Bifunctional proteins and uses thereof

Abstract

Provided herein are compositions and methods of treating muscular dystrophy by administering an effective amount of a composition comprising a bifunctional protein comprising a first binding moiety that binds to a muscle-specific molecule and a second functional domain that activates Notch signaling, thereby activating Notch signaling in muscle tissue in space and time to potentiate satellite cell in situ renewal, and thus enhance muscle repair / regeneration to improve muscle function, as a treatment for various muscular dystrophies and muscle-reducing conditions. Also provided are engineered DLL4 extracellular domains and protein constructs comprising the same.

Description

Cross-reference to related applications

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

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

[0003] This invention relates to bifunctional protein constructs comprising a first binding portion that specifically binds to a muscle-specific molecule and a second binding portion that specifically binds to and activates a Notch receptor. The invention also relates to methods for preparing such bifunctional protein constructs and the use of such bifunctional protein constructs for treating muscle-related diseases (e.g., muscular dystrophy and sarcopenia). An engineered DLL4 extracellular domain (ECD) and protein constructs comprising it are also provided. Background Technology

[0004] Muscular dystrophy (MD) is a group of different muscle diseases with many subtypes, characterized by varying degrees of muscle weakness and degeneration. MD is usually associated with defects in genes encoding proteins essential for muscle integrity or function. Degenerative MD is typically caused by defects in structural proteins that are directly or indirectly related to the dystrophin-associated glycoprotein complex (DGC), which is essential for maintaining the integrity of muscle cell membranes.

[0005] The direct link between MD and Notch signaling in the human body is established through limb-girdle muscular dystrophy (LGMD R21), which is caused by biallelic mutations in the protein O-glucosyltransferase 1 (POGLUT1), reduced Notch signaling, and loss of satellite cells (Servián-Morilla et al., 2016, EMBO MolMed. 8(11):1289-1309; Servián-Morilla et al., 2020, Acta Neuropathol. 139(3):565-582).

[0006] All references cited in this article (including patent applications, patent publications, and GenBank accessions) are incorporated herein by reference as if each individual reference were specifically and individually indicated to be incorporated in full by reference. Summary of the Invention

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

[0008] In some embodiments of the bifunctional protein construct described above, the muscle-specific molecule is a target antigen on, between, or within the sarcomere and basement membrane. In some embodiments, the target antigen is selected from the group consisting of: laminin, agrin, nestin, 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-dystrophin glycan (α-DG), β-DG, laminin-211, perlecan, collagen, α-caryogamic acid, β-caryogamic acid, γ-caryogamic acid, δ-caryogamic acid, ε-caryogamic acid, ζ-caryogamic acid, biglycan, sarcospan, and matrix glycans on α-DG. In some implementations, the target antigen is a matrix glycan on laminin subunit α-2 (LAMA2), CDH15, α-DG, or α-DG.

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

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

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

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

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

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

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

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

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

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

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

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

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

[0022] In some embodiments of any of the bifunctional protein constructs described above, the anti-CDH15 antibody portion 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 of any of the above-described bifunctional protein constructs, the anti-CDH15 antibody portion is an anti-CDH15 Fab. In some embodiments, the anti-CDH15 Fab comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 111 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 112.

[0024] In some embodiments of any of the bifunctional protein constructs described above, the first binding portion comprises a non-antibody portion that specifically binds to a muscle-specific molecule. In some embodiments, the non-antibody portion comprises a protein domain selected from the group consisting of: a laminin G-like domain (LG domain) of laminin, an LG domain of aggregates, an LG domain of nestin, an LG domain of lecithin, a laminin coil-coil binding domain of aggregates, and a laminin γ-binding domain of nestin. In some embodiments, the non-antibody portion comprises the LG domain of laminin. In some embodiments, the non-antibody portion comprises the LG domain of laminin subunit α-1 (LAMA1), LAMA2, laminin subunit α-3 (LAMA3), laminin subunit α-4 (LAMA4), or laminin subunit α-5 (LAMA5). In some embodiments, the non-antibody portion comprises the amino acid sequence of any of SEQ ID NO: 113-117.

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

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

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

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

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

[0030] In some embodiments of any of the bifunctional protein constructs described above, the second binding portion comprises an extracellular domain (ECD) of a Notch ligand selected from the group consisting of: δ-sample 1 (DLL1), DLL3, DLL4, Jagged1 (Jag1), and Jag2.

[0031] In some embodiments of any of the bifunctional protein constructs described above, the second binding portion comprises DLL4 ECD or a variant thereof. In some embodiments, the second binding portion comprises DLL4 ECD, and wherein DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 125. In some embodiments, the second binding portion comprises the variant DLL4ECD, and wherein the variant DLL4 ECD comprises the MNNL domain, DSL domain, and EGF1-5 domain of DLL4, or a sequence having at least about 90% sequence identity with the sequences of the MNNL domain, DSL domain, and EGF1-5 domain of DLL4. In some embodiments, the variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NO: 126-129 and 260-267.

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

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

[0034] In some embodiments of any of the bifunctional protein constructs described above, the second binding portion comprises Jag1 ECD or a variant thereof. In some embodiments, the second binding portion comprises Jag1 ECD, wherein Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 132. In some embodiments, the second binding portion comprises the variant Jag1ECD, wherein the variant Jag1 ECD comprises the MNNL domain, DSL domain, and EGF1-6 domain of Jag1, or a sequence having at least about 90% sequence identity with the sequences of the MNNL domain, DSL domain, and EGF1-6 domain of Jag1. In some embodiments, the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134.

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

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

[0037] In some embodiments of any of the bifunctional protein constructs described above, the first binding portion is fused to the second binding portion via an optional linker. In some embodiments, the first binding portion is fused to the N-terminus of the second binding portion. In some embodiments, the first binding portion is fused to the C-terminus of the second binding portion. In some embodiments, the bifunctional protein construct comprises (i) a second binding portion comprising an ECD of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2; and (ii) a first binding portion comprising: (a) an antibody portion, wherein the antibody portion is a scFv, Fab, or sdAb that specifically binds to a muscle-specific molecule; or (b) a non-antibody portion, wherein the non-antibody portion comprises a protein domain selected from the group consisting of LG domains of laminin, LG domains of aggregates, LG domains of nestin, and LG domains of beaded glycans. In some embodiments, the first binding portion comprises two or more tandemly linked non-antibody portions.

[0038] In some embodiments of any of the bifunctional protein constructs described above, the first binding portion comprises LAMA2 LG4-5 containing the amino acid sequence of SEQ ID NO: 118 or 119. In some embodiments, the second binding portion comprises variant DLL4 ECD, wherein variant DLL4 ECD contains the amino acid sequence of any of SEQ ID NO: 126-129 and 260-267. In some embodiments, the bifunctional protein construct contains the amino acid sequence of any of SEQ ID NO: 136-139. In some embodiments, the second binding portion comprises DLL1 ECD, wherein DLL1 ECD contains the amino acid sequence of SEQ ID NO: 130. In some embodiments, the bifunctional protein construct contains the amino acid sequence of SEQ ID NO: 140. In some embodiments, the second binding portion comprises DLL3 ECD, wherein DLL3ECD contains the amino acid sequence of SEQ ID NO: 131. In some embodiments, the bifunctional protein construct contains the amino acid sequence of SEQ ID NO: 141. In some embodiments, the second binding portion comprises a variant Jag1 ECD, 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 portion comprises a Jag2 ECD, 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 of any of the above-described bifunctional protein constructs, the first binding portion comprises anti-LAMA2 scFv. In some embodiments, 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 NO: 146-154.

[0040] In some embodiments of any of the above-described bifunctional protein constructs, the first binding portion comprises an anti-macroglycan scFv. In some embodiments, the anti-macroglycan 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 NO: 156-164.

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

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

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

[0044] In some embodiments of any of the bifunctional protein constructs described above, the first binding portion is fused to the second binding portion via a carrier protein. In some embodiments, the carrier protein is selected from the group consisting of human serum albumin (HSA), an anti-HSA antibody moiety, and a subunit of the Fc domain. In some embodiments, the first binding portion is fused to the N-terminus of the carrier protein via an optional first linker, and the second binding portion is fused to the C-terminus of the carrier protein via an optional second linker. In some embodiments, the first binding portion is fused to the C-terminus of the carrier protein via an optional first linker, and the second binding portion is fused to the N-terminus of the carrier protein via an optional second linker. In some embodiments, the bifunctional protein construct comprises (i) a second binding portion comprising an ECD of a Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2; and (ii) a first binding portion comprising: (a) an antibody portion, wherein the antibody portion is an scFv, Fab, or sdAb that specifically binds to a muscle-specific molecule; or (b) a non-antibody portion, wherein the non-antibody portion comprises a protein domain selected from the group consisting of LG domains of laminin, aggregates, nestin, and bead-glycans. In some embodiments, the first binding portion comprises two or more tandemly linked non-antibody portions.

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

[0046] In some embodiments of any of the bifunctional protein constructs described above, the first subunit or the second subunit of the Fc domain contains the H435R / Y436F mutation (EU number). In some embodiments, the Fc domain contains a knock-in-hole mutation, wherein: i) the first subunit of the Fc domain contains a knock-in mutation and the second subunit of the Fc domain contains a hole mutation; or ii) the second subunit of the Fc domain contains a knock-in mutation and the first subunit of the Fc domain contains a hole mutation. In some embodiments, the knock-in mutation is T366W (EU number) and the hole mutation is T366S / L368A / Y407V (EU number). In some embodiments, the Fc domain comprises a charged-pair mutation, wherein: i) an amino acid residue in the first subunit of the Fc domain is replaced by a positively charged residue, and an amino acid residue in the second subunit of the Fc domain is replaced by a negatively charged residue; or ii) an amino acid residue in the first subunit of the Fc domain is replaced by a negatively charged residue, and an amino acid residue in the second subunit of the Fc domain is replaced by a positively charged residue. In some embodiments, the Fc domain comprises i) an amino acid residue at D399 (EU number) in the first subunit of the Fc domain being replaced by a positively charged residue, and an amino acid residue at K409 (EU number) in the second subunit of the Fc domain being replaced by a negatively charged residue; or ii) an amino acid residue at K409 (EU number) in the first subunit of the Fc domain being replaced by a negatively charged residue, and an amino acid residue at D399 (EU number) in the second subunit of the Fc domain being replaced by a positively charged residue.

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

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

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

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

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

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

[0053] In some embodiments of any of the bifunctional protein constructs described above, the bifunctional protein construct comprises scFv1 and scFv2, both specifically binding 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 the amino acid sequence of any of SEQ ID NO: 207-222. In some embodiments, the first subunit and the second subunit of the Fc domain each comprise the amino acid sequence of SEQ ID NO: 188. In some embodiments, the first unit of the second binding portion and / or the second unit of the second binding portion comprises variant DLL4 ECD, and wherein variant DLL4 ECD comprises the amino acid sequence of any of SEQ ID NO: 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 NO: 223-226. In some embodiments, the first unit of the second binding portion and / or the second unit of the second binding portion comprises DLL1 ECD, wherein 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 portion and / or the second unit of the second binding portion comprises DLL3 ECD, wherein 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 portion and / or the second unit of the second binding portion comprises a variant Jag1 ECD, 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 portion and / or the second unit of the second binding portion comprises Jag2 ECD, wherein 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 containing the amino acid sequence of SEQ ID NO: 231.

[0054] In some embodiments of any of the bifunctional protein constructs described above, the bifunctional protein construct comprises scFv1 and scFv2, both of which specifically bind to the matrix glycan. 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 NO: 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 of any of the above-described bifunctional protein constructs, the first antibody portion is a first Fab (Fab1), and the second antibody portion 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 – first subunit of the Fc domain – optional linker – first unit specifically binding to the second binding portion of a first Notch receptor; ii) a second polypeptide comprising, from N' to C': VH2-(H2-CH1) –Optional linker – Second subunit of Fc domain –Optional linker – Second unit that specifically binds to the second binding portion of the second Notch receptor; 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.

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

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

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

[0060] In some embodiments of any of the bifunctional protein constructs described above, the first antibody portion is a first Fab (Fab1), and the second antibody portion is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': a first unit—optionally a linker—VH1-(H1-CH1)—optionally a linker—a first subunit of an Fc domain that specifically binds to a second binding portion of a first Notch receptor; ii) a second polypeptide comprising, from N' to C': a second unit—optionally a linker—VH2-(H2-CH1)—that specifically binds to a second binding portion of a second Notch receptor. –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 of any of the bifunctional protein constructs described above, the bifunctional protein construct further comprises a third unit of a second binding portion specifically binding to a third Notch receptor and a fourth unit of a second binding portion specifically binding to a fourth Notch receptor, wherein the third unit of the second binding portion and the fourth unit of the second binding portion each independently comprise an ECD 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 portion and the fourth unit of the second binding portion each independently comprise an ECD or a DLL4 variant thereof. In some embodiments, the third unit of the second binding portion and / or the fourth unit of the second binding portion comprises a DLL4 ECD, wherein the DLL4 ECD comprises the amino acid sequence of any of SEQ ID NO: 125. In some embodiments, the third unit of the second binding portion and / or the fourth unit of the second binding portion comprises a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the MNNL domain, DSL domain, and EGF1-5 domain of DLL4. In some embodiments, 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 second binding portion are identical. In some embodiments, at least one of the four units of the second binding portion is different from the other units.

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

[0063] In some embodiments of any of the bifunctional protein constructs described above, the first unit of the first binding portion comprises a first non-antibody portion specifically binding to the first muscle-specific molecule, and the second unit of the first binding portion comprises a second non-antibody portion specifically binding to the second muscle-specific molecule, wherein the first non-antibody portion and the second non-antibody portion are each independently selected from the group consisting of: the LG domain of laminin, the LG domain of aggregates, the LG domain of nestin, and the LG domain of bead-glycans. In some embodiments, the first unit of the first binding portion comprises two or more tandemly linked first non-antibody portions, and the second unit of the first binding portion comprises two or more tandemly linked second non-antibody portions. In some embodiments, the first non-antibody portion and / or the second non-antibody portion comprises LAMA2 LG4-5 containing the amino acid sequence SEQ ID NO: 118 or 119.

[0064] In some embodiments of 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 first binding portion – optional linker – a first subunit of the Fc domain – optional linker – a first unit of a second binding portion specifically binding to a first Notch receptor; and ii) a second polypeptide comprising, from N' to C': a second unit of the first binding portion – optional linker – a second subunit of the Fc domain – optional linker – a second unit of a second binding portion specifically binding to a second Notch receptor.

[0065] In some embodiments of 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 specifically binding to a second binding portion of a first Notch receptor – optional linker – a first subunit of an Fc domain – optional linker – a first unit of the first binding portion; and ii) a second polypeptide comprising, from N' to C': a second unit specifically binding to a second binding portion of a second Notch receptor – optional linker – a second subunit of the Fc domain – optional linker – a second unit of the first binding portion.

[0066] In some embodiments of any of the bifunctional protein constructs described above, the first unit of the first binding portion comprises two tandemly linked LAMA2 LG4-5 molecules, and the second unit of the first binding portion comprises two tandemly linked LAMA2 LG4-5 molecules. In some embodiments, the first unit and / or the second unit of the second binding portion comprises a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NO: 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 NO: 241-244. In some embodiments, the first unit and / or the second unit of the second binding portion comprises DLL1 ECD, 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 portion and / or the second unit of the second binding portion comprises DLL3 ECD, wherein 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 portion and / or the second unit of the second binding portion comprises variant Jag1 ECD, wherein 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 portion and / or the second unit of the second binding portion comprises Jag2 ECD, wherein 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: 249.

