Anti-TfR: acidic sphingomyelinase for treatment of acidic sphingomyelinase deficiency

Abstract

In one embodiment, a multi-domain therapeutic protein is provided, the multi-domain therapeutic protein comprising a TfR binding delivery domain fused to an acidic sphingomyelinase polypeptide; and nucleic acid constructs and compositions that allow insertion and / or expression of the multi-domain therapeutic protein coding sequence into a target genomic locus, such as an endogenous ALB locus. The multi-domain therapeutic proteins and nucleic acid constructs and compositions can be administered to cells, populations of cells, or subjects, the present invention relates to multi-domain therapeutic protein nucleic acids, and can be used in methods of integrating multi-domain therapeutic protein nucleic acids into target genomic loci, methods of expressing multi-domain therapeutic proteins in cells, methods of treating acidic sphingomyelinase deficiency in subjects, and methods of preventing or reducing signs or onset of symptoms of acidic sphingomyelinase deficiency in subjects.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Application No. 63 / 516,380, filed July 28, 2023, which is incorporated herein by reference in its entirety for all purposes.

[0002] References to the sequence list are submitted as an XML file. The sequence list written to file 616959SEQLIST.xml is 1,131,940 bytes long, created on July 24, 2024, and incorporated by reference. Background Technology

[0003] Acid sphingomyelinase deficiency (ASMD, also known as Niemann-Pick disease type A / B) is a lysosomal storage disorder caused by sphingomyelin phosphodiesterase 1 (Sphingomyelin phosphodiesterase 1 (SPD)). SMPD1 Loss-of-function mutations in the gene encoding acid sphingomyelinase (ASM) cause ASMD. ASM breaks down sphingomyelin in lysosomes; its absence leads to lysosomal lipid accumulation, cytotoxicity, and ultimately, histopathological changes. Visceral ASMD manifests as liver failure, hepatosplenomegaly, lung infection, hemorrhage, and an atherogenic lipid profile, resulting in early to late adulthood. Patients with more severe infantile neurovisceral ASMD also experience severe neurotic ataxia and hypotonia, and die before the age of 3. Alpha-oligophosphatase is an enzyme replacement therapy and the only approved treatment for ASMD, but it does not treat central nervous system symptoms and requires frequent infusions. Summary of the Invention

[0004] It provides multi-domain therapeutic proteins containing a TfR-binding delivery domain fused to an acid sphingomyelinase (ASM) peptide; and allows insertion of multi-domain therapeutic protein coding sequences into target genomic loci (such as endogenous). ALB The multidomain therapeutic protein (a gene locus) and / or nucleic acid constructs and compositions expressing the coding sequence of the multidomain therapeutic protein are provided. Cells comprising the multidomain therapeutic protein or nucleic acid constructs are also provided. The multidomain therapeutic protein, as well as the nucleic acid constructs and compositions, can be applied to cells, cell populations, or subjects, and can be used for methods of integrating multidomain therapeutic protein nucleic acids into target genomic loci, methods of expressing multidomain therapeutic proteins in cells, methods of treating acid sphingomyelinase deficiency (ASMD) in subjects, and methods of preventing or alleviating the onset of ASMD signs or symptoms in subjects.

[0005] In one aspect, a multi-domain therapeutic protein is provided comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide. In some such multi-domain therapeutic proteins, the C-terminus of the TfR-binding delivery domain is fused to the N-terminus of the acid sphingomyelinase polypeptide. In some such multi-domain therapeutic proteins, the C-terminus of the acid sphingomyelinase polypeptide is fused to the N-terminus of the TfR-binding delivery domain. In some such multi-domain therapeutic proteins, the TfR-binding delivery domain is fused to the acid sphingomyelinase polypeptide via a peptide linker, optionally wherein the linker comprises, is substantially composed of, or is composed of sequences listed in any of SEQ ID NO: 808, 617, and 616; optionally wherein the linker comprises, is substantially composed of, or is composed of sequences listed in any of SEQ ID NO: 808 and 617. In some of these multidomain therapeutic proteins, the adapter comprises, is substantially composed of, or is composed of the sequence listed in SEQ ID NO: 808. In some of these multidomain therapeutic proteins, the adapter comprises, is substantially composed of, or is composed of the sequence listed in SEQ ID NO: 617. In some of these multidomain therapeutic proteins, the adapter comprises, is substantially composed of, or is composed of the sequence listed in SEQ ID NO: 616. In some of these multidomain therapeutic proteins, the acid sphingomyelinase polypeptide lacks the acid sphingomyelinase signal peptide. In some of these multidomain therapeutic proteins, the acid sphingomyelinase polypeptide comprises, is substantially composed of, or is composed of the sequence listed in SEQ ID NO: 728, 731, or 733; optionally, the acid sphingomyelinase polypeptide comprises, is substantially composed of, or is composed of the sequence listed in SEQ ID NO: 733.

