) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96; (xvi) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and an LC- CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97; or (xvii) an HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and an LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98.

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

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

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

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

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

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

[0088] In another aspect, 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 wherein the amino acid position is in reference 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 comprises a mutation selected from the group consisting of: (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 (viii) G2S, T52N, R66T, P80L, F81L, T135N, H168Y, Q220H, and N260D. In some embodiments, the engineered DLL4 ECD comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 261-264.

[0089] In another aspect according to any of the engineered DLL4 ECD described above, the present invention provides a protein construct comprising the engineered DLL4 ECD. Insome embodiments, the protein construct further comprises a binding moiety that specifically binds to a muscle-specific molecule.

[0090] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] FIGs. 1A-1M depict different configurations of bifunctional proteins comprising an anti-muscle target protein and a Notch ligand or agonist. FIG. 1A depicts a monovalent Notch ligand with an anti-muscle protein antibody (Ab) in “Knob-into-Hole” format. FIGs. 1B and 1C depict additional formats and configurations of bifunctional proteins, of which R1, R2, R3, and R4 can be an anti-muscle target protein or a Notch ligand / agonist. FIG. 1D depicts a bivalent fusion protein with Notch ligands fused to the N-terminus of the Fc and an anti-muscle protein antibody Fab fused to the C-terminus of the Fc. FIG. 1E depicts a bivalent fusion protein with Notch ligands fused to the N-terminus of the Fc and an anti- muscle protein antibody scFv fused to the C-terminus of the Fc. FIG. 1F depicts a bivalent fusion protein Notch ligands fused to the N-terminus of the Fc and an anti-muscle target protein antibody single chain Ab (VHH or nanobody) fused to the C-terminus of the Fc. FIG. 1G depicts a bivalent fusion protein with Notch ligands fused to the C-terminus of the Fc and an anti-muscle protein antibody Fab fused to the N-terminus of the Fc. FIG. 1H depicts a bivalent fusion protein with Notch ligands fused to the C-terminus of the Fc and an anti- muscle protein scFv fused to the N-terminus of the Fc. FIG. 1I depicts a bivalent fusion protein with Notch ligands fused to the C-terminus of Fc and an anti-muscle protein VHH / nanobody fused to the N-terminus of Fc. FIG. 1J depicts a bivalent fusion protein with Notch ligands fused to the C-terminus of the antibody light chains. FIG. 1K depicts a bivalent fusion protein with Notch ligands fused to the N-terminus of the antibody light chains. FIG. 1L depicts a bivalent fusion protein with Notch ligands fused to the N-terminus of the antibody heavy chains. FIG. 1M depicts a tetravalent fusion protein with Notch ligands fused to the N-terminus of the antibody light and heavy chains.

[0092] FIGs. 2A-2J depict different configurations of bifunctional proteins comprising a non-antibody moiety (e.g., an LG domain of laminin, an LG domain of agrin, an LG domain of nidogen, and an LG domain of perlecan) and a Notch ligand or agonist. FIG. 2A depicts a monovalent protein with a Notch ligand fused to the N-terminus of a laminin G-like domain (LG domain) monomer, or alternatively, a scFv, sdAb, or Fab to a muscle specific protein.FIG. 2B depicts a monovalent protein with a Notch ligand fused to the N-terminus of an LG domain dimer, or alternatively, a scFv, sdAb, or Fab specifically recognizing a muscle specific protein. FIG. 2C depicts a monovalent protein with a Notch ligand fused to the N- terminus of an LG domain trimer or multimers in tandem, or alternatively, a scFv, sdAb, or Fab to a muscle specific protein. FIG. 2D depicts a monovalent protein from N’-to C’ terminus: a Notch ligand – an albumin, anti-albumin scFv or sdAb, or a monomeric human Fc – an LG domain monomer. FIG. 2E depicts a monovalent protein from N’-to C’ terminus: a Notch ligand – an albumin, anti-albumin scFv or sdAb, or a monomeric human Fc – an LG domain dimer. FIG. 2F depicts a monovalent protein from N’-to C’ terminus: a Notch ligand – an albumin, anti-albumin scFv or sdAb, or a monomeric human Fc – an LG domain trimer or multimers in tandem. FIGs. 2G and 2H depict bivalent fusion proteins with Notch ligands fused to the N-terminus of the Fc and LG domain fused to the C-terminus of Fc as monomer, dimer, or multimers in tandem, respectively. FIGs. 2I and 2J depict bivalent fusion proteins with Notch ligands fused to the C-terminus of the Fc and LG domain fused to the N-terminus of Fc as monomer, dimer, or multimers in tandem, respectively.

[0093] FIG. 3A depicts an SDS-PAGE of the reduced and non-reduced DLL4v-Fc-LG4-5 construct. FIG. 3B depicts a size exclusion chromatography (SEC) profile for protein standards (upper profile) and the DLL4v-Fc-LG4-5 construct (lower profile).

[0094] FIG. 4A depicts an SDS-PAGE of the reduced and non-reduced DLL4wt-Fc-LG4- 5 construct. FIG. 4B depicts a size exclusion chromatography (SEC) profile for protein standards (upper profile) and the DLL4wt-Fc-LG4-5 construct (lower profile).

[0095] FIG. 5A depicts an SDS-PAGE of the reduced and non-reduced DLL4v-Fc- LAMA2scFv construct. FIG. 5B depicts a size exclusion chromatography (SEC) profile for protein standards (upper profile) and the DLL4v-Fc-LAMA2scFv construct (lower profile).

[0096] FIG. 6 depicts binding of bifunctional proteins DLL4v-Fc-LG4-5, DLL4wt-Fc- LG4-5, and DLL4v-Fc-LAMA2scFv to Notch-1 using a 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] FIG. 7A depicts a kinetic analysis of the DLL4v-Fc-LG4-5 construct binding to different concentrations of purified matriglycan using Biacore system. The y-axis represents the absolute response (RU), and the x-axis represents the time in seconds. The arrows indicate the injection points of purified matriglycan. FIG. 7B depicts Western blot results of a laminin-overlay assay that shows DLL4v-Fc-LG4-5 binds to matriglycan.

[0098] FIG. 8 depicts a kinetic analysis of the DLL4v-Fc-LAMA2scFv construct binding to different concentrations of recombinant human LG4-5 using Biacore system, confirming that LAMA2scFv binds to LG4-5. The arrows indicate the injection points of recombinant human LG4-5.

[0099] FIG. 9A depicts a study schema. Male D2.mdx mice at approximately 7-9 weeks of age were assessed for muscle function by forelimb grip strength. The mice were randomized into two groups (n=10 / group). The control group was treated triweekly with 20 mg / kg of an isotype control antibody against an irrelevant antigen trinitrophenol (TNP). The experimental group was treated triweekly with 5 mg / kg of DLL4v-Fc-LAMA2scFv. The wildtype (WT) group received no treatment. Forelimb grip strength was assessed monthly over 7 months. FIG. 9B depicts the forelimb grip strength of WT, DLL4v-Fc-LAMA2scFv- treated, and isotype control-treated D2.mdx mice over 7 months. FIG. 9C depicts the bodyweight of WT, DLL4v-Fc-LAMA2scFv-treated, and isotype control-treated D2.mdx mice over 7 months.

[0100] FIG. 10A depicts a size exclusion chromatography (SEC) profile for protein standards (upper profile), huD4v11_2N-FcAAG-LG21DS, huD4v12_2N_A2-FcAAG- LG21DS, huD4v13_2N_-FcAAG-LG21DS, and huD4v14_2N_H2-FcAAG-LG21DS. FIG. 10B depicts a table outlining the yield and percentage of main peak as determined by analytical size exclusion chromatography (aSEC).

[0101] FIG. 11A depicts binding affinity of huD4v11_2N-FcAAG-LG21DS, huD4v12_2N_A2-FcAAG-LG21DS, huD4v13_2N_-FcAAG-LG21DS, and huD4v14_2N_H2-FcAAG-LG21DS to a murine Notch1 receptor, compared to that of the huD4v_G2S_F81L_H168Y-Fc(AAG)-LG21scFvDS (parental) and mud4v constructs as determined by an ELISA assay. FIG. 11B depicts a table outlining the binding affinity (KD) of each construct measured in nM. DETAILED DESCRIPTION

[0102] The present application provides bifunctional protein constructs that specifically target and delivered to muscle tissues with Notch activation in a spatiotemporally controlled manner. Binding of the bifunctional protein construct to muscle tissue, either via a protein on the muscle (cell) sarcolemma or a protein in the extracellular matrix (basal lamina), anchors the bifunctional protein construct and Notch activating moiety comprised therein (e.g., a Notch ligand or agonistic antibody specifically recognizing Notch 1-4), which is arequirement for Notch signaling. Therefore, the bifunctional protein constructs disclosed in this disclosure fulfill the three requirements for specific Notch signaling in muscles: 1) muscle targeting to ensure tissue specificity; 2) Notch ligand anchoring required for trans- activation; and 3) timely control of Notch activation for satellite cells renewal while permitting myoblast differentiation during muscle repair.

[0103] The present application thus in one aspect provides a bifunctional protein construct comprising a first binding moiety and a second binding moiety, wherein the first binding moiety specifically binds to a muscle-specific molecule, and wherein the second binding moiety specifically binds to a Notch receptor and activates the Notch receptor.

[0104] In another aspect, there is provided a method of making the bifunctional protein constructs described herein. Isolated nucleic acids, vectors, and host cells encoding any of the bifunctional protein constructs are also provided. Also provided are methods of treating a muscle-related disease in an individual (e.g., human), by administering to the individual an effective amount of the bifunctional protein construct described herein or pharmaceutical composition thereof.