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

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

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

[0070] In some embodiments of any of the bifunctional protein constructs described above, the second binding portion 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 portion specifically binding to one or more muscle-specific molecules; and wherein each of the one or more units of the first binding portion 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 of any of the bifunctional protein constructs described above, the bifunctional protein construct comprises: i) a first unit specifically binding to a second binding portion of a first Notch receptor, and ii) a second unit specifically binding to a second binding portion of a second Notch receptor; wherein the first unit of the second binding portion is fused to the C-terminus of a first subunit of an Fc domain via an optional first linker, and the second unit of the second binding portion is fused to the C-terminus of a second subunit of an Fc domain via an optional second linker; and wherein the first binding portion is fused to the N-terminus of either a first subunit of an Fc domain or a second subunit of an Fc domain via an optional third linker. In some embodiments, the bifunctional protein construct comprises: i) a first unit specifically binding to a first binding portion of a first muscle-specific molecule, and ii) a second unit specifically binding to a first binding portion of a second muscle-specific molecule; wherein the first unit of the first binding portion is fused to the N-terminus of a first subunit of an Fc domain via an optional third linker, and the second unit of the first binding portion is fused to the N-terminus of a second subunit of an Fc domain via an optional fourth linker.

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

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

[0074] In some embodiments of any of the bifunctional protein constructs described above, i) one or more units of the second binding portion each independently comprises an ECD of a Notch ligand or a variant thereof selected from the group consisting of: DLL1, DLL3, DLL4, Jag1, and Jag2; and ii) one or more units of the first binding portion each independently comprises an antibody portion that specifically binds to a muscle-specific molecule, wherein the antibody portion is independently selected from the group consisting of: scFv, Fab, and sdAb. In some embodiments, i) the two units of the second binding portion are identical; and / or ii) two or more units of the first binding portion are identical. In some embodiments, i) the two units of the second binding portion are different; and / or ii) at least one of the two or more units of the first binding portion is different from the other units. In some embodiments, the Fc domain comprises a club-and-socket structural mutation and / or a charged pair mutation. 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 acid sequence of SEQ ID NO: 259. The amino acid sequence of SEQ ID NO: 259, and the second subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 258.

[0075] On the other hand, this article provides one or more isolated nucleic acids that encode any of the aforementioned bifunctional protein constructs.

[0076] On the other hand, this document provides one or more vectors containing the aforementioned one or more isolated nucleic acids. In some embodiments, the one or more vectors are viral vectors. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector.

[0077] On the other hand, this document provides a host cell that expresses any of the above-described bifunctional protein constructs, one or more of the above-described isolated nucleic acids, or one or more of the above-described vectors. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell or a HEK293 cell.

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

[0079] On the other hand, this document provides a method for generating a bifunctional protein construct, the method comprising: i) culturing a host cell containing one or more isolated nucleic acids or one or more vectors described above under conditions suitable for expressing 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 one or more isolated nucleic acids or one or more vectors into the host cell.

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

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

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

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

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

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

[0086] In some embodiments of any of the antibody constructs described above, the anti-macroglycan antibody portion is a full-length anti-macroglycan antibody. In some embodiments, the full-length anti-macroglycan antibody comprises an Fc domain 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 SEQ ID NO: 187. In some embodiments, each subunit of the Fc domain comprises an L234A / L235A mutation (EU number). In some embodiments, each subunit of the Fc domain comprises an H435A mutation (EU number). In some embodiments, each subunit of the Fc domain comprises an L234A / L235A / P329G mutation (EU number). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence SEQ ID NO: 188. In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence SEQ ID NO: 277.

[0087] In some embodiments of the antibody constructs described above, the antibody construct further comprises a second binding portion that specifically binds to a second target molecule. In some embodiments, the second binding portion specifically binds to and activates the Notch receptor. In some embodiments, the second binding portion comprises an ECD 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 positions refer to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, the engineered DLL4 ECD further comprises mutations 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 mutations are selected from the group consisting of: G2S, E14H, R66S, R66T, P80L, F81L, H168Y, Q220H, N231D, and N260D. In some implementations, the engineered DLL4 ECD contains mutations selected from the following groups: (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 of SEQ ID NO: 261-264.

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

[0090] It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of the invention. Attached Figure Description

[0091] Figures 1A-1M Different conformations of bifunctional proteins containing anti-muscle target proteins and Notch ligands or agonists were depicted. Figure 1A A monovalent Notch ligand with anti-muscle protein antibody (Ab) was described in the form of a "mortar and pestle structure". Figure 1B and Figure 1C Alternative forms and conformations of bifunctional proteins are described, in which R1, R2, R3, and R4 can be anti-muscle target proteins or Notch ligands / agonists. Figure 1D A bivalent fusion protein of an antimuscle protein antibody Fab, fused to the N-terminus of Fc, is depicted. Figure 1E A bivalent fusion protein, scFv, of an antimuscle protein antibody fused to the N-terminus of Fc and the C-terminus of Fc, is depicted. Figure 1F A bivalent fusion protein was depicted, consisting of a Notch ligand fused to the N-terminus of Fc and an anti-muscle target protein single-chain Ab (VHH or nanobody) fused to the C-terminus of Fc. Figure 1G A bivalent fusion protein of an anti-muscle protein antibody Fab, fused to the C-terminus of Fc, is depicted. Figure 1H A bivalent fusion protein, consisting of a Notch ligand fused to the C-terminus of Fc and an antimuscle protein scFv fused to the N-terminus of Fc, was described. Figure 1I A bivalent fusion protein of antimuscle protein VHH / nanobody with Notch ligand fused to the C-terminus of Fc and N-terminus fused to Fc is depicted. Figure 1J A bivalent fusion protein with a Notch ligand fused to the C-terminus of an antibody light chain was depicted. Figure 1K A bivalent fusion protein with a Notch ligand fused to the N-terminus of an antibody light chain was depicted. Figure 1L A bivalent fusion protein with a Notch ligand fused to the N-terminus of the antibody heavy chain was depicted. Figure 1M A tetravalent fusion protein with Notch ligands fused to the N-terminus of the antibody light and heavy chains was depicted.

[0092] Figures 2A-2J Different conformations of bifunctional proteins containing non-antibody portions (e.g., the LG domain of laminin, the LG domain of aggregates, the LG domain of nestin, and the LG domain of beaded glycans) and Notch ligands or agonists were depicted. Figure 2AIt depicts monovalent proteins with Notch ligands fused to the N-terminus of laminin G-like domain (LG domain) monomers or alternatively targeting muscle-specific proteins such as scFv, sdAb, or Fab. Figure 2B Monovalent proteins, such as scFv, sdAb, or Fab, that have N-terminal fusion with a Notch ligand that dimers the LG domain or alternatively specifically recognize muscle-specific proteins, are described. Figure 2C It describes monovalent proteins having Notch ligands fused to the N-terminus of tandem LG domain trimers or multimers, or alternatively targeting muscle-specific proteins such as scFv, sdAb, or Fab. Figure 2D Monovalent proteins from the N' to the C' end are depicted: Notch ligand-albumin, antialbumin scFv or sdAb or monomeric human Fc-LG domain monomer. Figure 2E The monovalent proteins from the N' to the C' end are depicted: Notch ligand-albumin, antialbumin scFv or sdAb or monomeric human Fc-LG domain dimer. Figure 2F The monovalent proteins from the N' to the C' end are depicted: Notch ligands – albumin, antialbumin scFv or sdAb or monomeric human Fc – tandem LG domain trimers or multimers. Figure 2G and Figure 2H Bivalent fusion proteins with Notch ligands fused to the N-terminus of Fc and LG domains fused to the C-terminus of Fc, respectively, were described as monomers, dimers, or tandem multimers. Figure 2I and Figure 2J Bivalent fusion proteins with Notch ligands fused to the C-terminus of Fc and LG domains fused to the N-terminus of Fc, respectively, were described as monomers, dimers, or tandem multimers.

[0093] Figure 3A The SDS-PAGE depicts the reduced and non-reduced prototypes of the DLL4v-Fc-LG4-5 construct. Figure 3B Size exclusion chromatography (SEC) chromatograms of protein standards (top) and the DLL4v-Fc-LG4-5 construct (bottom) are depicted.

[0094] Figure 4A The SDS-PAGE depicts the reduced and non-reduced prototypes of the DLL4wt-Fc-LG4-5 construct. Figure 4B Size exclusion chromatography (SEC) chromatograms of protein standards (top) and the DLL4wt-Fc-LG4-5 construct (bottom) are depicted.

[0095] Figure 5A The SDS-PAGE depicts the reduced and non-reduced prototypes of the DLL4v-Fc-LAMA2scFv construct. Figure 5B Size exclusion chromatography (SEC) chromatograms of protein standards (top) and the DLL4v-Fc-LAMA2scFv construct (bottom) are depicted.

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

[0097] Figure 7A The binding kinetics of the DLL4v-Fc-LG4-5 construct to different concentrations of purified matrix glycan were depicted using the Biacore system. The y-axis represents the absolute response value (RU), and the x-axis represents time in seconds. Arrows indicate the injection points of the purified matrix glycan. Figure 7B The results of a Western blot assay depicting laminin coverage of DLL4v-Fc-LG4-5 bound to matrix glycan are described.

[0098] Figure 8 Kinetic analysis of the binding of the DLL4v-Fc-LAMA2scFv construct to different concentrations of recombinant human LG4-5 was depicted using the Biacore system, confirming that LAMA2scFv binds to LG4-5. Arrows indicate the injection sites of recombinant human LG4-5.

[0099] Figure 9A The research protocol was described. Muscle function in approximately 7–9 weeks-old male D2.mdx mice was assessed by forelimb grip strength. Mice were randomly assigned to two groups (n=10 / group). The control group was treated triweekly with an isotype control antibody against the irrelevant antigen trinitrophenol (TNP). The experimental group was treated triweekly with DLL4v-Fc-LAMA2scFv at 5 mg / kg. The wild-type (WT) group received no treatment. Forelimb grip strength was assessed monthly for 7 months. Figure 9B The forelimb grip strength of D2.mdx mice treated with WT, DLL4v-Fc-LAMA2scFv and isotype control mice was depicted over a period of 7 months. Figure 9C The body weights of D2.mdx mice treated with WT, DLL4v-Fc-LAMA2scFv, and isotype control mice were depicted over a 7-month period.

[0100] Figure 10A Size exclusion chromatograms (SEC) of protein standards (top), huD4v11_2N-FcAAG-LG21DS, huD4v12_2N_A2-FcAAG-LG21DS, huD4v13_2N_-FcAAG-LG21DS, and huD4v14_2N_H2-FcAAG-LG21DS are depicted. Figure 10B A table is presented outlining the yields and percentages of the main peaks, as determined by analytical size exclusion chromatography (aSEC).

[0101] Figure 11A The binding affinities of huD4v11_2N-FcAAG-LG21DS, huD4v12_2N_A2-FcAAG-LG21DS, huD4v13_2N_-FcAAG-LG21DS, and huD4v14_2N_H2-FcAAG-LG21DS with the mouse Notch1 receptor, as determined by ELISA assays, were described compared to the binding affinity of huD4v_G2S_F81L_H168Y-Fc(AAG)-LG21scFvDS (parental) and mud4v constructs. Figure 11B The text describes the binding affinity (K0) of each construct, measured in nM. D ) table. Detailed Implementation

[0102] This application provides a bifunctional protein construct that specifically targets and delivers to muscle tissue with Notch activation in a spatiotemporally controlled manner. The bifunctional protein construct binds to muscle tissue via proteins on the muscle (cell) membrane or in the extracellular matrix (basement membrane), anchoring the bifunctional protein construct and the Notch-activating portion contained therein (e.g., Notch ligands or agonist antibodies that specifically recognize Notch 1–4), which is essential for Notch signaling. Therefore, the bifunctional protein construct disclosed in this disclosure satisfies three requirements for specific Notch signaling in muscle: 1) muscle targeting to ensure tissue specificity; 2) Notch ligand anchoring required for transactivation; and 3) timely control of Notch activation for satellite cell renewal during muscle repair, while allowing myoblast differentiation.

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

[0104] On the other hand, a method for preparing the bifunctional protein constructs described herein is provided. Also provided are isolated nucleic acids, vectors, and host cells encoding any of the bifunctional protein constructs. A method for treating a muscle-related disease in an individual (e.g., a human) by administering an effective amount of the bifunctional protein constructs described herein or a pharmaceutical composition thereof to the individual is also provided.

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

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

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

[0108] An "antibody" can refer to an immunoglobulin molecule or fragment thereof (including the basic 4-chain antibody unit) that can specifically bind to a specific epitope of an antigen. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, and can be the immunoreactive portion of a complete immunoglobulin. The antibodies used in this invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intramolecular antibodies”), antigen-binding fragments (such as Fv, Fab, Fab', F(ab)2, and F(ab')2), as well as single-chain antibodies (scFv), heavy-chain antibodies (such as camel 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] Full-length antibodies consist of two heavy chains and two light chains. Variable regions of the light and heavy chains are responsible for antigen binding. These variable domains of the heavy and light chains may be referred to as “VH” and “VL”, respectively. The variable regions of the two chains typically contain three highly variable loops called 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. The CDR boundaries of the antibody and antigen-binding fragments disclosed herein can be defined or identified according to the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three core-residue junctions (CDRs) of either the heavy or light chain are interspersed between flanking segments called framework regions (FRs), which are more conserved than the CDRs and form a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified according to the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are further subdivided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0110] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, biantibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized biantibodies (ds biantibodies), single-chain Fv (scFv), scFv dimers (bivalent biantibodies), multispecific antibodies formed from portions of antibodies containing one or more CDRs, single-domain antibodies (sdAbs) (e.g., camelid single-domain antibodies), nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen bound to a parent antibody or a parent antibody fragment (e.g., a parental scFv). In some embodiments, the antigen-binding fragment may contain one or more CDRs from a specific human antibody that have been grafted into a frame region from one or more different human antibodies.

[0111] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain in tight, non-covalent association. The folding of these two domains generates six hypervariable rings (three from each of the heavy and light chains), which contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, although the affinity is lower than the entire binding site, even a single variable domain (or half of the Fv containing only three antigen-specific CDRs) can recognize and bind to the antigen.

[0112] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing VH and VL antibody domains linked together as a single polypeptide chain. In some embodiments, the scFv polypeptide also includes a polypeptide linker between the VH and VL domains, which enables the scFv to form the structure required for antigen binding. For a review of scFv, see Plückthunin. The Pharmacology of Monoclonal Antibodies Volume 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0113] A basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units plus an additional polypeptide called the J chain and contain 10 antigen-binding sites, while IgA antibodies contain 2–5 basic 4-chain units that can polymerize to form multivalent assemblies combined with the J chains. In the case of IgG, the 4-chain unit is typically about 150,000 Daltons. Each L chain is linked to an H chain by a covalent disulfide bond, while two H chains are linked to each other by one or more disulfide bonds depending on the H chain isoform. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for the α and γ chains and four CH domains for the μ and ε isoforms. Each L chain has a variable domain (VL) at its N-terminus, followed by a constant domain at its other end. VL is aligned with VH, and CL is aligned with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are thought to form the interface between the variable domains of the light chain and the variable domains of the heavy chain. VH and VL pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th ed., Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, Conn., 1994, p. 71 and Chapter 6. Based on the amino acid sequence of its constant domain, the L chain from any vertebrate species can be designated as one of two distinct types, called κ and λ. Immunoglobulins can be designated as different classes or isotypes based on the amino acid sequence of the constant domain of their heavy chain (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated as α, δ, ε, γ, and μ, respectively. Based on relatively small differences in CH sequence and function, the γ and α classes are further divided into subclasses, such as the following subclasses expressed by humans: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0114] The Fc fragment comprises the carboxyl-terminal portion of two H chains held together by disulfide bonds. The effector function of an antibody is determined by the sequence in the Fc region, which is also recognized by the Fc receptor (FcR) found on certain cell types.

[0115] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domains of the heavy and light chains are referred to as "VH" and "VL," respectively. These domains are typically the largest variable portion of an antibody (relative to other antibodies of the same class) and contain the antigen-binding site. Antibodies from camel species that consist only of a heavy chain have a single heavy-chain variable region, which is called a "VHH." Therefore, VHH is a special type of VH.

[0116] The term "variability" refers to the fact that the sequences of certain segments of a variable domain differ widely between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the entire span of the variable domain. Rather, in both the light and heavy chain variable domains, variability is concentrated in three segments called hypervariable regions (HVRs). The more conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions, predominantly employing a β-sheet configuration, linked by three HVRs that form loops connecting β-sheet structures and, in some cases, forming part of a β-sheet structure. The HVRs in each chain are held together closely adjacent to each other by the FR regions and, together with HVRs from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda, Md. (1991)). Constant domains do not directly participate in antibody-antigen binding, but exhibit various effector functions, such as antibody participation in antibody-dependent cytotoxicity.