[0006] In some of these multi-domain therapeutic proteins, the TfR binding and delivery domain contains an anti-TfR antigen-binding protein. Optionally, this antigen-binding protein is delivered at approximately 41 nM K. D Or, with a stronger affinity, it binds to the human transferrin receptor. Optionally, the antigen-binding protein binds at approximately 3 nM K. D Or, with a stronger affinity, it binds to the human transferrin receptor. Optionally, the antigen-binding protein binds at a K+ of approximately 0.45 nM to 3 nM. DBinds to the human transferrin receptor. Among some of these multidomain therapeutic proteins, anti-TfR antigen-binding proteins comprise: (i) HCVR comprising HCDR1, HCDR2, and HCDR3, and containing an amino acid sequence (or a variant thereof) listed in SEQ ID NO: 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, or 481; and / or (ii) LCVR comprising LCDR1, LCDR2, and LCDR ... The amino acid sequences (or variants thereof) listed in NO:176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, or 486.

[0007] Among some of these multidomain therapeutic proteins, anti-TfR antigen-binding proteins include: (1) HCVR, which comprises HCDR1, HCDR2, and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 171; and LCVR, which comprises LCDR1, LCDR2, and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 176; (2) HCVR, which comprises HCDR1, HCDR2, and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 181; and LCVR, which comprises LCDR1, LCDR2, and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 186; (3) HCVR, which comprises HCDR1, HCDR2, and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 191; and LCVR, which comprises LCDR1, LCDR2, and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 191. (4) HCVR, comprising HCDR1, HCDR2 and HCDR3, and comprising the amino acid sequence listed in SEQ ID NO: 201 (or a variant thereof); and LCVR, comprising LCDR1, LCDR2 and LCDR3, and comprising the amino acid sequence listed in SEQ ID NO: 206 (or a variant thereof); (5) HCVR, comprising HCDR1, HCDR2 and HCDR3, and comprising the amino acid sequence listed in SEQ ID NO: 211 (or a variant thereof); and LCVR, comprising LCDR1, LCDR2 and LCDR3, and comprising the amino acid sequence listed in SEQ ID NO: 216 (or a variant thereof); (6) HCVR, comprising HCDR1, HCDR2 and HCDR3, and comprising the amino acid sequence listed in SEQ ID NO: 221 (or a variant thereof); and LCVR, comprising LCDR1, LCDR2 and LCDR3, and comprising the amino acid sequence listed in SEQ ID NO: 206 (or a variant thereof). (7) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence listed in SEQ ID NO: 231 (or a variant thereof); and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence listed in SEQ ID NO: 236 (or a variant thereof).(8) HCVR comprising HCDR1, HCDR2, and HCDR3, and comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 241; and LCVR comprising LCDR1, LCDR2, and LCDR3, and comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 246; (9) HCVR comprising HCDR1, HCDR2, and HCDR3, and comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 251; and LCVR comprising LCDR1, LCDR2, and LCDR3, and comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 256; (10) HCVR comprising HCDR1, HCDR2, and HCDR3, and comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 261; and LCVR comprising LCDR1, LCDR2, and LCDR3, and comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 261; (11) An HCVR comprising HCDR1, HCDR2, and HCDR3 and comprising the amino acid sequence listed in SEQ ID NO: 271 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 and comprising the amino acid sequence listed in SEQ ID NO: 276 (or a variant thereof); (12) An HCVR comprising HCDR1, HCDR2, and HCDR3 and comprising the amino acid sequence listed in SEQ ID NO: 281 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 and comprising the amino acid sequence listed in SEQ ID NO: 286 (or a variant thereof); (13) An HCVR comprising HCDR1, HCDR2, and HCDR3 and comprising the amino acid sequence listed in SEQ ID NO: 291 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 and comprising the amino acid sequence listed in SEQ ID NO: 291 (or a variant thereof); (296) amino acid sequences (or variants thereof); (14) HCVR comprising HCDR1, HCDR2 and HCDR3 and comprising the amino acid sequence (or variant thereof) listed in SEQ ID NO: 301; and LCVR comprising LCDR1, LCDR2 and LCDR3 and comprising the amino acid sequence (or variant thereof) listed in SEQ ID NO: 306; (15) HCVR comprising HCDR1, HCDR2 and HCDR3 and comprising the amino acid sequence (or variant thereof) listed in SEQ ID NO: 311.(16) HCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 316; (17) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 331; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 336; and (18) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 316. The amino acid sequence (or a variant thereof) listed in NO:341; and LCVR, which comprises LCDR1, LCDR2, and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:346; (19) HCVR, which comprises HCDR1, HCDR2, and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:351; and LCVR, which comprises LCDR1, LCDR2, and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:356; (20) HCVR, which comprises HCDR1, HCDR2, and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:361; and LCVR, which comprises LCDR1, LCDR2, and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:366; (21) HCVR, which comprises HCDR1, HCDR2, and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:351; The amino acid sequence (or a variant thereof) listed in NO:371; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:376; (22) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:381; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:386.(23) HCVR comprising HCDR1, HCDR2, and HCDR3, and comprising the amino acid sequence listed in SEQ ID NO: 391 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3, and comprising the amino acid sequence listed in SEQ ID NO: 396 (or a variant thereof); (24) HCVR comprising HCDR1, HCDR2, and HCDR3, and comprising the amino acid sequence listed in SEQ ID NO: 401 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3, and comprising the amino acid sequence listed in SEQ ID NO: 406 (or a variant thereof); (25) HCVR comprising HCDR1, HCDR2, and HCDR3, and comprising the amino acid sequence listed in SEQ ID NO: 411 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3, and comprising the amino acid sequence listed in SEQ ID NO: 411 (or a variant thereof); (26) HCVR comprising HCDR1, HCDR2, and HCDR3 and comprising the amino acid sequence listed in SEQ ID NO: 421 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 and comprising the amino acid sequence listed in SEQ ID NO: 426 (or a variant thereof); (27) HCVR comprising HCDR1, HCDR2, and HCDR3 and comprising the amino acid sequence listed in SEQ ID NO: 431 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 and comprising the amino acid sequence listed in SEQ ID NO: 436 (or a variant thereof); (28) HCVR comprising HCDR1, HCDR2, and HCDR3 and comprising the amino acid sequence listed in SEQ ID NO: 441 (or a variant thereof); and LCVR comprising LCDR1, LCDR2, and LCDR3 and comprising the amino acid sequence listed in SEQ ID NO: 441 (or a variant thereof); (29) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence listed in SEQ ID NO: 451 (or a variant thereof); and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence listed in SEQ ID NO: 456 (or a variant thereof); (30) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence listed in SEQ ID NO: 461 (or a variant thereof).(31) HCVR, comprising LCDR1, LCDR2, and LCDR3, and containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 466; and LCVR, comprising HCDR1, HCDR2, and HCDR3, and containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 471; and LCVR, comprising LCDR1, LCDR2, and LCDR3, and containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 476; or (32) HCVR, comprising HCDR1, HCDR2, and HCDR3, and containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 481; and LCVR, comprising LCDR1, LCDR2, and LCDR3, and containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 486.