[0105] In another aspect, there is provided an engineered DLL4 extracellular domain (ECD) comprising a mutation selected from the group consisting of T52N and T135N, and wherein the amino acid position is in reference to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126. Also provided are protein constructs comprising the DLL4 ECD described herein. I. Definitions

[0106] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

[0107] The term “antibody” herein is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. The term “antibody moiety” refers to a full-length antibody or an antigen-binding fragment thereof.

[0108] An “antibody” may refer to an immunoglobulin molecule or a fragment thereof which is able to specifically bind to a specific epitope of an antigen (including the basic 4- chain antibody unit). Antibodies can be intact immunoglobulins derived from natural sources, or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”), antigen-binding fragments (such as Fv, Fab, Fab’, F(ab)2and 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 chain and light chain may be referred to as “VH” and “VL”, respectively. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC- CDR2, and HC-CDR3). CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al- Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved 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 chain. 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. Several of the major antibody classes are divided into subclasses such as lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3 (γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2 (α2 heavy chain).

[0110] The term “antigen-binding fragment” as used herein refers to an antibody fragment including, for example, a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv’), a disulfidestabilized diabody (ds diabody), a single-chain Fv (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a single domain antibody (sdAb) (e.g., a camelized 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 comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds. In some embodiments, an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.

[0111] “Fv” is the minimum antibody fragment, which contains a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light- chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0112] “Single-chain Fv,” also abbreviated as “sFv” or “scFv,” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0113] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4- chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 Daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while 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 bridges. Each H chain has at the N- terminus, a variable domain (VH) followed by three constant domains (CH) for each of the αand γ chains and four CH domains for μ and ε isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen- binding site. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8thEdition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated α, δ, ε, γ and μ, respectively. The γ and α classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.

[0114] The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.

[0115] The “variable region” or “variable domain” of an antibody refers to the amino- terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites. Heavy-chain only antibodies from the Camelidae species have a single heavy chain variable region, which is referred to as “VHH”. VHH is thus a special type of VH.

[0116] The term “variable” refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0117] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application may be made by a variety of techniques, including, for example, 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, 2nded. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, 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 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096;WO 1996 / 33735; WO 1991 / 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); U.S. Pat. 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. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[0118] The terms “full-length antibody,” “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically full-length 4-chain antibodies include those with heavy and light chains including an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions.

[0119] The term “diabodies” refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10) residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen- binding sites. Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in greater detail in, for example, EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

[0120] The monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include PRIMATTZFACTOR D® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest. As used herein, “humanized antibody” is used as a subset of “chimeric antibodies.”

[0121] As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. 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 M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. 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 entireties for use in the present application and for possible inclusion in one or more claims herein. Table 1: CDR Definitions Kabat1Chothia2MacCallum3IMGT4Aho5VH CDR1 31-35 26-32 30-35 27-38 25-40 VH CDR2 50-65 53-55 47-58 56-65 58-77 VH CDR3 95-102 96-101 93-101 105-117 109-137 VL CDR1 24-34 26-32 30-36 27-38 25-40 VL CDR2 50-56 50-52 46-55 56-65 58-77 VL CDR3 89-97 91-96 89-96 105-117 109-137 1Residue numbering follows the nomenclature of Kabat et al., supra 2Residue numbering follows the nomenclature of Chothia et al., supra 3Residue numbering follows the nomenclature of MacCallum et al., supra 4Residue numbering follows the nomenclature of Lefranc et al., supra 5Residue numbering follows the nomenclature of Honegger and Plückthun, supra

[0122] The expression “variable-domain residue-numbering as in Kabat” or “amino-acid- position numbering as in Kabat,” and variations thereof, refers to the numbering system usedfor heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or hypervariable region (HVR) of the variable domain. For example, a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

[0123] Unless indicated otherwise herein, the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., supra with minor modification. The “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody.

[0124] “Framework” or “FR” residues are those variable-domain residues other than the CDR residues as herein defined.

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

[0126] “Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. A suitable human acceptor antibody may be one selected from aconventional database, e.g., the KABAT database, Los Alamos database, the AbM, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody. A human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and / or a heavy chain variable framework region for insertion of the donor CDRs. A suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody. The prior art describes several ways of producing such humanized antibodies (see, for example, EP-A-0239400 and EP-A-054951). For further details, see, e.g., 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. Pat. 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 has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising 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). Also available for the preparation of human monoclonal antibodies are methods 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). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

[0128] The term “donor antibody” refers to an antibody (monoclonal, and / or recombinant) which contributes the amino acid sequences of its variable regions, CDRs, or other functional fragments or analogs thereof to a first immunoglobulin partner, so as toprovide the altered immunoglobulin coding region and resulting expressed altered antibody with the antigenic specificity and neutralizing activity characteristic of the donor antibody.

[0129] The term “acceptor antibody” refers to an antibody (monoclonal and / or recombinant) heterologous to the donor antibody, which contributes all (or any portion, but in some embodiments all) of the amino acid sequences encoding its heavy and / or light chain framework regions and / or its heavy and / or light chain constant regions to the first immunoglobulin partner. In certain embodiments a human antibody is the acceptor antibody.

[0130] The term “attach,” “attached,” “fuse,” or “fused” as used herein, refers to connecting or uniting by a bond, link, force or tie in order to keep two or more components together, which encompasses either direct or indirect attachment such that, for example, where a first polypeptide is directly bound to a second polypeptide or material, and, for example, where one or more intermediate compounds (e.g., amino acids, peptides, polypeptides, etc.) are disposed between the first polypeptide and the second polypeptide or material.

[0131] “Percent (%) amino acid sequence identity” or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, 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 algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0132] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent 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 times 100. For example, if 6 of 10 of the positions in twosequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0133] The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the CH1, CH2 and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.

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

[0135] The “CH1 domain” (also referred to as “C1” of “H1” domain) usually extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0136] “Hinge region” is generally defined as a 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” of a human IgG Fc domain (also referred to as “C2” domain) usually 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 a substitute for the domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22:161- 206 (1985).

[0138] The “CH3 domain” (also referred to as “C3” domain) comprises the stretch of residues C-terminal to a CH2 domain in an Fc domain (i.e. from about amino acid residue 341 to the C-terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG).

[0139] The term “Fc domain” or “fragment crystallizable region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc domains and variant Fc domains. Although the boundaries of the Fc domain of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc domain is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EUnumbering system) of the Fc domain may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native-sequence Fc domains for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0140] “Fc receptor” or “FcR” describes a receptor that binds the Fc domain of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See M. Daëron, Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed 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 the specific group of atoms or amino acids on an antigen to which an antibody or antibody moiety binds. Two antibodies or antibody moieties may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.

[0142] As use herein, the terms “specifically binds,” “specifically recognizing,” and “is specific for” refer to measurable and reproducible interactions, such as binding between a target and an antibody or antibody moiety, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody or antibody moiety that specifically recognizes a target (which can be an epitope) is an antibody or antibody moiety that binds this target with greater affinity, avidity, more readily, and / or with greater duration than its bindings to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by aradioimmunoassay (RIA). In some embodiments, an antibody that specifically binds a target has a dissociation constant (KD) of ≤10-5M, ≤10-6M, ≤10-7M, ≤10-8M, ≤10-9M, ≤10-10M, ≤10-11M, ≤10-12, ≤10-13, or ≤10-14M. In some embodiments, an antibody specifically binds an epitope on a protein that is conserved among the protein from different species. In some embodiments, specific binding can include, but does not require exclusive binding. Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORETM-tests and peptide scans.

[0143] The term “specificity” refers to selective recognition of an antigen binding protein or antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. The term “multispecific” as used herein denotes that an antigen binding protein or an antibody has two or more antigen-binding sites of which at least two bind a different antigen or a different epitope of the same antigen. “Bispecific” as used herein denotes that an antigen binding protein or an antibody has two different antigen-binding specificities. The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind the same epitope of the same antigen.

[0144] “Effector cells” are leukocytes which express one or more FcRs and perform effector functions. In one aspect, the effector cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils. The effector cells may be isolated from a native source, e.g., blood. Effector cells generally are lymphocytes associated with the effector phase, and function to produce cytokines (helper T cells), killing cells in infected with pathogens (cytotoxic T cells) or secreting antibodies (differentiated B cells).

[0145] “Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell 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) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996), may be performed. Antibody variants with altered Fc region amino acid sequences and increased or decreased C1q binding capability are described in U.S. Pat. No. 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” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present application. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

[0147] An “on-rate,” “rate of association,” “association rate,” or “kon” as used herein can also be determined as described above using methods such as biolayer interferometry and surface plasmon resonance (SPR).

[0148] An “isolated” polypeptide is one that has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide’s natural environment will not be present. Ordinarily, however, an isolated polypeptide will be prepared by at least one purification step.

[0149] An “isolated” nucleic acid molecule encoding a construct, antibody, or antigen- binding fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acidis free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies described herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies described herein existing naturally in cells. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0150] Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0151] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in its normal context in a living subject is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural context is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, 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. A hybridoma can be cloned and maintained indefinitely in cell culture and is able to produce monoclonal antibodies. A hybridoma can also be considered to be a hybrid cell.

[0153] The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide” may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or unnatural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide. An “isolated nucleic acid” refers to anucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

[0154] “Complementary” as used herein to refer to a nucleic acid, refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand 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 fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and or at least about 75%, or at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In some embodiments, all nucleotide residues of the first portion are capable of base pairing 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 linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. A vector may be a viral vector, plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector maybe 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 which integrates into a host genome. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0156] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting there from. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0157] The terms “polypeptide” and “peptide” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or unnatural amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, a “polypeptide” includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the polypeptide maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0158] As used herein, “conjugated” refers to covalent attachment of one molecule to a second molecule.