[0117] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., the individual antibodies constituting said group are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) present in small amounts. Monoclonal antibodies are highly specific (targeting a single antigenic site). Unlike polyclonal antibody preparations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on an antigen. In addition to their specificity, monoclonal antibodies are advantageous because they are synthesized via hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the production of said antibody by any particular method. For example, the monoclonal antibody to be used according to this application can be prepared by a variety of techniques, including, for example, hybridoma methods (e.g., Kohler and Milstein). Nature256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd edition, 1988); Hammerling et al., in: Monoclonal Antibodies and T - Cell Hybridomas 563-681 (Elsevier, NY, 1981), recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567), phage display technology (see, for example, 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 techniques for generating human antibodies or human-like antibodies in animals possessing partial or complete human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, for example, 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. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016; Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368: 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 "complete antibody" are used interchangeably to refer to an antibody in essentially its complete form, rather than an antibody fragment. Specifically, full-length 4-chain antibodies include antibodies having a heavy chain and a light chain that include an Fc region. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or a variant of its amino acid sequence. In some cases, an intact antibody may have one or more effector functions.

[0119] The term "biantibody" refers to a antibody constructed by combining a VH domain with a V... L Small antibody fragments are prepared from sFv fragments (see previous section) with short linkers (approximately 5-10 residues) between their domains. These short linkers enable interchain rather than intrachain pairing of the V domain, resulting in bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific biantibodies are heterodimers of two “crossover” sFv fragments, where the VH domain and V... L The domains are present on different polypeptide chains. For example, in EP 404,097, WO 93 / 11161, Hollinger et al., Proc. Natl. Acad. Sci. USA The biantibody is described in more detail in 90: 6444-6448 (1993).

[0120] Monoclonal antibodies as used herein explicitly include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chains is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of one or more chains is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include PRIMATTZ FACTOR D® antibodies, wherein the antigen-binding region of the antibody is derived from antibodies produced by immunizing macaques, for example, with the antigen of interest. As used herein, “humanized antibody” is used as a subset of “chimeric antibody”.

[0121] As used herein, the term “CDR” or “complementarity-determining region” is intended to refer to a non-continuous antigenic combination site found within the variable region of both heavy and light chain polypeptides. These specific regions have been described in the following literature: Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US 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 MP et al., Dev. Comp. Immunol. , 27: 55-77 (2003); and Honegger and Plückthun, J. Mol. Biol. , 309:657-670 (2001), where the definition includes overlap or subset of amino acid residues when compared with each other. However, the application of any definition to refer to the CDR of an antibody or its grafted antibody or variant is intended to fall within the scope of the terminology as defined and used herein. The amino acid residues covering the CDR defined by each of the foregoing references are listed in Table 1 below for comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, those by 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. References cited in this paragraph are incorporated herein by reference in their entirety for use in this application and may be included in one or more of the claims herein.

[0122] Table 1: CDR Definition

[0123] 1 Residue numbering follows the nomenclature of Kabat et al., as above. 2 Residue numbering follows the nomenclature of Chothia et al., as above. 3 Residue numbering follows the nomenclature of MacCallum et al., as above. 4 Residue numbering follows the nomenclature of Lefranc et al., as above. 5 Residue numbering follows the Honegger and Plückthun nomenclature, as described above. The expressions “as in Kabat variable domain residue numbering” or “as in Kabat amino acid position numbering” and their variations refer to the numbering system used by Kabat et al. (ibid.) for antibody editing of heavy chain or light chain variable domains. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening or insertion into the FR or hypervariable region (HVR) of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (according to residue 52a in Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c in Kabat). For a given antibody, the Kabat number of the residues can be determined by comparing the homologous regions of the antibody sequence with the “standard” Kabat numbered sequence.

[0124] Unless otherwise indicated herein, the residue numbers in the immunoglobulin heavy chain are those of the EU index as described in Kabat et al. (ibid.), with minor modifications. "EU index as described in Kabat" refers to the residue numbers of human IgG1 EU antibodies.

[0125] “Frame” or “FR” residues are those variable domain residues other than CDR residues as defined in this paper.

[0126] As used in this article, “immunoassay” refers to any assay that uses antibodies that can specifically bind to target molecules to detect and quantify any binding of target molecules.

[0127] "Humanized" nonhuman (e.g., rodent) antibodies are chimeric antibodies containing a minimal sequence derived from a nonhuman antibody. To a large extent, humanized antibodies are human immunoglobulins (receptor antibodies) in which residues from the hypervariable region (HVR) of the receptor are replaced by residues from the hypervariable region of a nonhuman species (donor antibody), such as mice, rats, rabbits, or nonhuman primates possessing the desired antibody specificity, affinity, and capability. In some cases, the frame region (FR) residues of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies may contain residues not present in the receptor antibody or the donor antibody. These modifications are made to further improve antibody performance. Generally, humanized antibodies will contain substantially all at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of the nonhuman immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. Humanized antibodies will also optionally contain at least a portion of the immunoglobulin constant region (Fc) (typically the constant region of human immunoglobulins). Suitable human receptor antibodies can be selected from common databases, such as the KABAT database, Los Alamos database, AbM, and Swiss protein database, based on their homology with the nucleotide and amino acid sequences of the donor antibody. Human antibodies characterized by homology (based on amino acids) with the frame regions of the donor antibody may be suitable for providing heavy chain constant regions and / or heavy chain variable frame regions for insertion into the donor CDR. Suitable receptor antibodies capable of providing light chain constant regions or variable frame regions can be selected in a similar manner. It should be noted that the heavy and light chains of the receptor antibody do not need to be derived from the same receptor antibody. Several methods for generating such humanized antibodies are described in the prior art (see, for example, EP-A-0239400 and EP-A-054951). For further details, see, for example, Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). Also see, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0128] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of human-produced antibodies and / or prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Various techniques known in the art, including phage display libraries, can be used to generate human antibodies. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Cole et al. also used this method to prepare human monoclonal antibodies. Monoclonal Antibodies and Cancer Therapy Alan R. Liss, 77 (1985); Boerner et al., J. Immunol., The method described in 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 an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen stimulation, but whose endogenous loci have been disabled, for example, by immunizing xenogeneic mice (see, for example, U.S. Patents 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). Also see, for example, Li et al., regarding human antibodies generated via human B-cell hybridoma technology. Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0129] The term "donor antibody" refers to an antibody (monoclonal and / or recombinant) that contributes its variable region, CDR, or other functional fragments or analogues of the amino acid sequence to a first immunoglobulin partner to provide an altered immunoglobulin coding region and to give the resulting altered antibody the antigen specificity and neutralizing activity characteristic of the donor antibody.

[0130] The term "receptor antibody" refers to an antibody (monoclonal and / or recombinant) heterologous to a donor antibody that contributes all (or any portion, but in some embodiments, all) of its amino acid sequence 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 some embodiments, the human antibody is a receptor antibody.

[0131] As used herein, the terms “attach,” “attached,” “fuse,” or “fused” refer to a connection or union by bonds, links, forces, or ties that holds two or more components together. This covers direct or indirect connections, such that, for example, a first polypeptide is directly bound to a second polypeptide or material, and, for example, one or more intermediate compounds (e.g., amino acids, peptides, polypeptides, etc.) are disposed between the first polypeptide and the second polypeptide or material.

[0132] The "percentage of amino acid sequence identity (%)" or "homology" for the peptide and antibody sequences identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the compared peptide, after sequence alignment and taking into account any conserved substitutions as part of sequence identity. The alignment used to determine the percentage of amino acid sequence identity can be performed in various ways within the capabilities of those skilled in the art, for example, 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 the alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences. However, for the purposes of this document, the amino acid sequence identity value % (Edgar, RC, ...) is generated using the sequence comparison computer program MUSCLE. Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0133] "Homologous" refers to the sequence similarity or identity between two polypeptides or two nucleic acid molecules. Two compared sequences are homologous when positions at those positions are occupied by the same base or amino acid monomer subunit. For example, if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions being compared, multiplied by 100. For example, if six out of ten positions in two sequences are matching or homologous, then the two sequences are 60% homologous. For instance, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, comparisons are made when two sequences are aligned to give the maximum homology.

[0134] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to another part of the immunoglobulin (the variable domain, which contains the antigen-binding site). The constant domain contains the C16 heavy chain. H 1. C H 2 and C H 3. Structural domains (collectively referred to as C) H ) and light chain CHL (or C L ) structural domain.

[0135] The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two distinct types (called kappa (“κ”) and lamuda (“λ”)) based on the amino acid sequence of their constant domains.

[0136] The “CH1 domain” (also known as the “C1” of the “H1” domain) typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0137] The "hinge region" is generally defined as the region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). The hinge region of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain SS bond in the same position.

[0138] The "CH2 domain" (also known as the "C2 domain") of the human IgG Fc domain typically extends from approximately amino acid 231 to approximately amino acid 340. The CH2 domain is unique because it does not pair tightly with another domain. Instead, two N-linked branched carbohydrate chains are intercalated between the two CH2 domains of the intact native IgG molecule. It is speculated that the carbohydrates provide an alternative for domain-domain pairing and contribute to the stability of the CH2 domain. (Burton) Molec Immunol. 22:161-206 (1985).

[0139] The “CH3 domain” (also known as the “C3 domain”) contains the extension of the residues in the Fc domain from the C-terminus to the CH2 domain (i.e. from approximately amino acid residue 341 of the antibody sequence to the C-terminal end (usually at amino acid residues 446 or 447 of IgG)).

[0140] The term "Fc domain" or "crystallizable fragment region" used herein is used to define the C-terminal region of the immunoglobulin heavy chain, including native sequence Fc domains and variant Fc domains. Although the boundaries of immunoglobulin heavy chain Fc domains may vary, the human IgG heavy chain Fc domain is generally defined as extending from an amino acid residue at position Cys226 or from position Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc domain (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, compositions of complete antibodies may comprise antibody populations with all K447 residues removed, antibody populations without K447 residues removed, and antibody populations having mixtures with or without K447 residues. Suitable native sequence Fc domains for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0141] The term "Fc receptor" or "FcR" describes a receptor that binds to the Fc domain of an antibody. Preferred FcRs are native human FcR sequences. Furthermore, preferred FcRs are receptors that bind IgG antibodies (γ receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an activation motif (ITAM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an inhibitory motif (ITIM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. (See M. Daëron, Annu. Rev. Immunol. 15:203-234 (1997). FcR is 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 covered by the term "FcR" in this paper.

[0142] As used herein, the term "epitope" refers to a specific atom or amino acid group on an antigen to which an antibody or antibody moiety binds. If two antibodies or antibody moiety exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen.

[0143] As used herein, the terms “specifically bind,” “specifically recognize,” and “specific to” refer to measurable and reproducible interactions, such as the binding between a target and an antibody or antibody moiety, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody or antibody moiety that specifically recognizes a target (which may be an epitope) binds to that target with greater affinity, cohesion, ease of binding, and / or longer duration of binding than it does to other targets. In some embodiments, the degree to which an antibody binds to an irrelevant target is less than about 10% of the antibody's binding to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, antibodies that specifically bind to a target have ≤10% binding. -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 - 9 M, ≤10 -10 M, ≤10 -11 M, ≤10 -12 ≤10 -13 or ≤10 -14 The dissociation constant of M (K) D In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved across proteins from different species. In some embodiments, specific binding may include, but is not required to be, exclusive binding. The binding specificity of the antibody or antigen-binding domain can be determined experimentally using methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, EIA, BIACORE™ assay, and peptide scanning.

[0144] The term "specificity" refers to the selective recognition of a specific epitope of an antigen by an antigen-binding protein or antibody. For example, natural antibodies are monospecific. The term "multispecific," as used herein, means that an antigen-binding protein or antibody has two or more antigen-binding sites, at least two of which bind to different antigens or different epitopes of the same antigen. The term "bispecific," as used herein, means that an antigen-binding protein or antibody has two different antigen-binding specificities. The term "monospecific," as used herein, means an antibody having one or more binding sites, each binding to the same epitope of the same antigen.

[0145] Effector cells are leukocytes that express one or more FcRs and perform effector functions. In one respect, effector cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, such as blood. Effector cells are typically lymphocytes associated with the effector phase and function to produce cytokines (helper T cells), kill cells infected with pathogens (cytotoxic T cells), or secrete antibodies (differentiated B cells).

[0146] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of appropriate subclasses) that bind to their homologous antigens. To assess complement activation, a CDC assay can be performed, for example, as described by Gazzano-Santoro et al. J. Immunol. Methods As described in 202:163 (1996). Antibody variants with altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in U.S. Patent Nos. 6,194,551B1 and WO99 / 51642. The contents of those patent publications are specifically incorporated herein by reference. See also Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0147] "Binding affinity" generally refers to the strength of the sum 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 otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is generally expressed as a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies typically bind antigens slowly and tend to dissociate easily, while high-affinity antibodies typically bind antigens faster and tend to remain bound for longer periods. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this application. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0148] As used in this article, "on-rate", "rate of association", "association rate" or "k" on"It can also be determined using methods such as biolayer interferometry and surface plasmon resonance (SPR) as described above."

[0149] "Isolated" polypeptides are polypeptides (e.g., native or recombinant) that have been identified, separated, and / or recovered from components of their environment of origin. Preferably, isolated polypeptides do not associate with any other components from their environment of origin. Contaminating components of their environment of origin (such as those produced by recombinant transfected cells) are materials that typically interfere with the research, diagnostic, or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteins or non-protein solutes. In a preferred embodiment, the polypeptide is purified to: (1) greater than 95% by weight of antibody, as determined, for example, by the Lowry method, and in some embodiments, greater than 99% by weight; (2) sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues using a rotary cup sequencer; or (3) homogeneity, determined by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferably silver staining. Isolated polypeptides include in situ polypeptides within recombinant cells, since at least one component of the polypeptide's native environment will be absent. However, isolated polypeptides are typically prepared by at least one purification step.

[0150] An "isolated" nucleic acid molecule encoding the constructs, antibodies, or antigen-binding fragments thereof described herein is a nucleic acid molecule that has been identified and isolated from at least one contaminant nucleic acid molecule typically associated with it in the environment in which it is produced. Preferably, the isolated nucleic acid is not associated with any components related to the environment in which it is produced. The isolated nucleic acid molecule encoding the polypeptides and antibodies described herein is in a form different from the form or environment in which it is found in nature. Therefore, the isolated nucleic acid molecule is different from the nucleic acids naturally present in cells that encode the polypeptides and antibodies described herein. The isolated nucleic acid includes nucleic acid molecules that are normally found in cells containing nucleic acid molecules, but which are located extrachromosomally or at chromosomal locations other than their natural chromosomal locations.

[0151] When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked." For example, if the DNA of a pre-sequence or secretory leader sequence is expressed as a pre-protein involved in polypeptide secretion, then the DNA of the pre-sequence or secretory leader sequence is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to that sequence; or, if a ribosome binding site is positioned to facilitate translation, then the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, they are contiguous and located within the reading frame. However, enhancers do not necessarily have to be contiguous. Ligation is accomplished by connecting at a convenient restriction site. If such a site is not available, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

[0152] "Isolated" means altered or removed from its natural state. For example, nucleic acids or peptides that are naturally present in a living subject in their normal background are not "isolated," but the same nucleic acids or peptides that are partially or completely isolated from coexisting material in their natural background are "isolated." Isolated nucleic acids or proteins may exist in substantially purified forms or may exist in non-natural environments, such as, for example, host cells.

[0153] As used herein, the term "hybridoma" refers to a cell resulting from the fusion of a B lymphocyte and a fusion partner, such as a myeloma cell. Hybridomas can be cloned and maintained indefinitely in cell culture and are capable of producing monoclonal antibodies. Hybridomas can also be considered hybrid cells.