[0159] “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typicallylimited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by 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%, at least 85% identical to the reference sequence.

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

[0161] The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include 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, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a “pharmaceutical composition” for administration to a subject. A pharmaceutically acceptable carrier is non- toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed.

[0163] The terms “pharmaceutical formulation” and “pharmaceutical composition” refer to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components which areunacceptably toxic to a subject to which the formulation would be administered. Such formulations may be sterile.

[0164] A “sterile” formulation is aseptic or essentially free from living microorganisms and their spores.

[0165] A “reconstituted” formulation is one which has been prepared by dissolving a lyophilized protein or antibody formulation in a diluent such that the protein is dispersed throughout. The reconstituted formulation is suitable for administration (e.g. subcutaneous administration) to a patient to be treated with the protein of interest and, in certain embodiments, may be one which is suitable for parenteral or intravenous administration.

[0166] An “isotonic” formulation is one which has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm. The term “hypotonic” describes a formulation with an osmotic pressure below that of human blood. Correspondingly, the term “hypertonic” is used to describe a formulation with an osmotic pressure above that of human blood. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example. The formulations of the present application can be hypertonic as a result of the addition of salt and / or buffer.

[0167] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0168] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0169] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease),preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and / or prolonging survival. The methods of the application contemplate any one or more of these aspects of treatment.

[0170] The terms “effective amount” and “pharmaceutically effective amount” as used herein refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and / or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system.

[0171] The terms “inhibition” or “inhibit” refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and / or amount as compared to that of a reference. In certain embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20% or greater (e.g., at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%). In another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater. In yet another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.

[0172] As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, an antibody which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody.

[0173] As used herein, the terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, in some embodiments a mammal, and in some embodiments a human, having a complement system, including a human in need of therapy for, or susceptible to, a condition or its sequelae. The individual may include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, mice, and humans. In some embodiments, the individual is a human.

[0174] An “affinity-matured” antibody is one with one or more alterations in one or more CDRs thereof that result in an improvement in the affinity of the antibody for antigen,compared to a parent antibody that does not possess those alteration(s). In some embodiments, an affinity-matured antibody has nanomolar or even picomolar affinities for 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) describes affinity maturation by VH- and VL-domain shuffling. Random mutagenesis of CDR and / or framework residues is described by, for example: 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 the application described herein include “consisting of” and / or “consisting essentially of” embodiments.

[0176] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0177] As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat disease of type X means the method is used to treat disease of types other than X.

[0178] The term “about X-Y” used herein has the same meaning as “about X to about Y.”

[0179] As used herein and in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise. II. Bifunctional Protein Constructs

[0180] The present application in one aspect provides a bifunctional protein construct comprising a first binding moiety and a second binding moiety, wherein the first binding moiety specifically binds to a muscle-specific molecule, and wherein the second binding moiety specifically binds to a Notch receptor and activates the Notch receptor. It is to be understood that the term “bifunctional protein construct” used here refers to a protein construct which comprises binding functions to both a muscle-specific molecule and a Notch receptor, and can encompass constructs having additional functionalities. For example, in some embodiments, the bifunctional protein construct is multispecific. In some embodiments, the bifunctional protein construct comprises a first binding moiety and a second binding moiety, wherein the first binding moiety comprises a means for binding to a muscle-specific molecule, and wherein the second binding moiety comprises a means for binding to a Notch receptor and activates the Notch receptor.

[0181] In some embodiments, the first binding moiety comprising an antibody moiety, which includes, but is not limited to, full length antibody, scFv, Fab, or sdAb. In some embodiments, the first binding moiety comprises a non-antibody moiety. Exemplary first binding moieties include, but are not limited to, anti-LAMA2 antibody moieties (e.g., any of the anti-human LAMA2 antibody moieties described herein), anti-matriglycan antibody moieties (e.g., any of the anti-human ADG41 antibody moieties described herein), and anti- CDH15 antibodies (e.g., any of the anti-human CDH15 antibody moieties described herein). These antibody moieties are described in more details in sections below. Means for binding to a muscle-specific molecules described herein can be any of the antibody moieties described herein and functional equivalents thereof.

[0182] In some embodiments, the first binding moiety comprises a non-antibody binding moiety specifically binding to the muscle-specific molecule, for example, a protein domain (e.g., a laminin G-like domain (LG domain)) of an extracellular matrix (ECM) protein that can bind to matriglycan. In some embodiments, the non-antibody moiety comprises a protein domain selected from the group consisting of an LG domain of laminin, an LG domain of agrin, an LG domain of nidogen, and an LG domain of perlecan. In some embodiments, the first binding moiety comprises two or more non-antibody moieties connected in tandem. Means for binding to a muscle-specific molecule described herein can be any of the non- antibody binding moieties described herein and functional equivalents thereof.

[0183] The second binding moiety described herein specifically binds to a Notch receptor and activates the Notch receptor. In some embodiments, the second binding moiety comprises an extracellular domain (ECD) or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jagged1 (Jag1), and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding moiety comprises a DLL1 ECD comprising the amino acid sequence of SEQ ID NO: 130, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 130. In some embodiments, the second binding moiety comprises a DLL3 ECD comprising the amino acid sequence of SEQ ID NO: 131, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 131. In some embodiments, the second binding moiety comprises a DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 125, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 125. In some embodiments, the second binding moiety comprises a variant DLL4 ECD comprisingthe amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 126-129 and 260-267. In some embodiments, the second binding moiety comprises a Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 132, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 132. In some embodiments, the second binding moiety comprises a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133 or 134, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 133 or 134. In some embodiments, the second binding moiety comprises a Jag2 ECD comprising the amino acid sequence of SEQ ID NO: 135, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 135. Means for binding to a Notch receptor (and optionally activates the Notch receptor) described herein can be any of the non-antibody moieties described herein and functional equivalents thereof. In some embodiments, the second binding moiety is an anti-Notch antibody moiety that activates the Notch receptor. Means for binding to a Notch receptor (and optionally activates the Notch receptor) described herein can be any of the anti-Notch antibody moieties described herein and functional equivalents thereof.

[0184] The first binding moiety and the second binding moiety described herein may be directly or indirectly fused (i.e., covalently linked) to each other. In some embodiments, the first binding moiety is fused to the second binding moiety via a carrier protein (e.g., an albumin, an anti-human serum albumin antibody, or a monomeric Fc domain). In some embodiments, the first binding moiety is fused to the second binding moiety via an optional peptide linker (e.g., any of the peptide linkers described under the “Linkers” subsection), for example a peptide linker that is no more than about 30 (such as no more than about any one of 25, 20, or 15) amino acids long. 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 can be used with any one of a variety of bispecific or multispecific antibody construct formats known in the art. Numerous formats have been developed in the art to address therapeutic opportunities afforded by molecules with multiple binding specificities. Several approaches have been described to prepare bi-specific antibody constructs in which specific antibody light chains or fragment pair with specific antibody heavy chains or fragments.

[0186] For example, International Patent Application No. PCT / EP2011 / 056388 (WO2011 / 131746) describes an in vitro method for generating a heterodimeric protein in which asymmetrical mutations are introduced into the CH3 regions of two monospecific starting proteins in order to drive directional “Fab-arm” or “half-molecule” exchange between two monospecific IgG4 or IgG4-like antibodies upon incubation under reducing conditions.

[0187] Schaefer et al. (Roche Diagnostics GmbH), describe a method to assemble two heavy and two light chains, derived from two existing antibodies, into human bivalent bispecific IgG antibodies without use of artificial linkers (PNAS (2011) 108(27): 11187- 11192 and US 2009 / 0232811). The method involves exchanging one or more heavy chain and light chain domains within the antigen-binding fragment (Fab) of one half of the bi- specific antibody (CrossMab). Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This “crossover” retains the antigen-binding affinity but makes the two arms so different that 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. 1. Direct Fusions or Fusion via Carrier Proteins

[0188] The first binding moiety and the second binding moiety can be directly or indirectly linked (i.e., via a peptide linker or a carrier protein, such as a monomeric carrier protein) to each other. The first binding moiety can be fused to the second binding moiety via an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). The first binding moiety and the second binding moiety may also be fused directly to each other. The first binding moiety may be fused at the N-terminus or the C-terminus of the second binding moiety, such as at either the N-terminus or the C-terminus of any one of the polypeptide(s) of the second binding moiety.

[0189] In some embodiments, the first binding moiety and the second binding moiety are linked to each other via a carrier protein. In some embodiments, the carrier protein is selected from the group consisting of a human serum albumin (HSA), an anti-HSA antibody moiety, and a subunit of an Fc domain. Anti-HSA antibody moieties are known in the art, including, 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 moiety is fused tothe N-terminus of the carrier protein via an optional first linker, and the second binding moiety is fused to the C-terminus of the carrier protein via an optional second linker. In some embodiments, the first binding moiety is fused to the C-terminus of the carrier protein via an optional first linker, and the second binding moiety 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., extending at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or longer).