[0154] The terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” are used interchangeably and refer to polymers of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear nucleotide sequence that constitutes a nucleic acid molecule or polynucleotide. “Isolated nucleic acid” refers to a segment or fragment of nucleic acid that has been isolated from a sequence flanking it in its naturally occurring state, i.e., a DNA fragment removed from the sequence normally adjacent to the fragment (i.e., the sequence adjacent to the fragment in its naturally occurring genome). The term also applies to nucleic acids that have been substantially purified from other components naturally accompanying the nucleic acid (i.e., RNA, DNA, or proteins naturally accompanying the nucleic acid in cells). Therefore, the term includes, for example, recombinant DNA incorporated into vectors, autonomously replicating plasmids or viruses, or incorporated into the genomic DNA of prokaryotes or eukaryotes, or existing as a separate molecule independent of other sequences (i.e., as cDNA or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion). The term also includes recombinant DNA that is part of a heterozygous gene encoding an additional polypeptide sequence.

[0155] As used herein, "complementary" for nucleic acids refers to a 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 adenine residues in the first nucleic acid region can form specific hydrogen bonds ("base pairing") with residues in the second nucleic acid region. If the residue is thymine or uracil, then the second nucleic acid region is antiparallel to the first region. Similarly, it is known that cytosine residues in the first nucleic acid strand can base pair with residues in the second nucleic acid strand. If the residue is guanine, then the second nucleic acid strand is antiparallel to the first strand. If, when the two regions are arranged in an antiparallel manner, at least one nucleotide residue in the first region can base pair with a residue in the second region, then the first region of the nucleic acid is complementary to the second region of the same or different nucleic acids. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, such that, when the first and second portions are arranged in an antiparallel manner, at least about 50%, and / or at least about 75%, or at least about 90%, or at least about 95% of the nucleotide residues in the first portion can base pair with the nucleotide residues in the second portion. In some implementations, all nucleotide residues in the first part are capable of base pairing with nucleotide residues in the second part.

[0156] As used herein, the term "vector" refers to a nucleic acid molecule capable of proliferating another nucleic acid it is linked to. This term includes vectors in the form of self-replicating nucleic acid structures as well as vectors incorporated into the genome of a host cell to which it has been introduced. Vectors can be viral vectors, plasmids, bacteriophages, bacterial artificial chromosomes, or yeast artificial chromosomes. Vectors can be adeno-associated virus (AAV) vectors or lentiviral vectors. Vectors can be DNA vectors or RNA vectors. Vectors can be autonomously replicating extrachromosomal vectors or vectors integrated into the host genome. Some 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."

[0157] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to act as a template for the synthesis of other polymers and macromolecules in biological processes. These polymers and macromolecules have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences, and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene encodes a protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is typically provided in sequence listings, and the non-coding strand, which serves as a template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0158] The terms "polypeptide" and "peptide" are used interchangeably and refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues can contain either native or non-native amino acid residues. Full-length proteins and fragments thereof are included in this definition. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Furthermore, "polypeptide" includes modifications to the native sequence (such as deletions, additions, and substitutions (which are generally conserved in nature)) as long as the polypeptide maintains its desired activity. These modifications can be intentional, such as through site-directed mutagenesis, or accidental, such as by generating host mutations in the protein or errors due to PCR amplification.

[0159] As used in this article, "combined" refers to the covalent connection of one molecule to a second molecule.

[0160] When used herein, a “variant” is a nucleic acid or peptide sequence that differs in sequence from a reference nucleic acid or peptide sequence, respectively, but retains the essential biological properties of the reference molecule. Sequence changes in nucleic acid variants may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions, and truncations. Sequence changes in peptide variants are generally limited or conserved, so the sequences of the reference peptide and the variant are very similar overall and identical in many regions. Variants and reference peptides may differ in amino acid sequence by one or more combinations of substitutions, additions, or deletions. Variants of nucleic acids or peptides may be naturally occurring (such as allelic variants) or may be variants not known to exist naturally. Non-naturally occurring variants of nucleic acids and peptides may be prepared by mutagenesis or by direct synthesis. In various embodiments, the variant sequence has 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%, or at least 85% identity with the reference sequence.

[0161] As used herein, the term "regulation" can refer to any method that alters the level or activity of a substrate. Non-limiting examples of protein regulation include influencing expression (including transcription and / or translation), folding, degradation or protein turnover, and protein localization. Non-limiting examples of enzyme regulation also include influencing enzyme activity. A "regulatory factor" is a molecule whose activity includes influencing the level or activity of a substrate. Regulatory factors can be direct or indirect. Regulatory factors can act to activate or inhibit, or otherwise regulate, their substrates.

[0162] The term "control sequence" refers to the DNA sequence necessary for the expression of an operable coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, promoters, optional operon sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0163] "Pharmaceutically acceptable carrier" refers to non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, formulation adjuvants, or carriers conventional in the art, used together with a therapeutic agent to form a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used and is compatible with other components of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation used.

[0164] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to preparations that are in a form that allows for the effective bioactivity of one or more active ingredients and do not contain any additional components that would cause unacceptable toxicity to a subject to whom the formulation will be administered. Such formulations may be sterile.

[0165] "Sterile" preparations are sterile or substantially free of living microorganisms and their spores.

[0166] A "reconstituted" formulation is a formulation prepared by dissolving a lyophilized protein or antibody formulation in a diluent to disperse the protein therein. Reconstituted formulations are suitable for administration to patients seeking treatment with the protein of interest (e.g., subcutaneous administration), and in some embodiments, may be suitable for parenteral or intravenous administration.

[0167] An "isotonic" formulation is one that has substantially the same osmotic pressure as human blood. Isotonic formulations typically have an osmotic pressure of approximately 250-350 mOsm. The term "hypotonic" describes a formulation with an osmotic pressure lower than that of human blood. Correspondingly, the term "hypertonic" is used to describe a formulation with an osmotic pressure higher than that of human blood. For example, isotonicity can be measured using a vapor pressure or cryo-osmometer. The formulations of this application may be hypertonic due to the addition of salts and / or buffer solutions.

[0168] As used herein, the terms “transfection,” “transformation,” or “transduction” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. This includes primary subject cells and their progeny.

[0169] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of the number of passages. The nucleic acid content of progeny cells cannot be exactly the same as that of parental cells and may contain mutations. This article includes screened or selected mutant progeny that have the same function or biological activity as the initially transformed cells.

[0170] As used herein, “treatment” or “treating” is a method for obtaining a beneficial or desired outcome (including clinical outcomes). For the purposes of this application, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, reducing the severity of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing remission of the disease (partial or complete), reducing the dosage of one or more other medications required to treat the disease, delaying the progression of the disease, improving or enhancing quality of life, increasing weight gain, and / or prolonging survival. The methods of this application contemplate any one or more of these aspects of treatment.

[0171] As used herein, the terms “effective amount” and “pharmaceutically effective amount” refer to an amount of a pharmaceutical agent sufficient to provide the desired biological outcome. This outcome may be a reduction (e.g., a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and / or relief of the signs, symptoms, or cause of a disease or disorder, or any other desired alteration of a biological system.

[0172] The terms “inhibition” or “inhibit” refer to a reduction or cessation of any phenotypic trait, or a reduction or cessation of the occurrence, extent, or likelihood of that trait. “Reduction” or “inhibit” means a decrease, reduction, or suppression of activity, function, and / or amount compared to a reference. In some embodiments, “reduction” or “inhibit” means the ability to cause an overall reduction of 20% or greater (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%). In another embodiment, “reduction” or “inhibit” means the ability to cause an overall reduction of 50% or greater. In yet another embodiment, “reduction” or “inhibit” means the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or greater.

[0173] As used herein, a "suppressive" function or activity is one that reduces the function or activity when compared to conditions that are otherwise identical except for the condition or parameter of interest, or alternatively, when compared to another condition. For example, an antibody that suppresses tumor growth reduces the rate of tumor growth compared to the rate of tumor growth in the absence of the antibody.

[0174] As used herein, the terms “patient,” “subject,” “individual,” etc., are used interchangeably and refer to any animal with a complement system, which in some embodiments is a mammal and in some embodiments is a human, including those who require treatment or are susceptible to a disease or its sequelae. An individual may include, for example, dogs, cats, pigs, cattle, sheep, goats, horses, rats, monkeys, mice, and humans. In some embodiments, the individual is a human.

[0175] Affinity-matured antibodies are antibodies with one or more alterations in one or more CDRs that enhance the antibody's affinity for the antigen compared to the parent antibody without these alterations. In some embodiments, affinity-matured antibodies have nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies are produced using procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation via VH and VL domain shuffling. Random mutagenesis of CDRs and / or framework residues has been described in references such as the following: 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).

[0176] It should be understood that the embodiments of this application described herein include “consisting of embodiments” and / or “substantially consisting of embodiments”.

[0177] In this document, references to "about" a value or parameter include (and descriptions) changes to that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0178] As used in this article, referring to a value or parameter as "not" generally means and describes something that is "different from" a value or parameter. For example, "the method is not used to treat type X disease" means that the method is used to treat types of diseases other than X.

[0179] The term “about XY” as used in this article has the same meaning as “about X to about Y”.

[0180] Unless the context clearly specifies otherwise, as used herein and in the appended claims, the singular forms “a,” “or,” and “this” include a plurality of indicators.

[0181] II. Bifunctional Protein Constructs In one aspect, this application provides a bifunctional protein construct comprising a first binding portion and a second binding portion, wherein the first binding portion specifically binds to a muscle-specific molecule, and wherein the second binding portion specifically binds to and activates a Notch receptor. It should be understood that the term "bifunctional protein construct" as used herein refers to a protein construct comprising binding functionality with both a muscle-specific molecule and a Notch receptor, and may encompass constructs having additional functions. For example, in some embodiments, the bifunctional protein construct is multispecific. In some embodiments, the bifunctional protein construct comprises a first binding portion and a second binding portion, wherein the first binding portion comprises a tool for binding to a muscle-specific molecule, and wherein the second binding portion comprises a tool for binding to and activating a Notch receptor.

[0182] In some embodiments, the first binding portion comprises an antibody portion, which includes, but is not limited to, a full-length antibody, scFv, Fab, or sdAb. In some embodiments, the first binding portion comprises a non-antibody portion. Exemplary first binding portions include, but are not limited to, anti-LAMA2 antibody portions (e.g., any of the anti-human LAMA2 antibody portions described herein), anti-matrixan antibody portions (e.g., any of the anti-human ADG41 antibody portions described herein), and anti-CDH15 antibodies (e.g., any of the anti-human CDH15 antibody portions described herein). These antibody portions are described in more detail in the following sections. Tools for binding to the muscle-specific molecules described herein can be any of the antibody portions described herein and their functional equivalents.

[0183] In some embodiments, the first binding portion comprises a non-antibody binding portion that specifically binds to a muscle-specific molecule, such as a protein domain of an extracellular matrix (ECM) protein that can bind to matrix glycans (e.g., a laminin G-like domain (LG domain)). In some embodiments, the non-antibody portion comprises a protein domain selected from the group consisting of: the LG domain of laminin, the LG domain of aggregates, the LG domain of nestin, and the LG domain of bead-like glycans. In some embodiments, the first binding portion comprises two or more tandemly linked non-antibody portions. The tool for binding to the muscle-specific molecule described herein can be any of the non-antibody binding portions described herein or their functional equivalents.

[0184] The second binding moiety described herein specifically binds to and activates the Notch receptor. In some embodiments, the second binding moiety comprises an extracellular domain (ECD) of a Notch ligand selected from the group consisting of delta-sample 1 (DLL1), DLL3, DLL4, Jagged1 (Jag1), and Jag2, or a variant thereof, 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 containing the amino acid sequence of SEQ ID NO: 130, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 130. In some embodiments, the second binding portion comprises a DLL3 ECD containing the amino acid sequence of SEQ ID NO: 131, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 131. In some embodiments, the second binding portion comprises a DLL4 ECD containing the amino acid sequence of SEQ ID NO: 125, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 125. In some embodiments, the second binding portion comprises a variant DLL4 ECD containing the amino acid sequence of any one of SEQ ID NO: 126-129 and 260-267, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 126-129 and 260-267. In some embodiments, the second binding portion comprises a Jag1 ECD containing the amino acid sequence of SEQ ID NO: 132, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 132. In some embodiments, the second binding portion comprises a variant Jag1 ECD containing the amino acid sequence of SEQ ID NO: 133 or 134, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 133 or 134.In some embodiments, the second binding portion comprises a Jag2 ECD containing the amino acid sequence of SEQ ID NO: 135, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 135. The tool for binding to (and optionally activating) the Notch receptor described herein can be any of the non-antibody portions described herein or their functional equivalents. In some embodiments, the second binding portion is an anti-Notch antibody portion that activates the Notch receptor. The tool for binding to (and optionally activating) the Notch receptor described herein can be any of the anti-Notch antibody portions described herein or their functional equivalents.

[0185] The first and second binding portions described herein can be fused directly or indirectly to each other (i.e., covalently linked). In some embodiments, the first binding portion is fused to the second binding portion via a carrier protein (e.g., albumin, anti-human serum albumin antibody, or monomeric Fc domain). In some embodiments, the first binding portion is fused to the second binding portion via an optional peptide linker (e.g., any of the peptide linkers described under the "Linkers" section), such as a peptide linker with a length not exceeding about 30 amino acids (e.g., not exceeding about 25, 20, or 15 amino acids). In some embodiments, the bifunctional protein constructs provided herein comprise Fc domains containing both a first subunit and a second subunit.

[0186] The bifunctional protein constructs provided herein can be used with any of the various bispecific or multispecific antibody constructs known in the art. Various forms have been developed in the art to address therapeutic opportunities offered by molecules with multiple binding specificities. Several methods for preparing bispecific antibody constructs have been described, wherein a specific antibody light chain or fragment is paired with a specific antibody heavy chain or fragment.

[0187] For example, International Patent Application No. PCT / EP2011 / 056388 (WO2011 / 131746) describes an in vitro method for generating heterodimeric proteins, wherein an asymmetric mutation is introduced into the CH3 region of two monospecific initiating proteins to drive a directional “Fab arm” or “half molecule” exchange between two monospecific IgG4 or IgG4-like antibodies during incubation under reducing conditions.

[0188] Schaefer et al. (Roche Diagnostics GmbH) described a method for assembling two heavy chains and two light chains from two existing antibodies into a human bivalent bispecific IgG antibody without the use of artificial connectors (PNAS (2011) 108(27): 11187-11192 and US 2009 / 0232811). This method involves exchanging one or more heavy and light chain domains within the antigen-binding fragment (Fab) of one half of the bispecific antibody (CrossMab). Based on a mortar and pestle structure technique that enables heterodimerization of the heavy chain, the correct association of the light chain and its homologous heavy chain is achieved by exchanging the heavy and light chain domains within the antigen-binding fragment (Fab) of the half of the bispecific antibody. This “crossover” preserves antigen-binding affinity but makes the two arms so different that light chain mismatch no longer occurs. See WO2009 / 080251, WO2009 / 080252, WO2009 / 080253 and WO2009 / 080254, each of which is incorporated herein by reference in its entirety.

[0189] 1. Direct fusion or fusion via carrier proteins The first and second binding portions can be directly or indirectly connected to each other (i.e., via peptide linkers or carrier proteins, such as monomeric carrier proteins). The first binding portion can be fused to the second binding portion via an optional linker (e.g., any one of SEQ ID NO: 211, 212, 337, and 338). The first and second binding portions can also be directly fused to each other. The first binding portion can be fused at the N-terminus or C-terminus of the second binding portion, such as at the N-terminus or C-terminus of any of the polypeptides of the second binding portion.

[0190] In some embodiments, the first binding portion and the second binding portion are linked to each other via a carrier protein. In some embodiments, the carrier protein is selected from the group consisting of human serum albumin (HSA), an anti-HSA antibody moiety, and a subunit of the Fc domain. The anti-HSA antibody moiety is known in the art, including, for example, those by 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 portion is fused to the N-terminus of the carrier protein via an optional first linker, and the second binding portion is fused to the C-terminus of the carrier protein via an optional second linker. In some embodiments, the first binding portion is fused to the C-terminus of the carrier protein via an optional first linker, and the second binding portion is fused to the N-terminus of the carrier protein via an optional second linker. In some embodiments, the carrier protein (e.g., HSA) prolongs the half-life of the bifunctional protein construct (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or longer).