[0190] In some embodiments, the first binding moiety comprises an scFv comprising a VH and a VL specifically binding to a muscle-specific molecule, wherein the scFv is fused to a second binding moiety specifically binding to a Notch receptor. The second binding moiety can be fused to the N-terminus or C-terminus of the scFv specifically recognizing a muscle- specific molecule via an optional linker. In some embodiments, the optional linker between the second binding moiety and the scFv comprises a carrier protein, such as HSA, an anti- HSA antibody moiety, and a subunit of an Fc domain. In some embodiments, the optional linker between the second binding moiety and the scFv is a peptide linker. In some embodiments, the bifunctional protein construct comprises from the N’ to the C’: [VH – first optional linker (e.g., a peptide linker) – VL] forming the scFv – second optional linker (e.g., a peptide linker or a carrier protein) – a second binding moiety specifically binding to a Notch receptor. In some embodiments, the bifunctional protein construct comprises from the N’ to the C’: [VL – first optional linker (e.g., a peptide linker) – VH] forming the scFv – second optional linker (e.g., a peptide linker or a carrier protein) – a second binding moiety specifically binding to a Notch receptor. In some embodiments, the bifunctional protein construct comprises from the N’ to the C’: a second binding moiety specifically binding to a Notch receptor – first optional linker (e.g., a peptide linker or carrier protein) – [VH – second optional linker (e.g., a peptide linker) – VL] forming the scFv. In some embodiments, the bifunctional protein construct comprises from the N’ to the C’: a second binding moiety specifically binding to a Notch receptor – first optional linker (e.g., a peptide linker or carrier protein) – [VL – second optional linker (e.g., a peptide linker) – VH] forming the scFv. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and matriglycan on α-DG. In some embodiments, the first binding moiety comprises an anti-matriglycan scFv (e.g., SEQ ID NO: 155). In some embodiments, the first binding moiety comprises an anti-LAMA2 scFv (e.g., SEQ ID NO: 145 or 268). In some embodiments, the second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1,and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the first binding moiety comprises more than one (e.g., two or three) scFvs linked in tandem, optionally via a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).

[0191] In some embodiments, the first binding moiety comprises a Fab comprising a first polypeptide chain comprising VH and CH1 and a second polypeptide chain comprising VL and CL, wherein the Fab specifically binds to a muscle-specific molecule and is fused to a second binding moiety specifically binding to a Notch receptor via an optional linker. In some embodiments, the second binding moiety is fused to the N-terminus of the first polypeptide chain of the Fab. In some embodiments, the second binding moiety is fused to the C-terminus of the first polypeptide chain of the Fab. In some embodiments, the second binding moiety is fused to the N-terminus of the second polypeptide chain of the Fab. In some embodiments, the second binding moiety 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 from N’ to C’: VH-CH1 – optional linker (e.g., a peptide linker or a carrier protein) – a second binding moiety specifically binding to a Notch receptor; and ii) a second polypeptide comprising from N’ to C’: VL-CL; wherein VH- CH1 and VL–CL form an Fab that specifically binds to a muscle-specific molecule. In some embodiments, the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH-CH1; and ii) a fusion polypeptide comprising from N’ to C’: VL-CL – optional linker (e.g., a peptide linker or a carrier protein) – a second binding moiety specifically binding to a Notch receptor; wherein VH-CH1 and VL–CL form an Fab that specially binds to a muscle-specific molecule. In some embodiments, the bifunctional protein construct comprises: i) a fusion polypeptide comprising from N’ to C’: a second binding moiety specifically binding to a Notch receptor – optional linker (e.g., a peptide linker or a carrier protein) – VH-CH1; ii) a second polypeptide comprising from N’ to C’: VL-CL; wherein VH-CH1 and VL-CL form an Fab that specifically binds to a muscle-specific molecule. In some embodiments, the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH-CH1; and ii) a fusion polypeptide comprising from N’ to C’: a second binding moiety specifically binding to a Notch receptor – optional linker (e.g., a peptide linker or a carrier protein) – VL-CL; wherein VH-CH1 and VL–CL form a Fab that specially binds to a muscle-specific molecule. In some embodiments, the optional linker between the second binding moiety and the Fab comprises a carrier protein,such as HSA, an anti-HSA antibody moiety, and a subunit of an Fc domain. In some embodiments, the optional linker between the second binding moiety 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 matriglycan on α-DG. In some embodiments, the first binding moiety comprises an anti-matriglycan Fab, such as an 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 first binding moiety comprises an anti-LAMA2 Fab, such as an 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 second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the first binding moiety comprises more than one (e.g., two or three) Fabs linked in tandem, optionally via a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).

[0192] In some embodiments, the first binding moiety comprises an sdAb comprising a VHH domain specifically binding to a muscle-specific molecule, wherein the sdAb is fused to a second binding moiety specifically binding to a Notch receptor. The second binding moiety can be fused to the N-terminus or C-terminus of the sdAb. In some embodiments, the bifunctional protein construct comprises from N’ to C’: sdAb (e.g., VHH) specifically binding to a muscle-specific molecule – optional linker (e.g., a peptide linker or a carrier protein) – a second binding moiety specifically binding to a Notch receptor. In some embodiments, the bifunctional protein construct comprises from N’ to C’: a second binding moiety specifically binding to a Notch receptor – optional linker (e.g., a peptide linker or a carrier protein) – sdAb (e.g., VHH) specifically binding to a muscle-specific molecule. In some embodiments, the optional linker between the second binding moiety and the sdAb comprises a carrier protein, such as HSA, an anti-HSA antibody moiety, and a subunit of an Fc domain. In some embodiments, the optional linker between the second binding moiety 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 matriglycan on α-DG. In some embodiments, the second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, andJag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the first binding moiety comprises more than one (e.g., two or three) sdAbs linked in tandem, optionally via a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).

[0193] In some embodiments, the first binding moiety comprises a non-antibody moiety specifically binding to a muscle-specific molecule, wherein the non-antibody moiety comprises an LG domain, e.g., selected from the group consisting of an LG domain of laminin, an LG domain of agrin, an LG domain of nidogen, and an LG domain of perlecan. In some embodiments, the bifunctional protein construct comprises from N’ to C’: a non- antibody moiety specifically binding to a muscle-specific molecule – optional linker (e.g., a peptide linker or a carrier protein) – a second binding moiety specifically binding to a Notch receptor. In some embodiments, the bifunctional protein construct comprises from N’ to C’: a second binding moiety specifically binding to a Notch receptor – optional linker (e.g., a peptide linker or a carrier protein) – a non-antibody moiety specifically binding to a muscle- specific molecule. In some embodiments, the optional linker between the second binding moiety and the non-antibody moiety comprises a carrier protein, such as HSA, an anti-HSA antibody moiety, and a subunit of an Fc domain. In some embodiments, the optional linker between the second binding moiety and the non-antibody moiety is a peptide linker. In some embodiments, the first binding moiety comprises an LAMA2 LG4-5 comprising the amino acid sequence of SEQ ID NO: 118 or 119. In some embodiments, the second binding moiety comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the first binding moiety comprises more than one (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) non-antibody moieties (such a LG domain) linked in tandem, optionally via a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). Exemplary Direct Fusions

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

[0195] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising a LAMA2 LG4-5, wherein the 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 denoted as “DLL4wt-LG4-5”).

[0196] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising a LAMA2 LG4-5, wherein the 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 denoted as “DLL4v-LG4-5”).

[0197] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL4 variant ECD, wherein the DLL4 variant ECD comprises the amino acid sequence of SEQ ID NO: 128; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising a LAMA2 LG4-5, wherein the 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 denoted as “DLL4max-LG4-5”).

[0198] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising a LAMA2 LG4-5, wherein the 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 denoted as “DLL4deimmune (DLL4di)-LG4-5”).

[0199] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL1 ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising an LG domain of LAMA2 (e.g., LAMA2 LG4-5). In some embodiments, there is provided a bifunctionalprotein construct comprising from N’ to C’: a second binding moiety comprising a DLL1 ECD, wherein the 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 moiety comprising a LAMA2 LG4-5, wherein the 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 denoted as “DLL1wt-LG4-5”).

[0200] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL3 ECD or a variant thereof; an optional peptide linker ; and a first binding moiety comprising an LG domain of LAMA2 (e.g., LAMA2 LG4-5). In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL3 ECD, wherein the 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 moiety comprising a LAMA2 LG4-5, wherein the 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 denoted as “DLL3wt-LG4-5”).

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

[0202] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising a LAMA2 LG4-5, wherein the 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 denoted as “Jag1wt-LG4-5”).

[0203] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising a LAMA2 LG4-5, wherein the 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 denoted as “Jag1v-LG4-5”).

[0204] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising an LG domain of LAMA2 (e.g., LAMA2 LG4-5). In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD, wherein the 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 moiety comprising a LAMA2 LG4-5, wherein the 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 denoted as “Jag2wt-LG4-5”).

[0205] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL4 ECD or a variant thereof (such as any of the DLL4 ECD or engineered DLL4 ECDs described herein, e.g., any of SEQ ID NOs: 126-129 and 260-267); an optional peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an anti-LAMA2 scFv (e.g., SEQ ID NO: 145 or 268).

[0206] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety 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 denoted as “DLL4wt-LG21scFv”).

[0207] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety 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 denoted as “DLL4v-LG21scFv”).

[0208] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD,wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 128; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety 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 denoted as “DLL4max-LG21scFv”).

[0209] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety 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 denoted as “DLL4di-LG21scFv”).

[0210] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL1 ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising an anti-LAMA2 scFv. In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL1 ECD, wherein the 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 moiety 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: 150 (hereinafter denoted as “DLL1wt-LG21scFv”).

[0211] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL3 ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising an anti-LAMA2 scFv. In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL3 ECD, wherein the 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 moiety 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: 151 (hereinafter denoted as “DLL3wt-LG21scFv”).

[0212] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag1 ECD or a variantthereof (such as any of the Jag1 ECD described herein, e.g., any of SEQ ID NOs: 132-134); an optional peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an anti-LAMA2 scFv (e.g., SEQ ID NO: 145 or 268).

[0213] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety 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 denoted as “Jag1wt-LG21scFv”).

[0214] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety 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 denoted as “Jag1v-LG21scFv”).

[0215] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising an anti-LAMA2 scFv. In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD, wherein the 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 moiety 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: 154 (hereinafter denoted as “Jag2wt-LG21scFv”).

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

[0217] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety 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 denoted as “DLL4wt-ADG41scFv”).

[0218] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety 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 denoted as “DLL4v-ADG41scFv”).

[0219] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 128; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety 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 denoted as “DLL4max-ADG41scFv”).

[0220] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety 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 denoted as “DLL4di-ADG41scFv”).

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

[0222] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL3 ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising an anti-matriglycan scFv. In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL3 ECD, wherein the 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 moiety 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: 161 (hereinafter denoted as “DLL3wt-ADG41scFv”).

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

[0224] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety 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 denoted as “Jag1wt-ADG41scFv”).

[0225] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety 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 denoted as “Jag1v-ADG41scFv”).

[0226] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD or a variant thereof; an optional peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an anti-matriglycan scFv. In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD, wherein the 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 moiety 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: 164 (hereinafter denoted as “Jag2wt- ADG41scFv”).

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

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

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

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

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

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

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

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

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

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

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

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

[0239] In some embodiments, there is provided a bifunctional protein construct, wherein the bifunctional protein construct comprises a first binding moiety comprising an anti- matriglycan Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety 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), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126, and wherein the anti-matriglycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and asecond 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 moiety and the second polypeptide of the anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 178 (hereinafter denoted as “DLL4wt-ADG41-Fab”).

[0240] In some embodiments, there is provided a bifunctional protein construct, wherein the bifunctional protein construct comprises a first binding moiety comprising an anti- matriglycan Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety 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), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127, and wherein 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 comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 179 (hereinafter denoted as “DLL4v-ADG41-Fab”).

[0241] In some embodiments, there is provided a bifunctional protein construct, wherein the bifunctional protein construct comprises a first binding moiety comprising an anti- matriglycan Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety 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), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 128, and wherein 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 comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 180 (hereinafter denoted as “DLL4max-ADG41-Fab”).

[0242] In some embodiments, there is provided a bifunctional protein construct, wherein the bifunctional protein construct comprises a first binding moiety comprising an anti-matriglycan Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety 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), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129, and wherein 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 comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 181 (hereinafter denoted as “DLL4di-ADG41-Fab”).

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

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

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

[0246] In some embodiments, there is provided a bifunctional protein construct, wherein the bifunctional protein construct comprises a first binding moiety comprising an anti- matriglycan Fab and a second binding moiety comprising a variant Jag1 ECD, wherein the second binding moiety 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), wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133, and wherein 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 comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 184 (hereinafter denoted as “Jag1wt-ADG41-Fab”).

[0247] In some embodiments, there is provided a bifunctional protein construct, wherein the bifunctional protein construct comprises a first binding moiety comprising an anti- matriglycan Fab and a second binding moiety comprising a variant Jag1 ECD, wherein thesecond binding moiety 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), wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134, and wherein 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 comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising the second binding moiety and the second polypeptide of the anti-matriglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 185 (hereinafter denoted as “Jag1v-ADG41-Fab”).

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

[0249] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL4 ECD or a variant thereof (e.g., SEQ ID NO: 339); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0250] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety 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 moiety 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: 319 (hereinafter denoted as “DLL4(EGF1-6)-GSlinker-laminin-a2 LG4-5”).

[0251] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a 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 moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0252] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety 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 moiety 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: 320 (hereinafter denoted as “DLL4(EGF1-6)-HSA-laminin-a2 LG4-5”).

[0253] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a 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 moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0254] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety 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 moiety 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: 323 (hereinafter denoted as “DLL1(EGF1-6)-HSA-laminin-a2 LG4-5”).

[0255] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a 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 moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0256] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, 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 moiety 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: 325 (hereinafter denoted as “Jagged1(EGF1-6)-laminin- a2 LG4-5”).

[0257] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag1 ECD or a variant thereof (e.g., 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 moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0258] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag1 ECD, 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); a carrier protein (e.g., SEQ ID NO: 344); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety 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 denoted as “Jagged1(EGF1-6)-HSA- laminin-a2 LG4-5”).

[0259] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD or a variant thereof (e.g., SEQ ID NO: 342); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0260] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag2 ECD, 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 moiety comprising a laminin-a2LG4-5 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: 328 (hereinafter denoted as “Jagged2(EGF1-6)-laminin- a2 LG4-5”).

[0261] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a Jag2 ECD or a variant thereof (e.g., 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 moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0262] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a variant Jag2 ECD, 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 moiety 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 denoted as “Jagged2(EGF1-6)-HSA- laminin-a2 LG4-5”).

[0263] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety comprising a DLL1 ECD or a variant thereof (e.g., SEQ ID NO: 340); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0264] In some embodiments, there is provided a bifunctional protein construct comprising from N’ to C’: a second binding moiety 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); and a first binding moiety 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: 322 (hereinafter denoted as “DLL1(EGF1-6)-laminin-a2 LG4-5”). 2. Fc Domain Fusions

[0265] In some embodiments, the bifunctional protein construct comprises an Fc domain comprising a first subunit and a second subunit. The Fc domain can be a third moietyconnected to the first binding moiety and the second binding moiety. In some embodiments, when the first binding moiety or second binding moiety is a full-length antibody, the Fc domain can is part of the full-length antibody.

[0266] In some embodiments, the Fc domain is a variant Fc domain possessing minimal or no effector functions. 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 comprise a human Fc domain sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc domain) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0268] In some embodiments, the Fc domain possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody moiety in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In some embodiments, the Fc domain comprises one or more mutations that increase the half-life of the antibody moiety in vivo. In some embodiments, the variant Fc domain has increased FcRn binding at pH 6.0. In some embodiments, the variant Fc domain comprises an M252Y / S254T / T256E (“YTE”) mutation (EU numbering). See, e.g., 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 comprises an M428L / N434S mutation. See, e.g., 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 conducted to analyze CDC and / or ADCC activities of the Fc domain. For example, Fc receptor (FcR) binding assays can be conducted to determine whether the antibody possesses FcγR binding (hence likely ADCC activity), and / or retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas 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 to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. 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); 5,821,337 (see Bruggemann, M. Et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, CA; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12):1759-1769 (2006)).

[0270] Antibodies with reduced effector function include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include a variant Fc with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc variant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581). In some embodiments, the Fc domain of the bifunctional protein construct comprises one or more of these mutations. In some embodiments, the variant Fc domain comprises an N297A, N297Q, or N297G mutation. See, e.g., 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 comprises a L235E mutation. See, e.g., Alegre et al., J Immunol., 148:3461–3468 (1992) and Wang et al., Protein & Cell, 9(1):63-73 (2018).

[0271] Certain antibody variants with improved or diminished binding to FcRs are described. See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 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 an IgG1 Fc domain (e.g., human IgG1 Fc domain). In some embodiments, the human IgG1 Fc comprises the amino acid sequence of SEQ ID NO: 187. In some embodiments, each subunit of the Fc domain comprises a mutation that reduces or abolishes the effector function. In some embodiments, effector function is eliminated through a mutation in the constant region that eliminated glycosylation, e.g., “effector-less mutation.” In one aspect, the effector-less mutation is an N297A or DANA mutation (D265A+N297A) in the CH2 region (EUnumbering). See, e.g., Shields et al., J. Biol. Chem. 276 (9): 6591-6604 (2001). Alternatively or additionally, effector function can be reduced or eliminated through production techniques, such as expression in host cells that do not glycosylate (e.g., E. coli.) or in which result in an altered glycosylation pattern that is ineffective or less effective at promoting effector function (e.g., Shinkawa et al., J. Biol. Chem. 278(5): 3466-3473 (2003). In some embodiments, each subunit of the Fc domain comprises an L234A / L235A “LALA” mutation (EU numbering). In some embodiments, each subunit of the Fc domain comprises an L234A / L235A / P329G “LALALPG” mutation (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the Fc domain comprises a mutation that reduces half-life of the bifunctional protein construct in blood circulation, such as an H435A mutation (EU numbering). In some embodiments, each subunit of the Fc domain (e.g., human IgG1 Fc) comprises L234A / L235A and H435A mutations. In some embodiments, the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises 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 comprises a mutation that reduces Fab exchange, such as an S228P mutation. In some embodiments, the IgG4 Fc domain comprises an F234A / L235A mutations. See, e.g., Xu et al., Cell Immunol., 200:16–26 (2000) and Wang et al., Protein & Cell, 9(1):63-73 (2018). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 concerning other examples of Fc domain variants.

[0273] In some embodiments, when the bifunctional protein construct is a heterodimeric protein comprising two different polypeptide chains, the Fc domain can comprise one or more mutations that facilitate assembly of the heterodimeric protein. For example, knob-into- hole is a heterodimerization technology for the CH3 domain of an antibody. Previously, knobs-into-holes technology has been applied to the production of human full-length bispecific antibodies with 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 herein incorporated by reference in its entirety for all purposes. All these mutations can be incorporated into the bifunctional protein constructs described herein.