[0191] In some embodiments, the first binding portion comprises an scFv containing VH and VL that specifically bind to muscle-specific molecules, wherein the scFv is fused to a second binding portion that specifically binds to the Notch receptor. The second binding portion may be fused to the N-terminus or C-terminus of the scFv that specifically recognizes muscle-specific molecules via an optional linker. In some embodiments, the optional linker between the second binding portion and the scFv comprises a carrier protein, such as an HSA, an anti-HSA antibody moiety, and a subunit of an Fc domain. In some embodiments, the optional linker between the second binding portion and the scFv is a peptide linker. In some embodiments, the bifunctional protein construct comprises, from N' to C': [VH – first optional linker (e.g., peptide linker) – VL] forming the scFv – second optional linker (e.g., peptide linker or carrier protein) – second binding portion that specifically binds to the Notch receptor. In some embodiments, the bifunctional protein construct from N' to C' comprises: [VL – first optional linker (e.g., peptide linker) – VH] – second optional linker (e.g., peptide linker or carrier protein) – a second binding moiety specifically binding to the Notch receptor. In some embodiments, the bifunctional protein construct from N' to C' comprises: a second binding moiety specifically binding to the Notch receptor – a first optional linker (e.g., peptide linker or carrier protein) – [VH – second optional linker (e.g., peptide linker) – VL] – a second optional linker (e.g., peptide linker) – VH] – a second optional linker (e.g., peptide linker or carrier protein) – a second binding moiety specifically binding to the Notch receptor. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. In some embodiments, the first binding moiety comprises an anti-matrix glycan 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 of a Notch ligand selected from the group consisting of δsample 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NO: 126-129 and 260-267). In some embodiments, the second binding moiety comprises the amino acid sequence of any of SEQ ID NO: 126-135 and 260-267.In some embodiments, the first binding portion comprises more than one (e.g., two or three) tandemly linked scFv, optionally connected via peptide linkers (e.g., any one of SEQ ID NO: 211, 212, 337 and 338).

[0192] In some embodiments, the first binding moiety comprises Fab, which includes a first polypeptide chain containing VH and CH1 and a second polypeptide chain containing VL and CL, wherein Fab specifically binds to a muscle-specific molecule and is fused via an optional linker to a second binding moiety specifically bound to a Notch receptor. In some embodiments, the second binding moiety is fused to the N-terminus of the first polypeptide chain of Fab. In some embodiments, the second binding moiety is fused to the C-terminus of the first polypeptide chain of Fab. In some embodiments, the second binding moiety is fused to the N-terminus of the second polypeptide chain of Fab. In some embodiments, the second binding moiety is fused to the C-terminus of the second polypeptide chain of Fab. In some embodiments, the bifunctional protein construct comprises: i) a fusion polypeptide comprising, from N' to C': VH-CH1 – optional linker (e.g., peptide linker or carrier protein) – a second binding moiety specifically bound to a Notch receptor; and ii) a second polypeptide comprising, from N' to C': VL-CL; wherein VH-CH1 and VL-CL form Fab specifically bound 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 – an optional linker (e.g., a peptide linker or carrier protein) – a second binding moiety specifically binding to the Notch receptor; wherein VH-CH1 and VL-CL form a Fab specifically binding to muscle-specific molecules. In some embodiments, the bifunctional protein construct comprises: i) a fusion polypeptide comprising, from N' to C': a second binding moiety specifically binding to the Notch receptor – an optional linker (e.g., a peptide linker or carrier protein) – VH-CH1; and ii) a second polypeptide comprising, from N' to C': VL-CL; wherein VH-CH1 and VL-CL form a Fab specifically binding to muscle-specific molecules. 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 the Notch receptor – optionally a linker (e.g., a peptide linker or carrier protein) – VL-CL; wherein VH-CH1 and VL-CL form a Fab specifically binding 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 the 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 matrix glycans on α-DG.In some embodiments, the first binding portion comprises an anti-matrixan Fab, such as an anti-matrixan Fab comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 176 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 177. In some embodiments, the first binding portion comprises an anti-LAMA2 Fab, such as an anti-LAMA2 Fab comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 165 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 166. In some embodiments, the second binding portion comprises an ECD of a Notch ligand selected from the group consisting of δsample 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NO: 126-129 and 260-267). In some embodiments, the second binding portion comprises the amino acid sequence of any of SEQ ID NO: 126-135 and 260-267. In some embodiments, the first binding portion comprises more than one (e.g., two or three) tandemly linked Fabs, optionally linked via peptide linkers (e.g., any one of SEQ ID NO: 211, 212, 337 and 338).

[0193] In some embodiments, the first binding portion comprises an sdAb containing a VHH domain that specifically binds to a muscle-specific molecule, wherein the sdAb is fused to a second binding portion that specifically binds to a Notch receptor. The second binding portion may be fused to the N-terminus or C-terminus of the sdAb. In some embodiments, the bifunctional protein construct comprises, from N' to C': an sdAb (e.g., VHH) that specifically binds to a muscle-specific molecule – an optional linker (e.g., a peptide linker or carrier protein) – a second binding portion that specifically binds to a Notch receptor. In some embodiments, the bifunctional protein construct comprises, from N' to C': a second binding portion that specifically binds to a Notch receptor – an optional linker (e.g., a peptide linker or carrier protein) – an sdAb (e.g., VHH) that specifically binds to a muscle-specific molecule. In some embodiments, the optional linker between the second binding portion and the sdAb comprises a carrier protein, such as an HSA, an anti-HSA antibody portion, and a subunit of an Fc domain. In some embodiments, the optional linker between the second binding portion 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 matrix glycans on α-DG. In some embodiments, the second binding moiety comprises an ECD of a Notch ligand selected from the group consisting of δ-sample 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NO: 126-129 and 260-267). In some embodiments, the second binding moiety comprises an amino acid sequence of any of SEQ ID NO: 126-135 and 260-267. In some embodiments, the first binding moiety comprises more than one (e.g., two or three) tandemly linked sdAbs, optionally via peptide linkers (e.g., any of SEQ ID NO: 211, 212, 337, and 338).

[0194] In some embodiments, the first binding portion comprises a non-antibody portion that specifically binds to a muscle-specific molecule, wherein the non-antibody portion comprises, for example, an LG domain selected from the group consisting of: an LG domain of laminin, an LG domain of aggregates, an LG domain of nestin, and an LG domain of bead-glycans. In some embodiments, the bifunctional protein construct from N' to C' comprises: a non-antibody portion that specifically binds to a muscle-specific molecule – an optional linker (e.g., a peptide linker or carrier protein) – a second binding portion that specifically binds to a Notch receptor. In some embodiments, the bifunctional protein construct from N' to C' comprises: a second binding portion that specifically binds to a Notch receptor – an optional linker (e.g., a peptide linker or carrier protein) – a non-antibody portion that specifically binds to a muscle-specific molecule. In some embodiments, the optional linker between the second binding portion and the non-antibody portion comprises a carrier protein, such as an HSA, an anti-HSA antibody portion, and a subunit of an Fc domain. In some embodiments, the optional linker between the second binding portion and the non-antibody portion is a peptide linker. In some embodiments, the first binding portion comprises LAMA2 LG4-5 containing the amino acid sequence of SEQ ID NO: 118 or 119. In some embodiments, the second binding portion comprises an ECD of a Notch ligand selected from the group consisting of δ-sample 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NO: 126-129 and 260-267). In some embodiments, the second binding portion comprises the amino acid sequence of any of SEQ ID NO: 126-135 and 260-267. In some embodiments, the first binding portion comprises more than one (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) tandemly linked, optionally via a peptide linker (e.g., any of SEQ ID NO: 211, 212, 337, and 338) non-antibody portions (such as the LG domain).

[0195] Exemplary direct fusion In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion comprising DLL4ECD or a variant thereof (e.g., any one of SEQ ID NO: 126-135 and 260-267); an optional peptide linker (e.g., any one of SEQ ID NO: 211, 212, 337 and 338); and a first binding portion comprising the LG domain of LAMA2 (e.g., LAMA2 LG4-5).

[0196] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 136 (hereinafter referred to as "DLL4wt-LG4-5").

[0197] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 137 (hereinafter referred to as "DLL4v-LG4-5").

[0198] In some embodiments, a bifunctional protein construct is provided 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 LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 138 (hereinafter referred to as "DLL4max-LG4-5").

[0199] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 139 (hereinafter referred to as "DLL4 deimmunization (DLL4di)-LG4-5").

[0200] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion comprising DLL1ECD or a variant thereof; an optional peptide linker; and a first binding portion comprising the LG domain of LAMA2 (e.g., LAMA2 LG4-5). In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion comprising DLL1 ECD, wherein DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding portion comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 140 (hereinafter referred to as "DLL1wt-LG4-5").

[0201] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion comprising DLL3ECD or a variant thereof; an optional peptide linker; and a first binding portion comprising the LG domain of LAMA2 (e.g., LAMA2 LG4-5). In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion comprising DLL3 ECD, wherein DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding portion comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 141 (hereinafter referred to as "DLL3wt-LG4-5").

[0202] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion comprising Jag1ECD or a variant thereof (such as any of the Jag1 ECDs described herein, e.g., any of SEQ ID NO: 132-134); an optional peptide linker (e.g., any of SEQ ID NO: 211, 212, 337, and 338); and a first binding portion comprising the LG domain of LAMA2 (e.g., LAMA2 LG4-5) (e.g., SEQ ID NO: 119).

[0203] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 142 (hereinafter referred to as "Jag1wt-LG4-5").

[0204] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 143 (hereinafter referred to as "Jag1v-LG4-5").

[0205] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion comprising Jag2ECD or a variant thereof; an optional peptide linker; and a first binding portion comprising the LG domain of LAMA2 (e.g., LAMA2 LG4-5). In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion comprising Jag2 ECD, wherein Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding portion comprising LAMA2 LG4-5, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 144 (hereinafter referred to as "Jag2wt-LG4-5").

[0206] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising DLL4ECD or a variant thereof (such as any of the DLL4 ECD described herein or engineered DLL4 ECD, e.g., any of SEQ ID NO: 126-129 and 260-267); an optional peptide linker (e.g., any of SEQ ID NO: 211, 212, 337 and 338); and a first binding moiety comprising anti-LAMA2 scFv (e.g., SEQ ID NO: 145 or 268).

[0207] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 146 (hereinafter referred to as "DLL4wt-LG21scFv").

[0208] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 147 (hereinafter referred to as "DLL4v-LG21scFv").

[0209] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 148 (hereinafter referred to as "DLL4max-LG21scFv").

[0210] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 149 (hereinafter referred to as "DLL4di-LG21scFv").

[0211] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising DLL1ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising anti-LAMA2 scFv. In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising DLL1 ECD, wherein DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising anti-LAMA2 scFv, wherein anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 150 (hereinafter referred to as "DLL1wt-LG21scFv").

[0212] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising DLL3ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising anti-LAMA2 scFv. In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising DLL3 ECD, wherein DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising anti-LAMA2 scFv, wherein anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 151 (hereinafter referred to as "DLL3wt-LG21scFv").

[0213] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising Jag1ECD or a variant thereof (such as any of the Jag1 ECDs described herein, e.g., any of SEQ ID NO: 132-134); an optional peptide linker (e.g., any of SEQ ID NO: 211, 212, 337, and 338); and a first binding moiety comprising anti-LAMA2 scFv (e.g., SEQ ID NO: 145 or 268).

[0214] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising anti-LAMA2 scFv, wherein anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 152 (hereinafter referred to as "Jag1wt-LG21scFv").

[0215] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising anti-LAMA2 scFv, wherein anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 153 (hereinafter referred to as "Jag1v-LG21scFv").

[0216] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising Jag2ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising anti-LAMA2 scFv. In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising Jag2 ECD, wherein Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising anti-LAMA2 scFv, wherein anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 154 (hereinafter referred to as "Jag2wt-LG21scFv").

[0217] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising DLL4ECD or a variant thereof (such as any of the DLL4 ECD described herein or engineered DLL4 ECD, e.g., any of SEQ ID NO: 126-129 and 260-267); an optional peptide linker (e.g., any of SEQ ID NO: 211, 212, 337 and 338); and a first binding moiety comprising anti-matrix glycan scFv (e.g., SEQ ID NO: 155).

[0218] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising an anti-Matrix-Glycan scFv, wherein the anti-Matrix-Glycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 156 (hereinafter referred to as "DLL4wt-ADG41scFv").

[0219] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising an anti-Matrix-Glycan scFv, wherein the anti-Matrix-Glycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 157 (hereinafter referred to as "DLL4v-ADG41scFv").

[0220] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising an anti-Matrix-Glycan scFv, wherein the anti-Matrix-Glycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 158 (hereinafter referred to as "DLL4max-ADG41scFv").

[0221] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising an anti-Matrix-Glycan scFv, wherein the anti-Matrix-Glycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 159 (hereinafter referred to as "DLL4di-ADG41scFv").

[0222] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising DLL1ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising an anti-Matrix-Glycan scFv. In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising DLL1 ECD, wherein DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-Matrix-Glycan scFv, wherein the anti-Matrix-Glycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 160 (hereinafter referred to as "DLL1wt-ADG41scFv").

[0223] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising DLL3ECD or a variant thereof; an optional peptide linker; and a first binding moiety comprising an anti-Matrix-Glycan scFv. In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising DLL3 ECD, wherein DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-Matrix-Glycan scFv, wherein the anti-Matrix-Glycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 161 (hereinafter referred to as "DLL3wt-ADG41scFv").

[0224] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising Jag1ECD or a variant thereof (such as any of the Jag1 ECDs described herein, e.g., any of SEQ ID NO: 132-134); an optional peptide linker (e.g., any of SEQ ID NO: 211, 212, 337, and 338); and a first binding moiety comprising an anti-macroglycan scFv (e.g., SEQ ID NO: 155).

[0225] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising an anti-macroglycan scFv, wherein the anti-macroglycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 162 (hereinafter referred to as "Jag1wt-ADG41scFv").

[0226] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising an anti-macroglycan scFv, wherein the anti-macroglycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 163 (hereinafter referred to as "Jag1v-ADG41scFv").

[0227] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising Jag2ECD or a variant thereof; an optional peptide linker (e.g., any one of SEQ ID NO: 211, 212, 337, and 338); and a first binding moiety comprising an anti-macroglycan scFv. In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising Jag2 ECD, wherein Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135; an optional peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-macroglycan scFv, wherein the anti-macroglycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 164 (hereinafter referred to as "Jag2wt-ADG41scFv").

[0228] In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises a first binding portion containing anti-LAMA2 Fab (e.g., SEQ ID NO: 165) and a second binding portion containing DLL4 ECD or variants thereof (such as any of the DLL4 ECD described herein or engineered DLL4 ECDs, e.g., any of SEQ ID NO: 126-129 and 260-267), wherein the second binding portion is fused to the N-terminus of the VL of anti-LAMA2 Fab (e.g., SEQ ID NO: 165) via an optional peptide linker (e.g., any of SEQ ID NO: 211, 212, 337 and 338).

[0229] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-LAMA2 Fab and a second binding moiety containing 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 containing the anti-LAMA2 Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 167 (hereinafter referred to as "DLL4wt-LG21-Fab").

[0230] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-LAMA2 Fab and a second binding moiety containing 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: 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 containing the anti-LAMA2 Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 168 (hereinafter referred to as "DLL4v-LG21-Fab").

[0231] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-LAMA2 Fab and a second binding moiety containing 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 containing the anti-LAMA2 Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 169 (hereinafter referred to as "DLL4max-LG21-Fab").

[0232] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-LAMA2 Fab and a second binding moiety containing 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 containing the anti-LAMA2 Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 170 (hereinafter referred to as "DLL4di-LG21-Fab").

[0233] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing anti-LAMA2 Fab and a second binding moiety containing DLL1 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing anti-LAMA2 Fab and a second binding moiety containing DLL1 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of 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 containing the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide containing a second binding moiety and an anti-LAMA2 Fab second polypeptide, wherein the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 171 (hereinafter referred to as "DLL1wt-LG21-Fab").