[0274] Thus, for example, in some embodiments, the bifunctional protein construct comprises an Fc domain comprising a first CH3 domain and a second CH3 domaincomprising knob-into-hole (KIH) residues. In some embodiments the first CH3 domain is altered so that within the CH3 / CH3 interface, one or more amino acid residues are replaced with one or more amino acid residues having a larger side chain volume, thereby generating a protuberance on the surface of the first CH3 domain that interacts with the second CH3 domain; and the second CH3 domain is altered so that within the CH3 / CH3 interface, one or more amino acid residues are replaced amino acid residues having a smaller side chain volume, thereby generating a cavity on the surface of the second CH3 domain that interacts with the first CH3 domain. In some embodiments, the protuberance is a knob. In some embodiments, the alteration to generate the knob is T366W. In some embodiments, the cavity is a hole. In some embodiments, the alterations to generate the hole are T366S / L368A / Y407V. Hence in some embodiments, the Fc domain described herein comprises knob-into-hole mutations, wherein: 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 second subunit of the Fc domain comprises a knob mutation, and the first subunit of the Fc domain comprises a hole mutation. In some embodiments, the knob mutation is T366W (EU numbering), and the hole mutation is T366S / L368A / Y407V (EU numbering). Unless indicated otherwise, all amino acid positions within the Fc domain are numbered according to EU numbering system.

[0275] In some embodiments, the bifunctional protein construct comprises an Fc domain comprising a mutation allowing for purification of asymmetric Fc domains. In some embodiments, either the first subunit of the Fc domain or the second subunit of the Fc domain comprises an H435R / Y436F mutation (EU numbering). In some embodiments, each subunit of the Fc domain comprises an H435R / Y436F mutation (EU numbering).

[0276] Strop et al. (Rinat-Pfizer Inc.), describes a method of producing stable bi-specific antibodies by expressing and purifying two antibodies of interest separately, and then mixing them together under specified redox conditions (J. Mol. Biol. (2012) 420:204-19).

[0277] Other heterodimerization domains having a strong preference for forming heterodimers over homodimers can be incorporated into the instant bifunctional protein constructs. Illustrative examples include but are not limited to, for example, WO2007147901 (Kjærgaard et al.– Novo Nordisk: describing ionic interactions); WO2009 / 089004 (Kannan et al.– Amgen: describing electrostatic steering effects); WO2010 / 034605 (Christensen et al. – Genentech; describing coiled coils). See also, for example, Pack, P. & Plueckthun, A., Biochemistry 31, 1579-1584 (1992) describing leucine zipper or Pack et al., Bio / Technology 11, 1271-1277 (1993) describing the helix-turn-helix motif. The term “heterodimerizationdomain” does not exclude additional units in addition to 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 heterodimeric protein comprising two different polypeptide chains, the Fc domain can comprise one or more charged-pair mutations that facilitate assembly of the heterodimeric 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, an amino acid residue at D399 (EU numbering) in the first subunit of the Fc domain is replaced with a positively charged residue, and an 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, an amino acid residue at K409 (EU numbering) in the first subunit of the Fc domain is replaced with a negatively charged residue, and an 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 the amino acid sequence of any one of SEQ ID NO: 188, 254, and 277.

[0280] In some embodiments, there is provided a bifunctional protein construct comprising: i) one or more units of a first binding moiety specifically binding to a muscle- specific molecule; ii) a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; iii) a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and iv) an Fc domain comprising a first subunit and a second subunit; optionally wherein the first unit of the second binding moiety and the second unit of the second binding moiety each independently comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2, such as any of the engineered 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 moiety and the second unit of the second binding moiety are the same. In some embodiments, the first unit of the second binding moiety and the second unit of the second binding moiety are different. The one ormore units of the first binding moiety can be the same or different; and / or can bind to the same or different muscle-specific molecules. In some embodiments, the bifunctional protein construct further comprises a third unit of a second binding moiety specifically binding to a third Notch receptor, and a fourth unit of a second binding moiety specifically binding to a fourth Notch receptor. In some embodiments, the third unit of the second binding moiety and the fourth unit of the second binding moiety each independently comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). The first, second, third, and / or fourth unit of the second binding moiety can be the same or different; and / or can bind to the same or different muscle- specific molecules. In some embodiments, all four units of the second binding moiety are the same. In some embodiments, at least one of the four units of the second binding moiety is different from the others. The one or more units of the first binding moiety can comprise antibody moiety or non-antibody moiety. The various binding moieties can independently fuse to the N and / or C terminus of the Fc domain. The bifunctional protein construct can be homodimeric or heterodimeric.

[0281] In some embodiments, there is provided a bifunctional protein construct comprising: i) a first unit of a first binding moiety specifically binding to a first muscle- specific molecule; ii) a second unit of a first binding moiety specifically binding to a second muscle-specific molecule; iii) one or more units of a second binding moiety specifically binding to a Notch receptor and activating the Notch receptor; and iv) an Fc domain comprising a first subunit and a second subunit. In some embodiments, there is provided a bifunctional protein construct comprising: i) a first unit of a first binding moiety comprising a first antibody moiety specifically binding to a first muscle-specific molecule, ii) a second unit of a first binding moiety comprising a second antibody moiety specifically binding to a second muscle-specific molecule, iii) one or more units of a second binding moiety specifically binding to a Notch receptor and activating the Notch receptor; and iv) an Fc domain comprising a first subunit and a second subunit; wherein the first antibody moiety and the second antibody moiety are each independently selected from the group consisting of a Fab, an scFv, and an sdAb. In some embodiments, there is provided a bifunctional protein construct comprising: i) a first unit of a first binding moiety comprising a first non-antibody moiety specifically binding to a first muscle-specific molecule, ii) a second unit of a first binding moiety comprising a second non-antibody moiety specifically binding to a second muscle-specific molecule, iii) one or more units of a second binding moiety specificallybinding to a Notch receptor and activating the Notch receptor; and iv) an Fc domain comprising a first subunit and a second subunit; wherein the first non-antibody moiety and the second non-antibody moiety are each independently selected from the group consisting of an LG domain of laminin, an LG domain of agrin, an LG domain of nidogen, and an LG domain of perlecan. In some embodiments, the first non-antibody moiety and / or the second non-antibody moiety comprises LAMA2 LG4-5 comprising the amino acid sequence of SEQ ID NO: 118 or 119. In some embodiments, the first unit of the first binding moiety comprises two or more first non-antibody moieties connected in tandem, and the second unit of the first binding moiety comprises two or more second non-antibody moieties connected in tandem. In some embodiments, the first unit of the first binding moiety and the second unit of the first binding moiety are the same. In some embodiments, the first unit of the first binding moiety and the second unit of the first binding moiety are different. The one or more units of the second binding moiety can be the same or different; and / or can bind to the same or different Notch receptors. The various binding moieties can independently fuse to the N and / or C terminus of the Fc domain. The bifunctional protein construct can be homodimeric or heterodimeric.

[0282] In some embodiments, the one or more units of the first binding moiety in the bifunctional protein construct comprises (or is) a Fab, wherein the Fab can be connected to the C-terminus of a subunit of the Fc domain via VH or VL, such as through an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). For example, in some embodiments, there is provided a bifunctional protein construct comprising: a first Fab (Fab1) specifically binding to a first muscle-specific molecule comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) specifically binding to a second muscle-specific molecule comprising a VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2- CL); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – VH1-(H1-CH1); ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the Fc domain – optional linker – VH2-(H2-CH1); iii) a third polypeptide comprising from N’ to C’: VL1-(L1-CL); and iv) a fourth polypeptide comprising from N’ to C’: VL2-(L2-CL); and wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, there is provided a bifunctional protein construct comprising: a first Fab (Fab1) specifically binding to a first muscle-specific molecule comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) specifically binding to a second muscle-specific molecule comprising a VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2- CL); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – VL1-(L1-CL); ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the Fc domain – optional linker – VL2-(L2-CL); iii) a third polypeptide comprising from N’ to C’: VH1-(H1-CH1); and iv) a fourth polypeptide comprising from N’ to C’: VH2-(H2-CH1); and wherein VL1- (L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can 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 matriglycan on α-DG. Fab1 and Fab2 can be the same or different; and / or can bind to the same or different muscle-specific molecules. In some embodiments, Fab1 and Fab2 both specifically bind to matriglycan Fab. In some embodiments, anti-matriglycan Fab1 and / or anti-matriglycan Fab2 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, Fab1 and Fab2 both 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. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and / or can bind to the same or different Notch receptors. In some embodiments, the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

[0283] In some embodiments, the one or more units of the first binding moiety comprises (or is) an scFv, wherein the scFv can be connected to the C-terminus of a subunit of the Fc domain via VH or VL, such as through an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). For example, in some embodiments, there is provided a bifunctional protein construct comprising: a first scFv (scFv1) specifically binding to a first muscle- specific molecule comprising VH (VH1) and VL (VL1); a second scFv (scFv2) specifically binding to a second muscle-specific molecule comprising a VH (VH2) and VL (VL2); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – scFv1 (VH1 – optional linker – VL1, or VL1 – optional linker – VH1); and ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety– optional linker – the second subunit of the Fc domain – optional linker – scFv2 (VH2 – optional linker – VL2, or VL2 – optional linker – VH2). In some embodiments, the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can 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 matriglycan on α-DG. scFv1 and scFv2 can be the same or different; and / or can bind to the same or different muscle-specific molecules. In some embodiments, scFv1 and scFv2 both specifically bind to matriglycan. In some embodiments, anti-matriglycan scFv1 and / or anti-matriglycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, scFv1 and scFv2 both 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 moiety and the second unit of the second binding moiety can be the same or different; and / or can bind to the same or different Notch receptors. In some embodiments, the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In someembodiments, the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