[0234] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing anti-LAMA2 Fab and a second binding moiety containing DLL3 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing anti-LAMA2 Fab and a second binding moiety containing DLL3 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of 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 containing the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide containing a second binding moiety and an anti-LAMA2 Fab, wherein the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 172 (hereinafter referred to as "DLL3wt-LG21-Fab").

[0235] In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises a first binding moiety containing anti-LAMA2 Fab (e.g., SEQ ID NO: 165) and a second binding moiety containing Jag1 ECD or variants thereof (such as any of the Jag1 ECDs described herein, e.g., any of SEQ ID NO: 132-134), wherein the second binding moiety is fused to the N-terminus of the VL of anti-LAMA2 Fab (e.g., SEQ ID NO: 165) via an optional peptide linker (e.g., any of SEQ ID NO: 211, 212, 337, and 338).

[0236] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-LAMA2 Fab and a second binding moiety containing 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 containing the anti-LAMA2 Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 173 (hereinafter referred to as "Jag1wt-LG21-Fab").

[0237] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-LAMA2 Fab and a second binding moiety containing 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 containing the anti-LAMA2 Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 174 (hereinafter referred to as "Jag1v-LG21-Fab").

[0238] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing anti-LAMA2 Fab and a second binding moiety containing Jag2 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing anti-LAMA2 Fab and a second binding moiety containing Jag2 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135, and wherein 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 containing the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide containing a second binding moiety and an anti-LAMA2 Fab second polypeptide, wherein the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 175 (hereinafter referred to as "Jag2wt-LG21-Fab").

[0239] In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises a first binding portion containing an anti-macroglycan Fab (e.g., SEQ ID NO: 176) and a second binding portion containing a DLL4 ECD or a variant thereof (such as the DLL4 ECD described herein or any engineered DLL4 ECD, such as any one of SEQ ID NO: 126-129 and 260-267), wherein the second binding portion is fused to the N-terminus of the VL of the anti-macroglycan Fab (e.g., SEQ ID NO: 176) via an optional peptide linker (e.g., any one of SEQ ID NO: 211, 212, 337 and 338).

[0240] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-Matrix-glycan Fab and a second binding moiety containing a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-Matrix-glycan 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-Matrix-glycan 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 fusion polypeptide comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the second binding moiety and the second polypeptide of the anti-Matrix-glycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 178 (hereinafter referred to as "DLL4wt-ADG41-Fab").

[0241] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-Matrix-glycan Fab and a second binding moiety containing a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-Matrix-glycan 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-Matrix-glycan 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 fusion polypeptide comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a second polypeptide comprising the second binding moiety and the second polypeptide of the anti-Matrix-glycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 179 (hereinafter referred to as "DLL4v-ADG41-Fab").

[0242] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-Matrix-glycan Fab and a second binding moiety containing a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-Matrix-glycan 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-Matrix-glycan 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 fusion polypeptide comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the second binding moiety and the second polypeptide of the anti-Matrix-glycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 180 (hereinafter referred to as "DLL4max-ADG41-Fab").

[0243] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-Matrix-glycan Fab and a second binding moiety containing a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-Matrix-glycan 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-Matrix-glycan 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 fusion polypeptide comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the second binding moiety and the second polypeptide of the anti-Matrix-glycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 181 (hereinafter referred to as "DLL4di-ADG41-Fab").

[0244] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-macroglycan Fab and a second binding moiety containing a DLL1 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-macroglycan Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-macroglycan Fab and a second binding moiety containing a DLL1 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-macroglycan 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-macroglycan 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 containing the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide containing a second binding moiety and an anti-matrix glycan Fab, wherein the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 182 (hereinafter referred to as "DLL1wt-ADG41-Fab").

[0245] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-macroglycan Fab and a second binding moiety containing DLL3 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-macroglycan Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-macroglycan Fab and a second binding moiety containing DLL3 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-macroglycan 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-macroglycan 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 containing the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide containing a second binding moiety and an anti-matrix glycan Fab, wherein the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 183 (hereinafter referred to as "DLL3wt-ADG41-Fab").

[0246] In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises a first binding portion containing an anti-macroglycan Fab (e.g., SEQ ID NO: 176) and a second binding portion containing a Jag1 ECD or a variant thereof (such as any of the Jag1 ECDs described herein, e.g., any of SEQ ID NO: 132-134), wherein the second binding portion is fused to the N-terminus of the VL of the anti-macroglycan Fab (e.g., SEQ ID NO: 176) via an optional peptide linker (e.g., any of SEQ ID NO: 211, 212, 337, and 338).

[0247] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-macroglycan Fab and a second binding moiety containing a variant Jag1 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-macroglycan 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-macroglycan 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 fusion polypeptide comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the second binding moiety and the second polypeptide of the anti-macroglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 184 (hereinafter referred to as "Jag1wt-ADG41-Fab").

[0248] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-macroglycan Fab and a second binding moiety containing a variant Jag1 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-macroglycan 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-macroglycan 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 comprising the anti-macroglycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 185 (hereinafter referred to as "Jag1v-ADG41-Fab").

[0249] In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-macroglycan Fab and a second binding moiety containing Jag2 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-macroglycan Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, comprising a first binding moiety containing an anti-macroglycan Fab and a second binding moiety containing Jag2 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-macroglycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135, and wherein the anti-macroglycan 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 containing the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide containing a second binding moiety and an anti-matrix glycan Fab, wherein the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 186 (hereinafter referred to as "Jag2wt-ADG41-Fab").

[0250] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion comprising DLL4ECD or a variant thereof (e.g., SEQ ID NO: 339); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding portion comprising a laminin-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0251] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion 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 referred to as "DLL4(EGF1-6)-GS linker-laminin-a2 LG4-5").

[0252] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion comprising DLL4ECD 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 portion comprising a laminin-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0253] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion 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 referred to as "DLL4(EGF1-6)-HSA-laminin-a2 LG4-5").

[0254] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising DLL1ECD 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-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0255] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion 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 referred to as "DLL1(EGF1-6)-HSA-laminin-a2 LG4-5").

[0256] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion comprising Jag1ECD or a variant thereof (e.g., SEQ ID NO: 341); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding portion comprising a laminin-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0257] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion 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 referred to as "Jagged1(EGF1-6)-Laminin-a2 LG4-5").

[0258] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising Jag1ECD 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-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0259] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising a laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 326 (hereinafter referred to as "Jagged1(EGF1-6)-HSA-laminin-a2 LG4-5").

[0260] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion comprising Jag2ECD or a variant thereof (e.g., SEQ ID NO: 342); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding portion comprising a laminin-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0261] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion 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: 328 (hereinafter referred to as "Jagged2(EGF1-6)-Laminin-a2 LG4-5").

[0262] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding moiety comprising Jag2ECD 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-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0263] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion comprising a laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 329 (hereinafter referred to as "Jagged2(EGF1-6)-HSA-laminin-a2 LG4-5").

[0264] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion comprising DLL1ECD or a variant thereof (e.g., SEQ ID NO: 340); an optional peptide linker (e.g., SEQ ID NO: 337); and a first binding portion comprising a laminin-α2 LG4-5 domain (e.g., SEQ ID NO: 343).

[0265] In some embodiments, a bifunctional protein construct is provided comprising, from N' to C': a second binding portion 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 portion 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 referred to as "DLL1(EGF1-6)-Laminin-a2 LG4-5").

[0266] 2. Fc domain fusion In some embodiments, the bifunctional protein construct includes an Fc domain containing a first subunit and a second subunit. The Fc domain may be a third portion connected to the first and second binding portions. In some embodiments, when the first or second binding portion is a full-length antibody, the Fc domain may be part of the full-length antibody.

[0267] In some implementations, the Fc domain is a variant Fc domain with minimal or no effector function. In some implementations, the variant Fc domain is derived from IgG1 Fc or IgG4 Fc.

[0268] One or more amino acid modifications can be introduced into the Fc domain to generate Fc domain variants. Fc domain variants can contain human Fc domain sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc domains) with amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0269] In some embodiments, the Fc domain possesses some, but not all, effector functions that make the Fc fragment a desirable candidate for applications where the in vivo half-life of the antibody moiety is important, and certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In some embodiments, the Fc domain contains one or more mutations that extend the in vivo half-life of the antibody moiety. In some embodiments, the variant Fc domain has increased FcRn binding at pH 6.0. In some embodiments, the variant Fc domain contains the M252Y / S254T / T256E (“YTE”) mutation (EU number). See, for example, Dall'Acqua et al. J Immunol ., 169:5171–5180 (2002) and Wang et al., Protein & Cell , 9(1):63-73 (2018). In some implementations, the variant Fc domain contains the M428L / N434S mutation. See, for example, Zalevsky et al., Nat Biotechnol ., 28:157–159 (2010) and Wang et al., Protein & Cell , 9(1):63-73 (2018).

[0270] In vitro and / or in vivo cytotoxicity assays can be performed to analyze the CDC and / or ADCC activity of the Fc domain. For example, an Fc receptor (FcR) binding assay can be performed to determine whether the antibody has FcgR binding (and therefore potentially ADCC activity) and / or retains FcRn binding capacity. The main cells mediating ADCC (NK cells) express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet. Annu. Rev. Immunol. Table 2 on page 464 of 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al.). Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (See Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and CytoTox 96). ® Non-radioactive cytotoxicity assays (Promega, Madison, WI). Effector cells useful for this type of assay include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, assays can be performed in vivo, for example in animal models such as those used by Clynes et al. Proc. Nat'l Acad. Sci. USA The ADCC activity of the molecule of interest was evaluated in animal models disclosed in 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example, Petkova, SB, et al.). Int'l. Immunol. 18(12):1759-1769 (2006)).

[0271] Antibodies with reduced effector function include those with substitutions of one or more of the Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include variant Fc variants with substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc variants that substitute residues 265 and 297 for alanine (US Patent No. 7,332,581). In some embodiments, the Fc domain of the bifunctional protein construct contains one or more of these mutations. In some embodiments, the variant Fc domain contains the N297A, N297Q, or N297G mutation. See, for example, Bolt et al. Eur J Immunol ., 23:403–411 (1993); Leabman et al., Mabs , 5:896–903 (2013); Tao and Morrison, J Immunol ., 143:2595–2601 (1989); Walker et al., Biochem J. , 259:347–353 (1989); and Wang et al., Protein & Cell, 9(1):63-73 (2018). In some implementations, the variant Fc domain contains the L235E mutation. See, for example, Alegre et al., J Immunol ., 148:3461–3468 (1992) and Wang et al., Protein & Cell , 9(1):63-73 (2018).

[0272] Describe certain antibody variants that have improved or reduced binding to FcR. See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312 and Shields et al. , J. Biol. Chem. 9(2): 6591-6604 (2001).

[0273] In some embodiments, the Fc domain is derived from human IgG1, human IgG2, or human IgG4. In some embodiments, the Fc domain is derived from the IgG1 Fc domain (e.g., the human IgG1 Fc domain). In some embodiments, the human IgG1 Fc contains the amino acid sequence of SEQ ID NO: 187. In some embodiments, each subunit of the Fc domain contains a mutation that reduces or eliminates effector function. In some embodiments, effector function is eliminated by a mutation that eliminates glycosylation in the constant region (e.g., an "effector-less mutation"). In one aspect, the effector-less mutation is an N297A or DANA mutation (D265A+N297A) (EU number) in the CH2 region. See, for example, Shields et al. J. Biol. Chem 276 (9): 6591-6604 (2001). Alternatively or additionally, effector function can be reduced or eliminated by production techniques such as expression in host cells that do not undergo glycosylation (e.g., *E. coli*) or in host cells that result in glycosylation patterns that are ineffective or less effective in promoting effector function (e.g., Shinkawa et al.). J. Biol. Chem278(5): 3466-3473 (2003). In some embodiments, each subunit of the Fc domain contains the L234A / L235A “LALA” mutation (EU number). In some embodiments, each subunit of the Fc domain contains the L234A / L235A / P329G “LALALPG” mutation (EU number). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 277. In some embodiments, the Fc domain contains mutations that shorten the half-life of the bifunctional protein construct in circulation, such as the H435A mutation (EU number). In some embodiments, each subunit of the Fc domain (e.g., human IgG1 Fc) contains the L234A / L235A and H435A mutations. In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 188. In some embodiments, the Fc domain is the IgG4 (e.g., human IgG4) Fc domain. In some embodiments, the Fc domain contains mutations that reduce Fab exchange, such as the S228P mutation. In some embodiments, the IgG4 Fc domain contains the F234A / L235A mutation. See, for example, Xu et al. Cell Immunol ., 200:16–26 (2000) and Wang et al., Protein & Cell , 9(1):63-73 (2018). See also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351, which relates to other instances of variants of the Fc domain.

[0274] In some implementations, when the bifunctional protein construct is a heterodimeric protein comprising two distinct polypeptide chains, the Fc domain may contain one or more mutations that promote the assembly of the heterodimeric protein. For example, the mortar and pestle structure is a heterodimerization technique for the CH3 domain of antibodies. Previously, the mortar and pestle structure technique has been used to generate full-length human 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 incorporated herein by reference in its entirety for all purposes. All of these mutations can be incorporated into the bifunctional protein constructs described herein.

[0275] Therefore, for example, in some embodiments, the bifunctional protein construct comprises an Fc domain containing a first CH3 domain and a second CH3 domain having mortar and pestle (KIH) residues. In some embodiments, the first CH3 domain is modified such that within the CH3 / CH3 interface, one or more amino acid residues are replaced by one or more amino acid residues with a larger side chain volume, thereby generating a protrusion on the surface of the first CH3 domain that interacts with the second CH3 domain; and the second CH3 domain is modified such that within the CH3 / CH3 interface, one or more amino acid residues are replaced by amino acid residues with 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 protrusion is a mortar. In some embodiments, the modification used to generate the mortar is T366W. In some embodiments, the cavity is a mortar. In some embodiments, the modification used to generate the mortar is T366S / L368A / Y407V. Therefore, in some embodiments, the Fc domain described herein contains a club-and-mortar structure mutation, wherein: i) the first subunit of the Fc domain contains a club mutation, and the second subunit of the Fc domain contains a mortar mutation; or ii) the second subunit of the Fc domain contains a club mutation, and the first subunit of the Fc domain contains a mortar mutation. In some embodiments, the club mutation is T366W (EU number), and the mortar mutation is T366S / L368A / Y407V (EU number). Unless otherwise indicated, all amino acid positions within the Fc domain are numbered according to the EU numbering system.

[0276] In some embodiments, the bifunctional protein construct includes an Fc domain containing a mutation that allows for the purification of the asymmetric Fc domain. In some embodiments, the first subunit or the second subunit of the Fc domain contains an H435R / Y436F mutation (EU number). In some embodiments, each subunit of the Fc domain contains an H435R / Y436F mutation (EU number).

[0277] Strop et al. (Rinat-Pfizer Inc.) described a method for producing stable bispecific antibodies by separately expressing and purifying two antibodies of interest, and then mixing them together under specific redox conditions (J. Mol. Biol. (2012) 420:204-19).

[0278] Other heterodimerizing domains, which have a strong preference for forming heterodimers compared to homodimers, can be incorporated into the bifunctional protein constructs of this invention. Illustrative examples include, but are not limited to, WO2007147901 (Kjærgaard et al. – Novo Nordisk: describing ion interactions); WO2009 / 089004 (Kannan et al. – Amgen: describing electrostatic redirection effects); WO2010 / 034605 (Christensen et al. – Genentech: describing coiled helices). See also, for example, Pack, P. and Plueckthun, A., Biochemistry 31, 1579-1584 (1992) describing leucine zippers or Pack et al., Bio / Technology 11, 1271-1277 (1993) describing helical-turn-helical motifs. The term “heterodimerizing domain” does not exclude additional units besides the two heterodimers in the bifunctional construct. In some implementations, the bifunctional protein construct includes one or more heterodimerization domains.

[0279] In some embodiments, when the bifunctional protein construct is a heterodimeric protein comprising two distinct polypeptide chains, the Fc domain may contain one or more charged pair mutations that promote heterodimeric protein assembly. In some embodiments, amino acid residues in the first subunit of the Fc domain are substituted with positively charged residues (e.g., R, H, or K), and amino acid residues in the second subunit of the Fc domain are substituted with negatively charged residues (e.g., D or E). In some embodiments, amino acid residues in the first subunit of the Fc domain are substituted with negatively charged residues, and amino acid residues in the second subunit of the Fc domain are substituted with positively charged residues. In some embodiments, the amino acid residue at D399 (EU number) in the first subunit of the Fc domain is substituted with a positively charged residue, and the amino acid residue at K409 (EU number) in the second subunit of the Fc domain is substituted with a negatively charged residue. In some implementations, the amino acid residue at K409 (EU number) in the first subunit of the Fc domain is replaced by a negatively charged residue, and the amino acid residue at D399 (EU number) in the second subunit of the Fc domain is replaced by a positively charged residue.

[0280] In some embodiments, the Fc domain used in the bifunctional protein constructs described herein comprises the amino acid sequence of any one of SEQ ID NO: 188, 254, and 277.

[0281] In some embodiments, a bifunctional protein construct is provided comprising: i) one or more units of a first binding portion specifically binding to a muscle-specific molecule; ii) a first unit of a second binding portion specifically binding to a first Notch receptor and activating the first Notch receptor; iii) a second unit of a second binding portion 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 portion and the second unit of the second binding portion each independently comprise an ECD 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 NO: 126-129 and 260-267). In some embodiments, the first unit of the second binding portion and the second unit of the second binding portion are identical. In some embodiments, the first unit of the second binding portion is different from the second unit of the second binding portion. One or more units of the first binding portion may be identical or different; and / or may 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 portion specifically binding to a third Notch receptor and a fourth unit of a second binding portion specifically binding to a fourth Notch receptor. In some embodiments, the third unit of the second binding portion and the fourth unit of the second binding portion each independently comprise an ECD 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 units of the second binding portion may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, all four units of the second binding portion are the same. In some embodiments, at least one of the four units of the second binding portion is different from the other units. One or more units of the first binding portion may comprise an antibody portion or a non-antibody portion. The various binding portions may be independently fused to the N and / or C-terminus of the Fc domain. The bifunctional protein construct may be homodimer or heterodimer.

[0282] In some embodiments, a bifunctional protein construct is provided, comprising: i) a first unit that specifically binds to a first binding portion of a first muscle-specific molecule; ii) a second unit that specifically binds to a first binding portion of a second muscle-specific molecule; iii) one or more units that specifically bind to and activate a Notch receptor; and iv) an Fc domain comprising a first subunit and a second subunit. In some embodiments, a bifunctional protein construct is provided, comprising: i) a first unit of a first binding portion comprising a first antibody portion that specifically binds to a first muscle-specific molecule; ii) a second unit of the first binding portion comprising a second antibody portion that specifically binds to a second muscle-specific molecule; iii) one or more units that specifically bind to and activate a Notch receptor; and iv) an Fc domain comprising a first subunit and a second subunit; wherein the first antibody portion and the second antibody portion are each independently selected from the group consisting of Fab, scFv, and sdAb. In some embodiments, a bifunctional protein construct is provided, comprising: i) a first unit of a first binding portion comprising a first non-antibody portion specifically binding to a first muscle-specific molecule; ii) a second unit of the first binding portion comprising a second non-antibody portion specifically binding to a second muscle-specific molecule; iii) one or more units of a second binding portion specifically binding to and activating a Notch receptor; and iv) an Fc domain comprising a first subunit and a second subunit; wherein the first and second non-antibody portions are each independently selected from the group consisting of: the LG domain of laminin, the LG domain of aggregates, the LG domain of nestin, and the LG domain of bead-glycans. In some embodiments, the first and / or second non-antibody portions comprise LAMA2 LG4-5 containing the amino acid sequence SEQ ID NO: 118 or 119. In some embodiments, the first unit of the first binding portion comprises two or more tandemly linked first non-antibody portions, and the second unit of the first binding portion comprises two or more tandemly linked second non-antibody portions. In some embodiments, the first unit and the second unit of the first binding portion are identical. In some embodiments, the first unit of the first binding portion is different from the second unit of the first binding portion. One or more units of the second binding portion may be identical or different; and / or may bind to the same or different Notch receptors. Various binding portions may independently fuse to the N and / or C-terminus of the Fc domain. The bifunctional protein construct may be homodimer or heterodimer.

[0283] In some embodiments, one or more units of the first binding portion in the bifunctional protein construct include (or) Fab, wherein Fab may be attached to the C-terminus of the subunit of the Fc domain via VH or VL, such as through an optional adapter (e.g., any of SEQ ID NO: 211, 212, 337 and 338). For example, in some embodiments, a bifunctional protein construct is provided 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 VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit specifically binding to a first Notch receptor and activating a second binding portion of the first Notch receptor; a second unit specifically binding to a second Notch receptor and activating a second binding portion of 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': a first unit of the second binding portion – optional linker – a first subunit of the Fc domain – optional linker – VH1-(H1-CH1); ii) a second polypeptide comprising, from N' to C': a second unit of the second binding portion – optional linker – a 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, a bifunctional protein construct is provided 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 VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit specifically binding to a first Notch receptor and activating a second binding portion of the first Notch receptor; a second unit specifically binding to a second Notch receptor and activating a second binding portion of 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': a first unit of the second binding portion – optional linker – a first subunit of the Fc domain – optional linker – ii) a second polypeptide comprising, from N' to C': a second unit of the second binding moiety – optional linker – a 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 that may be the same or different in the bifunctional protein construct (e.g., any one of SEQ ID NO: 211, 212, 337, and 338). In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. Fab1 and Fab2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, both Fab1 and Fab2 specifically bind to matrix glycan Fab. In some embodiments, anti-matrix glycan Fab1 and / or anti-matrix glycan Fab2 comprise a first polypeptide containing the amino acid sequence of SEQ ID NO: 176 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 177. In some embodiments, both Fab1 and Fab2 specifically bind to LAMA2. In some embodiments, anti-LAMA2 Fab1 and / or anti-LAMA2 Fab2 comprise a first polypeptide containing the amino acid sequence of SEQ ID NO: 165 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 166.The first unit of the second binding portion and the second unit of the second binding portion may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding portion (one or both units) comprises an ECD of a Notch ligand selected from the group consisting of delta-sample 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NO: 126-129 and 260-267). In some embodiments, the second binding portion (one or both units) comprises the amino acid sequence of any of SEQ ID NO: 126-135 and 260-267.

[0284] In some embodiments, one or more units of the first binding portion comprise (or) scFv, wherein the scFv may be attached 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 NO: 211, 212, 337, and 338). For example, in some embodiments, a bifunctional protein construct is provided comprising: a first scFv (scFv1) specifically binding to a first muscle-specific molecule comprising VH (VH1) and VL (VL1); and specifically binding to a subunit comprising VH (VH2) and VL (VL1). The second scFv (scFv2) of the second muscle-specific molecule of (VL2); a first unit of the second binding moiety of the first Notch receptor specifically binding to and activating the first Notch receptor; a second unit of the second binding moiety of the second Notch receptor specifically binding to 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': a first unit of the second binding moiety – optional linker – a 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': a second unit of the second binding moiety – optional linker – a 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 that may be the same or different in the bifunctional protein construct (e.g., any one of SEQ ID NO: 211, 212, 337, and 338). In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. scFv1 and scFv2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, both scFv1 and scFv2 specifically bind to matrix glycans. In some embodiments, anti-matrix glycan scFv1 and / or anti-matrix glycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, both scFv1 and scFv2 specifically bind to LAMA2. In some embodiments, anti-LAMA2 scFv1 and / or anti-LAMA2 scFv2 comprises the amino acid sequence of SEQ ID NO: 145 or 268. The first unit of the second binding portion and the second unit of the second binding portion may be the same or different; and / or may bind to the same or different Notch receptors.In some embodiments, the second binding portion (one or two units) comprises an ECD of a Notch ligand selected from the group consisting of δ-sample 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NO: 126-129 and 260-267). In some embodiments, the second binding portion (one or two units) comprises an amino acid sequence of any of SEQ ID NO: 126-135 and 260-267.

[0285] In some embodiments, one or more units of the first binding portion include (or) sdAb, wherein the sdAb may be connected to the C-terminus of the subunit of the Fc domain, such as via an optional connector (e.g., any of SEQ ID NO: 211, 212, 337 and 338). For example, in some embodiments, a bifunctional protein construct is provided 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 specifically binding to a first Notch receptor and activating a second binding portion of the first Notch receptor; a second unit specifically binding to a second Notch receptor and activating a second binding portion of 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': a first unit of the second binding portion – optional linker – a first subunit of the Fc domain – optional linker – sdAb1; ii) a second polypeptide comprising, from N' to C': a second unit of the second binding portion – optional linker – a second subunit of the Fc domain – optional linker – sdAb2. In some embodiments, the optional linker is a peptide linker that may be the same or different in the bifunctional protein construct (e.g., any one of SEQ ID NO: 211, 212, 337, and 338). In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. sdAb1 and sdAb2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. The first unit of the second binding portion and the second unit of the second binding portion may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding portion (one or both units) comprises an ECD of a Notch ligand selected from the group consisting of δ-sample 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any one of the engineered DLL4ECDs described herein (e.g., any one of SEQ ID NO: 126-129 and 260-267). In some embodiments, the second binding portion (one or two units) comprises the amino acid sequence of either SEQ ID NO: 126-135 or 260-267.

[0286] In some embodiments, one or more units of the first binding portion comprise (or) Fab, wherein Fab may be connected to the N-terminus of the subunit of the Fc domain via CH1 or CL, such as through an optional connector (e.g., any of SEQ ID NO: 211, 212, 337 and 338). In some embodiments, a bifunctional protein construct is provided 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 VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit specifically binding to a first Notch receptor and activating a second binding portion of the first Notch receptor; a second unit specifically binding to a second Notch receptor and activating a second binding portion of 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 – first subunit of the Fc domain – optional linker – second binding portion; ii) a second polypeptide comprising, from N' to C': VH2-(H2-CH1) –Optional linker – Second subunit of Fc domain –Optional linker – Second unit of second binding portion; iii) Third polypeptide comprising from N' to C': VL1-(L1-CL); and iv) 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, a bifunctional protein construct is provided 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 VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit specifically binding to a first Notch receptor and activating a second binding portion of the first Notch receptor; a second unit specifically binding to a second Notch receptor and activating a second binding portion of 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 – first subunit of the Fc domain – optional linker – second binding portion; ii) a second polypeptide comprising, from N' to C': VL2-(L2-CL) –Optional linker – Second subunit of the Fc domain –Optional linker – Second unit that specifically binds to the second binding portion of the 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 that may be the same or different in the bifunctional protein construct (e.g., any one of SEQ ID NO: 211, 212, 337, and 338). In some embodiments, the first muscle-specific molecule and the second muscle-specific molecule are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. Fab1 and Fab2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, both Fab1 and Fab2 specifically bind to matrix glycan Fab. In some embodiments, anti-matrix glycan Fab1 and / or anti-matrix glycan Fab2 comprise a first polypeptide containing the amino acid sequence of SEQ ID NO: 176 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 177. In some embodiments, both Fab1 and Fab2 specifically bind to LAMA2. In some embodiments, anti-LAMA2 Fab1 and / or anti-LAMA2 Fab2 comprise a first polypeptide containing the amino acid sequence of SEQ ID NO: 165 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 166.The first unit of the second binding portion and the second unit of the second binding portion may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding portion (one or both units) comprises an ECD of a Notch ligand selected from the group consisting of delta-sample 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NO: 126-129 and 260-267). In some embodiments, the second binding portion (one or both units) comprises the amino acid sequence of any of SEQ ID NO: 126-135 and 260-267.

[0287] In some embodiments, a bifunctional protein construct is provided comprising: i) a first binding portion comprising a full-length antibody that specifically binds to a muscle-specific molecule, wherein the full-length antibody comprises an Fc domain containing a first subunit and a second subunit; ii) a first unit of a second binding portion 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 the second binding portion 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 portion specifically binds to and activates a first Notch receptor, and the second unit of the second binding portion specifically binds to and activates a second Notch receptor. In some embodiments, the optional linker is a peptide linker that may be the same or different within the bifunctional protein construct (e.g., any one of SEQ ID NO: 211, 212, 337, and 338). In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. The first unit of the second binding portion and the second unit of the second binding portion may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding portion (one or both units) comprises an ECD of a Notch ligand selected from the group consisting of delta-sample 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NO: 126-129 and 260-267). In some embodiments, the second binding portion (one or both units) comprises the amino acid sequence of any of SEQ ID NO: 126-135 and 260-267.

[0288] In some embodiments, one or more units of the first binding portion comprise (or) scFv, wherein the scFv may be attached 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 NO: 211, 212, 337, and 338). In some embodiments, a bifunctional protein construct is provided 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 VH (VH2) and VL (VL2); a first unit specifically binding to a second binding portion of a first Notch receptor and activating the first Notch receptor; a second unit specifically binding to a second Notch receptor and activating the second binding portion of 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 – first subunit of the Fc domain – optional linker – 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 – second subunit of the Fc domain – optional linker – second unit of the second binding moiety. In some embodiments, the optional linker is a peptide linker that may be the same or different in the bifunctional protein construct (e.g., any one of SEQ ID NO: 211, 212, 337, and 338). In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. scFv1 and scFv2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, both scFv1 and scFv2 specifically bind to matrix glycans. In some embodiments, anti-matrix glycan scFv1 and / or anti-matrix glycan scFv2 comprise the amino acid sequence of SEQ ID NO: 155. In some embodiments, both scFv1 and scFv2 specifically bind to LAMA2. In some embodiments, anti-LAMA2 scFv1 and / or anti-LAMA2 scFv2 comprise the amino acid sequence of SEQ ID NO: 145 or 268. The first unit of the second binding portion and the second unit of the second binding portion may be the same or different; and / or may bind to the same or different Notch receptors.In some embodiments, the second binding portion (one or two units) comprises an ECD of a Notch ligand selected from the group consisting of δ-sample 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NO: 126-129 and 260-267). In some embodiments, the second binding portion (one or two units) comprises an amino acid sequence of any of SEQ ID NO: 126-135 and 260-267.

[0289] In some embodiments, one or more units of the first binding portion include (or) sdAb, wherein the sdAb may be connected to the N-terminus of the subunit of the Fc domain, such as via an optional connector (e.g., any of SEQ ID NO: 211, 212, 337 and 338). In some embodiments, a bifunctional protein construct is provided 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 specifically binding to a first Notch receptor and activating a second binding portion of the first Notch receptor; a second unit specifically binding to a second Notch receptor and activating a second binding portion of 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 – first subunit of the Fc domain – optional linker – first unit of the second binding portion; and ii) a second polypeptide comprising, from N' to C': sdAb2 – optional linker – second subunit of the Fc domain – optional linker – second unit of the second binding portion. In some embodiments, the optional linker is a peptide linker that may be the same or different in the bifunctional protein construct (e.g., any one of SEQ ID NO: 211, 212, 337, and 338). In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. sdAb1 and sdAb2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. The first unit of the second binding portion and the second unit of the second binding portion may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding portion (one or both units) comprises an ECD of a Notch ligand selected from the group consisting of δ-sample 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any one of the engineered DLL4ECDs described herein (e.g., any one of SEQ ID NO: 126-129 and 260-267). In some embodiments, the second binding portion (one or two units) comprises the amino acid sequence of either SEQ ID NO: 126-135 or 260-267.