[0284] In some embodiments, the one or more units of the first binding moieties comprises (or is) an sdAb, wherein the sdAb can be connected to the C-terminus of a subunit of the Fc domain, such as through an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). For example, in some embodiments, there is provided a bifunctional protein construct comprising: a first sdAb (sdAb1, e.g., VHH1) specifically binding to a first muscle- specific molecule; a second sdAb (sdAb2, e.g., VHH2) specifically binding to a second muscle-specific molecule; a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – the first subunit of the Fc domain – optional linker – sdAb1; ii) a second polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – the second subunit of the Fc domain – optional linker – sdAb2. In some embodiments, the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can 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 matriglycan on α-DG. sdAb1 and sdAb2 can be the same or different; and / or can bind to the same or different muscle-specific molecules. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and / or can bind to the same or different Notch receptors. In some embodiments, the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260- 267). In some embodiments, the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

[0285] In some embodiments, the one or more units of the first binding moieties comprises (or is) a Fab, wherein the Fab can be connected to the N-terminus of a subunit of the Fc domain via CH1 or CL, such as through an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). In some embodiments, there is provided a bifunctional protein construct comprising: a first Fab (Fab1) specifically binding to a first muscle-specificmolecule comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) specifically binding to a second muscle-specific molecule comprising a VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH1-(H1-CH1) – optional linker – the first subunit of the Fc domain – optional linker – the first unit of the second binding moiety; ii) a second polypeptide comprising from N’ to C’: VH2-(H2-CH1) – optional linker – the second subunit of the Fc domain – optional linker – the second unit of the second binding moiety; iii) a third polypeptide comprising from N’ to C’: VL1-(L1-CL); and iv) a fourth polypeptide comprising from N’ to C’: VL2-(L2-CL); and wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, there is provided a bifunctional protein construct comprising: a first Fab (Fab1) specifically binding to a first muscle-specific molecule comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) specifically binding to a second muscle-specific molecule comprising a VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VL1-(L1-CL) – optional linker – the first subunit of the Fc domain – optional linker – the first unit of the second binding moiety; ii) a second polypeptide comprising from N’ to C’: VL2-(L2-CL) – optional linker – the second subunit of the Fc domain – optional linker – the second unit of the second binding moiety specifically binding to a second Notch receptor; iii) a third polypeptide comprising from N’ to C’: VH1- (H1-CH1); and iv) a fourth polypeptide comprising from N’ to C’: VH2-(H2-CH1); and wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2- CL) form Fab2. In some embodiments, the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can 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 matriglycan on α-DG. Fab1 and Fab2 can be the same or different; and / or can bind to thesame or different muscle-specific molecules. In some embodiments, Fab1 and Fab2 both specifically bind to matriglycan Fab. In some embodiments, anti-matriglycan Fab1 and / or anti-matriglycan Fab2 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, Fab1 and Fab2 both 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. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and / or can bind to the same or different Notch receptors. In some embodiments, the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260- 267). In some embodiments, the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

[0286] In some embodiments, there is provided a bifunctional protein construct comprising: i) a first binding moiety comprising a full-length antibody specifically binding 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 moiety 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 moiety 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 moiety specifically binds to a first Notch receptor and activates the first Notch receptor, and the second unit of the second binding moiety 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 can 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 matriglycan on α-DG. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and / or can bind to the same or different Notch receptors. In some embodiments, the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267).In some embodiments, the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

[0287] In some embodiments, the one or more units of the first binding moiety comprises (or is) an scFv, wherein the scFv can be connected to the N-terminus of a subunit of the Fc domain via VH or VL, such as through an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). In some embodiments, there is provided a bifunctional protein construct comprising: a first scFv (scFv1) specifically binding to a first muscle-specific molecule comprising VH (VH1) and VL (VL1); a second scFv (scFv2) specifically binding to a second muscle-specific molecule comprising a VH (VH2) and VL (VL2); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: scFv1 (VH1 -optional linker – VL1 or VL1 -optional linker – VH1) -optional linker– the first subunit of the Fc domain – optional linker – the first unit of the second binding moiety; and ii) a second polypeptide comprising from N’ to C’: scFv2 (VH2 – optional linker – VL2 or VL2 -optional linker – VH2) – optional linker – the second subunit of the Fc domain – optional linker – the second unit of the second binding moiety. In some embodiments, the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can 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 matriglycan on α-DG. scFv1 and scFv2 can be the same or different; and / or can bind to the same or different muscle-specific molecules. In some embodiments, scFv1 and scFv2 both specifically bind to matriglycan. In some embodiments, anti-matriglycan scFv1 and / or anti- matriglycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, scFv1 and scFv2 both 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 moiety and the second unit of the second binding moiety can be the same or different; and / or can bind to the same or different Notch receptors. In some embodiments, the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In someembodiments, the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

[0288] In some embodiments, the one or more units of the first binding moieties comprises (or is) an sdAb, wherein the sdAb can be connected to the N-terminus of a subunit of the Fc domain, such as through an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). In some embodiments, there is provided a bifunctional protein construct comprising: a first sdAb (sdAb1, e.g., VHH1) specifically binding to a first muscle-specific molecule; a second sdAb (sdAb2, e.g., VHH2) specifically binding to a second muscle- specific molecule; a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: sdAb1 – optional linker– the first subunit of the Fc domain – optional linker – the first unit of the second binding moiety; and ii) a second polypeptide comprising from N’ to C’: sdAb2 – optional linker – the second subunit of the Fc domain – optional linker – the second unit of the second binding moiety. In some embodiments, the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can 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 matriglycan on α-DG. sdAb1 and sdAb2 can be the same or different; and / or can bind to the same or different muscle-specific molecules. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and / or can bind to the same or different Notch receptors. In some embodiments, the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260- 267). In some embodiments, the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

[0289] In some embodiments, there is provided a bifunctional protein construct comprising: a first Fab (Fab1) specifically binding to a first muscle-specific molecule comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) specifically binding to a second muscle-specific molecule comprising a VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit of a second binding moiety specificallybinding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH1-(H1-CH1) – optional linker – the first subunit of the Fc domain; ii) a second polypeptide comprising from N’ to C’: VH2-(H2-CH1) – optional linker – the second subunit of the Fc domain; iii) a third polypeptide comprising from N’ to C’: VL1-(L1-CL) – optional linker – the first unit of the second binding moiety; and iv) a fourth polypeptide comprising from N’ to C’: VL2-(L2-CL) – optional linker – the second unit of the second binding moiety; and wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can 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 matriglycan on α-DG. Fab1 and Fab2 can be the same or different; and / or can bind to the same or different muscle-specific molecules. In some embodiments, Fab1 and Fab2 both specifically bind to matriglycan Fab. In some embodiments, anti-matriglycan Fab1 and / or anti-matriglycan Fab2 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, Fab1 and Fab2 both 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. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and / or can bind to the same or different Notch receptors. In some embodiments, the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260- 267). In some embodiments, the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

[0290] In some embodiments, there is provided a bifunctional protein construct comprising: i) a first binding moiety comprising a full-length antibody specifically binding to a muscle-specific molecule; ii) a first unit of a second binding moiety fused (directly or via an optional linker) to the C-terminus of a first light chain of the full-length antibody; and iii) asecond unit of a second binding moiety 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 moiety specifically binds to a first Notch receptor and activates the first Notch receptor, and the second unit of the second binding moiety 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 can 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 matriglycan on α-DG. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and / or can bind to the same or different Notch receptors. In some embodiments, the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

[0291] In some embodiments, there is provided a bifunctional protein construct comprising: a first Fab (Fab1) specifically binding to a first muscle-specific molecule comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) specifically binding to a second muscle-specific molecule comprising a VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit of a second binding moiety specifically binding to a first Notch receptor and activating the first Notch receptor; a second unit of a second binding moiety specifically binding to a second Notch receptor and activating the second Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: VH1-(H1-CH1) – optional linker – the first subunit of the Fc domain; ii) a second polypeptide comprising from N’ to C’: VH2-(H2-CH1) – optional linker – the second subunit of the Fc domain; iii) a third polypeptide comprising from N’ to C’: the first unit of the second binding moiety – optional linker – VL1-(L1-CL); and iv) a fourth polypeptide comprising from N’ to C’: the second unit of the second binding moiety – optional linker – VL2-(L2-CL); and wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2- CH1) and VL2-(L2-CL) form Fab2. In some embodiments, the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can 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 matriglycan on α-DG. Fab1 and Fab2 can be the same or different; and / or can bind to the same or different muscle-specific molecules. In some embodiments, Fab1 and Fab2 both specifically bind to matriglycan Fab. In some embodiments, anti-matriglycan Fab1 and / or anti-matriglycan Fab2 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, Fab1 and Fab2 both 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. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and / or can bind to the same or different Notch receptors. In some embodiments, the second binding moiety (one or both units) comprises an ECD or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding moiety (one or both units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

[0292] In some embodiments, there is provided a bifunctional protein construct comprising: a first binding moiety comprising a full-length antibody specifically binding to a muscle-specific molecule, a first unit of a second binding moiety fused (directly or via an optional linker) to the N-terminus of a first light chain of the full-length antibody, a...

Claims

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

2. The bifunctional protein construct of claim 1, wherein the muscle-specific molecule is a target antigen on the sarcolemma, between the sarcolemma and the basal lamina, or in the basal lamina.

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

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

7. The bifunctional protein construct of any one of claims 1-6, wherein the first binding moiety comprises an antibody moiety specifically binding to the muscle-specific molecule.