[0290] In some embodiments, a bifunctional protein construct is provided 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 VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit specifically binding to a first Notch receptor and activating a second binding portion of the first Notch receptor; a second unit specifically binding to a second Notch receptor and activating a second binding portion of 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': a first subunit of a VH1-(H1-CH1) – optionally linker-Fc domain; ii) a second polypeptide comprising, from N' to C': VH2-(H2-CH1) domain. – Optional linker – second subunit of the Fc domain; iii) a third polypeptide comprising, from N' to C': VL1-(L1-CL) – optional linker – first unit of the second binding moiety; and iv) a fourth polypeptide comprising, from N' to C': VL2-(L2-CL) – optional linker – 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 that may be the same or different in the bifunctional protein construct (e.g., any one of SEQ ID NO: 211, 212, 337, and 338). In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. Fab1 and Fab2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, both Fab1 and Fab2 specifically bind to matrix glycan Fab. In some embodiments, anti-matrix glycan Fab1 and / or anti-matrix glycan Fab2 comprise a first polypeptide containing the amino acid sequence of SEQ ID NO: 176 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 177. In some embodiments, both Fab1 and Fab2 specifically bind to LAMA2. In some embodiments, anti-LAMA2 Fab1 and / or anti-LAMA2 Fab2 comprise a first polypeptide containing the amino acid sequence of SEQ ID NO: 165 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 166.The first unit of the second binding portion and the second unit of the second binding portion may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding portion (one or both units) comprises an ECD of a Notch ligand selected from the group consisting of delta-sample 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NO: 126-129 and 260-267). In some embodiments, the second binding portion (one or both units) comprises the amino acid sequence of any of SEQ ID NO: 126-135 and 260-267.

[0291] In some embodiments, a bifunctional protein construct is provided comprising: i) a first binding portion comprising a full-length antibody that specifically binds to a muscle-specific molecule; ii) a first unit of a second binding portion fused (directly or via an optional linker) to the C-terminus of a first light chain of the full-length antibody; and iii) a second unit of a second binding portion 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 portion specifically binds to and activates a first Notch receptor, and the second unit of the second binding portion specifically binds to and activates a second Notch receptor. In some embodiments, the optional linker is a peptide linker (e.g., any one of SEQ ID NO: 211, 212, 337, and 338) that may be the same or different within the bifunctional protein construct. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. The first unit of the second binding portion and the second unit of the second binding portion may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding portion (one or two units) comprises an ECD of a Notch ligand selected from the group consisting of δ-sample 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NO: 126-129 and 260-267). In some embodiments, the second binding portion (one or two units) comprises an amino acid sequence of any of SEQ ID NO: 126-135 and 260-267.

[0292] In some embodiments, a bifunctional protein construct is provided 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 VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit specifically binding to a first Notch receptor and activating a second binding portion of the first Notch receptor; a second unit specifically binding to a second Notch receptor and activating a second binding portion of 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': a first subunit of a VH1-(H1-CH1) – optionally linker-Fc domain; ii) a second polypeptide comprising, from N' to C': VH2-(H2-CH1) domain. – Optional linker – second subunit of the Fc domain; iii) a third polypeptide comprising, from N' to C': a first unit of the second binding moiety – optional linker – VL1-(L1-CL); and iv) a fourth polypeptide comprising, from N' to C': a 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 that may be the same or different in the bifunctional protein construct (e.g., any one of SEQ ID NO: 211, 212, 337, and 338). In some embodiments, the first and second muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. Fab1 and Fab2 may be the same or different; and / or may bind to the same or different muscle-specific molecules. In some embodiments, both Fab1 and Fab2 specifically bind to matrix glycan Fab. In some embodiments, anti-matrix glycan Fab1 and / or anti-matrix glycan Fab2 comprise a first polypeptide containing the amino acid sequence of SEQ ID NO: 176 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 177. In some embodiments, both Fab1 and Fab2 specifically bind to LAMA2. In some embodiments, anti-LAMA2 Fab1 and / or anti-LAMA2 Fab2 comprise a first polypeptide containing the amino acid sequence of SEQ ID NO: 165 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 166.The first unit of the second binding portion and the second unit of the second binding portion may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding portion (one or both units) comprises an ECD of a Notch ligand selected from the group consisting of delta-sample 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NO: 126-129 and 260-267). In some embodiments, the second binding portion (one or both units) comprises the amino acid sequence of any of SEQ ID NO: 126-135 and 260-267.

[0293] In some embodiments, a bifunctional protein construct is provided, comprising: a first unit comprising a first binding portion of a full-length antibody specifically binding to a muscle-specific molecule, a first unit fused (directly or via an optional linker) to the N-terminus of a second binding portion of a first light chain of the full-length antibody, and a second unit fused (directly or via an optional linker) to the N-terminus of a second light chain of the full-length antibody; wherein the first unit of the second binding portion specifically binds to and activates a first Notch receptor, and the second unit of the second binding portion specifically binds to and activates a second Notch receptor. In some embodiments, the optional linker is a peptide linker (e.g., any one of SEQ ID NO: 211, 212, 337, and 338) that may be the same or different within the bifunctional protein construct. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. The first unit of the second binding portion and the second unit of the second binding portion may be the same or different; and / or may bind to the same or different Notch receptors. In some embodiments, the second binding portion (one or two units) comprises an ECD of a Notch ligand selected from the group consisting of δ-sample 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NO: 126-129 and 260-267). In some embodiments, the second binding portion (one or two units) comprises an amino acid sequence of any of SEQ ID NO: 126-135 and 260-267.

[0294] In some embodiments, a bifunctional protein construct is provided 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 VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit specifically binding to a first Notch receptor and activating a second binding portion of the first Notch receptor; a second unit specifically binding to a second Notch receptor and activating a second binding portion of 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': a first unit of the second binding portion – optional linker – VH1-(H1-CH1) – optional linker – a first subunit of the Fc domain; ii) a second polypeptide comprising, from N' to C': a second unit of the second binding portion – optional linker – VH2-(H2-CH1) – optional linker – second subunit of the Fc do...

Claims

1. A bifunctional protein construct comprising a first binding portion and a second binding portion, wherein the first binding portion specifically binds to a muscle-specific molecule, and wherein the second binding portion 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 basement membrane, or in the basement membrane.

3. The bifunctional protein construct of claim 2, wherein the target antigen is selected from the group consisting of: laminin, aggregates, nestin, lecithin, 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-dystrophy proteoglycan (α-DG), β-DG, laminin-211, lecithin, collagen, α-caryogam, β-caryogam, γ-caryogam, δ-caryogam, ε-caryogam, ζ-caryogam, disaccharide proteoglycan, myosin, and matrix glycans on α-DG.

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

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

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

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

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

6.

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

50.

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

12.

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

65.

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

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

85.

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

95.

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

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

107.

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

109.

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

21. The bifunctional protein construct of claim 20, wherein the non-antibody portion comprises a protein domain selected from the group consisting of: laminin G-like domain (LG domain) of laminin, LG domain of aggregate protein, LG domain of nestin, LG domain of globin, laminin coil-coil binding domain of aggregate protein, and laminin γ binding domain of nestin.

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

23. The bifunctional protein construct of claim 22, wherein the non-antibody portion comprises the LG4-5 domain 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 to 24, wherein the second binding portion comprises an extracellular domain (ECD) of a Notch ligand selected from the group consisting of: δ-sample 1 (DLL1), DLL3, DLL4, Jagged1 (Jag1), and Jag2.

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

135.

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

28. The bifunctional protein construct of claim 27, wherein: a) The second binding portion comprises a variant DLL4 ECD containing the amino acid sequence of any one of SEQ ID NO: 126-129 and 260-267; b) The second binding portion comprises a DLL1 ECD containing the amino acid sequence of SEQ ID NO: 130; c) The second binding portion comprises DLL3 containing the amino acid sequence of SEQ ID NO: 131; d) The second binding portion comprises a variant Jag1 ECD containing the amino acid sequence of SEQ ID NO: 133 or 134; or e) wherein the second binding portion comprises a Jag2 ECD containing 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 one of SEQ ID NO: 136-144.

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

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

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

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

34. The bifunctional protein construct of claim 33, wherein the mortar mutation is T366W (EU number) and the acetabular mutation is T366S / L368A / Y407V (EU number).

35. The bifunctional protein construct according to any one of claims 32 to 34, wherein: a) The bifunctional protein construct comprises: i) a first unit that specifically binds to a second binding portion of a first Notch receptor, and ii) a second unit that specifically binds to a second binding portion of a second Notch receptor, wherein the first unit of the second binding portion and the second unit of the second binding portion each independently comprise an ECD 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 that specifically binds to a first binding portion of a first muscle-specific molecule, and ii) a second unit that specifically binds to a first binding portion of a second muscle-specific molecule.

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

37. The bifunctional protein construct of claim 36, wherein the first antibody portion is a first Fab (Fab1) and the second antibody portion is a second Fab (Fab2); The bifunctional protein construct comprises: i) A first polypeptide comprising, from N' to C': a first unit that specifically binds to a second binding portion of a first Notch receptor – an optional linker – the first subunit of the Fc domain – an optional linker – VH1-(H1-CH1); ii) A second polypeptide comprising, from N' to C': a second unit that specifically binds to a second binding portion of a second Notch receptor – an optional linker – the second subunit of the Fc domain – an optional linker – VH2-(H2-CH1); iii) A third polypeptide, comprising from N' to C': VL1-(L1-CL); and iv) The fourth polypeptide, comprising from N' to C': VL2-(L2-CL); and 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 portion is a first Fab (Fab1) and the second antibody portion is a second Fab (Fab2); The bifunctional protein construct comprises: i) A first polypeptide comprising, from N' to C': a first unit that specifically binds to a second binding portion of a first Notch receptor – an optional linker – the first subunit of the Fc domain – an optional linker – VL1-(L1-CL); ii) A second polypeptide comprising, from N' to C': a second unit that specifically binds to a second binding portion of a second Notch receptor – an optional linker – the second subunit of the Fc domain – an optional linker – VL2-(L2-CL); iii) The third polypeptide, comprising from N' to C': VH1-(H1-CH1); and iv) The fourth polypeptide, which comprises from N' to C': VH2-(H2-CH1); and 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 both Fab1 and Fab2 specifically bind to LAMA2, and wherein anti-LAMA2 Fab1 and / or anti-LAMA2 Fab2 comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 165 and a second polypeptide containing the amino acid sequence of SEQ ID NO:

166.

40. The bifunctional protein construct according to any one of claims 35 to 39, wherein: a) The first unit of the second binding portion and / or the second unit of the second binding portion comprises variant DLL4 ECD, and wherein the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NO: 126-129 and 260-267; b) The first unit of the second binding portion and / or the second unit of the second binding portion comprises 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 portion and / or the second unit of the second binding portion comprises 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 portion and / or the second unit of the second binding portion comprises 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 portion and / or the second unit of the second binding portion comprises 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 portion is a first sdAb (sdAb1) and the second antibody portion is a second sdAb (sdAb2); The bifunctional protein construct comprises: i) A first polypeptide comprising, from N' to C': a first unit specifically binding to a second binding portion of 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 that specifically binds to a second binding portion of a second Notch receptor – an optional linker – a second subunit of the Fc domain – an optional linker – sdAb2.

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

43. The bifunctional protein construct of claim 42, wherein both scFv1 and scFv2 specifically bind to LAMA2, and wherein the 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 according to any one of claims 41 to 43, wherein: a) The first unit of the second binding portion and / or the second unit of the second binding portion comprises a variant DLL4 ECD containing an amino acid sequence of any one of SEQ ID NO: 126-129 and 260-267; b) The first unit of the second binding portion and / or the second unit of the second binding portion comprises a DLL1 ECD containing the amino acid sequence of SEQ ID NO: 130; c) The first unit of the second binding portion and / or the second unit of the second binding portion comprises a DLL3 ECD containing the amino acid sequence of SEQ ID NO: 131; d) The first unit of the second binding portion and / or the second unit of the second binding portion comprises a variant Jag1 ECD containing the amino acid sequence of SEQ ID NO: 133 or 134; or e) The first unit of the second binding portion and / or the second unit of the second binding portion comprises a Jag2 ECD containing 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 having an amino acid sequence of any one of SEQ ID NO: 223-231 and 269-276.

46. ​​The bifunctional protein construct of claim 35 or 36, wherein the first antibody portion is a first Fab (Fab1) and the second antibody portion is a second Fab (Fab2); The bifunctional protein construct comprises: i) A first polypeptide comprising, from N' to C': a first unit that specifically binds to a second binding portion of a first Notch receptor – an optional linker – VH1-(H1-CH1) – an optional linker – the first subunit of the Fc domain; ii) A second polypeptide comprising, from N' to C': a second unit that specifically binds to a second binding portion of 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 – optional linker – VL1-(L1-CL) that specifically binds to the second binding portion of the third Notch receptor; and iv) The fourth polypeptide, from N' to C', comprises: a fourth unit – optional linker – VL2-(L2-CL) that specifically binds to the second binding portion of the fourth Notch receptor; and 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 to 35, wherein the first unit of the first binding portion comprises a first non-antibody portion specifically bound to the first muscle-specific molecule, the second unit of the first binding portion comprises a second non-antibody portion specifically bound to the second muscle-specific molecule, and wherein the first non-antibody portion and the second non-antibody portion are each independently selected from the group consisting of: the LG domain of laminin, the LG domain of aggregates, the LG domain of nestin, and the LG domain of bead-glycan.

48. The bifunctional protein construct of claim 47, wherein the first non-antibody portion and / or the second non-antibody portion comprises LAMA2 LG4-5 containing 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': a first unit of the first binding portion – an optional linker – a first subunit of the Fc domain – an optional linker – a first unit of the second binding portion specifically binding to a first Notch receptor; and ii) A second polypeptide comprising, from N' to C': the second unit of the first binding portion – an optional linker – the second subunit of the Fc domain – an optional linker – a second unit that specifically binds to the second binding portion of the 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 specifically binding to a second binding portion of a first Notch receptor – an optional linker – a first subunit of the Fc domain – an optional linker – the first unit of the first binding portion; and ii) A second polypeptide comprising, from N' to C': a second unit that specifically binds to a second binding portion of a second Notch receptor – an optional linker – a second subunit of the Fc domain – an optional linker – a second unit of the first binding portion.

51. The bifunctional protein construct according to any one of claims 47 to 50, wherein: a) The first unit of the second binding portion and / or the second unit of the second binding portion comprises variant DLL4 ECD, and wherein the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NO: 126-129 and 260-267; b) The first unit of the second binding portion and / or the second unit of the second binding portion comprises 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 portion and / or the second unit of the second binding portion comprises 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 portion and / or the second unit of the second binding portion comprises 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 portion and / or the second unit of the second binding portion comprises 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 bonding portion is fused to the N-terminus of the first subunit of the Fc structural domain via an optional first connector, and the second bonding portion is fused to the N-terminus of the second subunit of the Fc structural domain via an optional second connector; or ii) The first bonding portion is fused to the N-terminus of the second subunit of the Fc structural domain via an optional first connector, and the second bonding portion is fused to the N-terminus of the first subunit of the Fc structural domain via an optional second connector.

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

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

55. One or more vectors as described in claim 54, wherein the vector is a viral vector.

56. A host cell expressing a bifunctional protein construct as described in any one of claims 1 to 52, comprising one or more isolated nucleic acids as described in claim 53, or comprising one or more vectors as described in claim 54 or 55.

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

58. A method for generating a bifunctional protein construct, comprising: i) Culturing host cells containing one or more isolated nucleic acids as described in claim 53, or one or more vectors as described in claim 54 or 55, or host cells as described in claim 56, under conditions suitable for expressing the bifunctional protein construct; and ii) Recover the expressed bifunctional protein construct from the cultured host cells.

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

60. The method of claim 59, wherein the muscle-related disease is selected from the group consisting of: Pompe disease, central nucleus myopathy, progressive ossifying fibrous dysplasia (FOP), Friedreich ataxia (FRDA), familial hypertrophic cardiomyopathy, Lane distal myopathy, myofibril 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 includes one or more of the following: Dichené muscular dystrophy (DMD), Beck muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophy (CMD), facioscapulohumeral muscular dystrophy (FSHD), myotonic muscular dystrophy (DM), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), congenital myopathy, Charcot-Marie-Tuss (CMT) disease, and Emory-Dreyfus muscular dystrophy (EDMD).

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

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

126.

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

66. The 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 to 66, wherein the engineered DLL4ECD comprises mutations 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 NO: 261-264.

69. A protein construct comprising an engineered DLL4ECD as described in any one of claims 64 to 68.

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

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