8. The bifunctional protein construct of claim 7, wherein the antibody moiety is selected from the group consisting of a full-length antibody, a Fab, a Fab’, a F(ab’)2, an scFv, and an sdAb.

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

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

6.

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

50.

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

12.

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

65.

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

15. The bifunctional protein construct of claim 14, wherein the anti-matriglycan antibody moiety comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 75, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 83, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 84, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 85.

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

95.

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

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

107.

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

109.

20. The bifunctional protein construct of any one of claims 1-6, wherein the first binding moiety comprises a non-antibody moiety specifically binding to the muscle-specific molecule.

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

22. The bifunctional protein construct of claim 21, wherein the non-antibody moiety comprises an LG domain of laminin.

23. The bifunctional protein construct of claim 22, wherein the non-antibody moiety comprises LG4-5 domains of LAMA2 (LAMA2 LG4-5).

24. The bifunctional protein construct of claim 23, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 118 or 119.

25. The bifunctional protein construct of any one of claims 1-24, wherein the second binding moiety comprises an extracellular domain (ECD) or a variant thereof of a Notch ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jagged1 (Jag1), and Jag2.

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

135.

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

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

135.

29. The bifunctional protein construct of claim 28, wherein the bifunctional protein construct comprises the amino acid sequence of any of SEQ ID NOs: 136-144.

30. The bifunctional protein construct of claim 28, wherein the first binding moiety comprises a Fab specifically binding to a muscle-specific molecule.

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

32. The bifunctional protein construct of any one of claims 1-26, wherein the bifunctional protein construct further comprises an Fc domain comprising a first subunit and a second subunit.

33. The bifunctional protein construct of claim 32, wherein: a) wherein the Fc domain is derived from human IgG1 comprising the amino acid sequence of SEQ ID NO: 187; b) each subunit of the Fc domain comprises an L234A / L235A mutation (EU numbering); c) each subunit of the Fc domain comprises an H435A mutation (EU numbering); d) each subunit of the Fc domain comprises an 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 comprises an H435R / Y436F mutation (EU numbering); and / or g) the Fc domain comprises knob-into-hole mutations, and wherein: i) the first subunit of the Fc domain comprises a knob mutation, and the second subunit of the Fc domain comprises a hole mutation; orii) 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 of claim 33, wherein the knob mutation is T366W (EU numbering), and the hole mutation is T366S / L368A / Y407V (EU numbering).

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

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

37. The bifunctional protein construct of claim 36, wherein the first antibody moiety is a first Fab (Fab1), and the second antibody moiety is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – the first subunit of the Fc domain – optional linker – VH1-(H1-CH1); ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – the second subunit of the Fc domain – optional linker – VH2-(H2-CH1); iii) a third polypeptide comprising from N’ to C’: VL1-(L1-CL); andiv) a fourth polypeptide comprising from N’ to C’: VL2-(L2-CL); and wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2- (L2-CL) form Fab2.

38. The bifunctional protein construct of claim 36, wherein the first antibody moiety is a first Fab (Fab1), and the second antibody moiety is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – the first subunit of the Fc domain – optional linker – VL1-(L1-CL); ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – the second subunit of the Fc domain – optional linker – VL2-(L2-CL); iii) a third polypeptide comprising from N’ to C’: VH1-(H1-CH1); and iv) a fourth polypeptide comprising from N’ to C’: VH2-(H2-CH1); and wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2- (L2-CL) form Fab2.

39. The bifunctional protein construct of claim 37 or 38, wherein Fab1 and Fab2 both specifically bind to LAMA2, and wherein 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.

40. The bifunctional protein construct of any one of claims 35-39, wherein: a) the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a variant DLL4 ECD, and wherein the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267; b) the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a DLL1 ECD, and wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; c) the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a DLL3 ECD, and wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131;d) the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a variant Jag1 ECD, and 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 moiety and / or the second unit of the second binding moiety comprises a Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO:

135.

41. The bifunctional protein construct of claim 36, wherein the first antibody moiety is a first sdAb (sdAb1), and the second antibody moiety is a second sdAb (sdAb2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – the first subunit of the Fc domain – optional linker – sdAb1; and ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – the second subunit of the Fc domain – optional linker – sdAb2.

42. The bifunctional protein construct of claim 36, wherein the first antibody moiety is a first scFv (scFv1), and the second antibody moiety is a second scFv (scFv2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – the first subunit of the Fc domain – optional linker – scFv1; and ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – the second subunit of the Fc domain – optional linker – scFv2.

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

44. The bifunctional protein construct of any one of claims 41-43, wherein:a) the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a variant DLL4 ECD comprising the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267; b) the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a DLL1 ECD comprising the amino acid sequence of SEQ ID NO: 130; c) the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a DLL3 ECD comprising the amino acid sequence of SEQ ID NO: 131; d) the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133 or 134; or e) the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a Jag2 ECD comprising the amino acid sequence of SEQ ID NO:

135.

45. The bifunctional protein construct of claim 44, wherein the bifunctional protein construct comprises a first polypeptide and a second polypeptide each comprising the amino acid sequence of any of SEQ ID NOs: 223-231 and 269-276.

46. The bifunctional protein construct of claim 35 or 36, wherein the first antibody moiety is a first Fab (Fab1), and the second antibody moiety is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – VH1-(H1-CH1) – optional linker – the first subunit of the Fc domain; ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – VH2-(H2-CH1) – optional linker – the second subunit of the Fc domain; iii) a third polypeptide comprising from N’ to C’: a third unit of a second binding moiety specifically binding to a third Notch receptor – optional linker – VL1-(L1-CL); and iv) a fourth polypeptide comprising from N’ to C’: a fourth unit of a second binding moiety specifically binding to a fourth Notch receptor – optional linker – VL2-(L2-CL); andwherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2- (L2-CL) form Fab2.

47. The bifunctional protein construct of any one of claims 32-35, wherein the first unit of the first binding moiety comprises a first non-antibody moiety specifically binding to the first muscle-specific molecule, the second unit of the first binding moiety comprises a second non- antibody moiety specifically binding to the second muscle-specific molecule, and wherein the first non-antibody moiety and the second non-antibody moiety are each independently selected from the group consisting of an LG domain of laminin, an LG domain of agrin, an LG domain of nidogen, and an LG domain of perlecan.

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

49. The bifunctional protein construct of claim 47 or 48, wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: the first unit of the first binding moiety – optional linker – the first subunit of the Fc domain – optional linker – a first unit of a second binding moiety specifically binding to a first Notch receptor; and ii) a second polypeptide comprising from N’ to C’: the second unit of the first binding moiety – optional linker – the second subunit of the Fc domain – optional linker – a second unit of a second binding moiety specifically binding to a second Notch receptor.

50. The bifunctional protein construct of claim 47 or 48, wherein the bifunctional protein construct comprises: i) a first polypeptide comprising from N’ to C’: a first unit of a second binding moiety specifically binding to a first Notch receptor – optional linker – the first subunit of the Fc domain – optional linker – the first unit of the first binding moiety; and ii) a second polypeptide comprising from N’ to C’: a second unit of a second binding moiety specifically binding to a second Notch receptor – optional linker – the second subunit of the Fc domain – optional linker – the second unit of the first binding moiety.

51. The bifunctional protein construct of any one of claims 47-50, wherein:a) the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a variant DLL4 ECD, and wherein the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NOs: 126-129 and 260-267; b) the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a DLL1 ECD, and wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; c) the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a DLL3 ECD, and wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; d) the first unit of the second binding moiety and / or the second unit of the second binding moiety comprises a variant Jag1 ECD, and 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 moiety and / or the second unit of the second binding moiety comprises a Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO:

135.

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

53. One or more isolated nucleic acids encoding the bifunctional protein construct of any one of claims 1-52.

54. One or more vectors comprising the one or more isolated nucleic acids of claim 53.

55. The one or more vectors of claim 54, which is a viral vector.

56. A host cell expressing the bifunctional protein construct of any one of claims 1-52, comprising the one or more isolated nucleic acids of claim 53, or comprising the one or more vectors of claim 54 or 55.

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

58. A method of producing a bifunctional protein construct, comprising: i) culturing a host cell comprising the one or more isolated nucleic acids of claim 53 or the one or more vectors of claim 54 or 55, or the host cell of claim 56, under a condition suitable for the expression of the bifunctional protein construct; and ii) recovering the expressed bifunctional protein construct from the cultured host cell.

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

60. The method of claim 59, wherein the muscle-related disease is selected from the group consisting of Pompe disease, centronuclear myopathy, fibrodysplasia ossificans progressive (FOP), Friedreich’s ataxia (FRDA), familial hypertrophic cardiomyopathy, Laing distal myopathy, myofibrillar myopathy, and muscular dystrophy.

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

62. The method of claim 61, wherein the muscular dystrophy comprises one or more of: 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-Dreifuss muscular dystrophy (EDMD).

63. The method of any one of claims 59-62, 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 comprises a mutation selected from the group consisting of T52N and T135N, and wherein the amino acid position is in reference to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO:

126.

65. The engineered DLL4 ECD of claim 64, wherein 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.

66. The engineered DLL4 ECD of 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 engineered DLL4 ECD of any one of claims 64-66, wherein the engineered DLL4 ECD comprises a mutation selected from the group consisting of: (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 (viii) G2S, T52N, R66T, P80L, F81L, T135N, H168Y, Q220H, and N260D.

68. The engineered DLL4 ECD of claim 67, wherein the engineered DLL4 ECD comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 261-264.

69. A protein construct comprising the engineered DLL4 ECD of any one of claims 64- 68.

70. The protein construct of claim 69, wherein the protein construct further comprises a binding moiety that specifically binds to a muscle-specific molecule.

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