[0008] Among some of these multidomain therapeutic proteins, anti-TfR antigen-binding proteins include: (1) HCVR, which includes HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 391; and LCVR, which includes LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 396; or (2) HCVR, which includes HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 411; and LCVR, which includes LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 416. Among some of these multidomain therapeutic proteins, anti-TfR antigen-binding proteins include: HCVR, which comprises HCDR1, HCDR2, and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 391; and LCVR, which comprises LCDR1, LCDR2, and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 396.

[0009] Among some of these multidomain therapeutic proteins, anti-TfR antigen-binding proteins comprise: (a) HCVR, which comprises: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 172, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 173, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 174; and LCVR, which comprises: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 177, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 178, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 179; (b) HCVR, which comprises: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 182, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 183, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 174. HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 184; and LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 187, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 188, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 189; (c) HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 192, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 193, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 194; and LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 197, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 198, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 194; LCDR3 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 202; (d) HCVR comprising: HCDR1 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 203, HCDR2 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 204;and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 207, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 208, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 209; (e) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 212, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 213, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 214; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 217, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 218, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 209. (f) An HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 222, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 223, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 224; and an LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 227, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 228, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 229; (g) An HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 232, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 233, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 229. HCDR3 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 234; and LCVR comprising: LCDR1 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 237, LCDR2 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 238, and LCDR3 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 239;(h) an HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 242, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 243, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 244; and an LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 247, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 248, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 249; (i) an HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 252, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 253, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 244. HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 254; and LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 257, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 258, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 259; (j) HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 262, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 263, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 264; and LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 267, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 268, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 264; LCDR3 of the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 269; (k) HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 272, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 273, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 274;and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 277, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 278, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 279; (l) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 282, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 283, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 284; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 287, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 288, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 289; (m) HCVR, the HCVR comprising: comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 277, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 278, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 289; HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 292, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 293, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 294; and LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 297, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 298, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 299; (n) HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 302, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 303, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 304; and LCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 292, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 293, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 294; and LCVR comprising: LCDR1 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 307, LCDR2 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 308, and LCDR3 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 309;(o) HCVR, comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 312, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 313, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 314; and LCVR, comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 317, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 318, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 319; (p) HCVR, comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 322, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 323, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 314. HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 324; and LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 327, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 328, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 329; (q) HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 332, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 333, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 334; and LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 337, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 338, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 329. LCDR3 of the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 339; (r) HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 342, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 343, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 344;and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 347, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 348, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 349; (s) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 352, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 353, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 354; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 357, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 358, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 359. LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 369; (t) HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 362, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 363, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 364; and LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 367, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 368, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 369; (u) HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 372, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 373, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 379. HCDR3 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 374; and LCVR comprising: LCDR1 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 377, LCDR2 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 378, and LCDR3 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 379;(v) HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 382, ​​HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 383, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 384; and LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 387, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 388, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 389; (w) HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 392, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 393, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 394. HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 394; and LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 397, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 398, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 399; (x) HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 402, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 403, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 404; and LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 407, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 408, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 399; LCDR3 containing the amino acid sequence (or a variant thereof) listed in NO:409; (y) HCVR comprising: HCDR1 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO:412, HCDR2 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO:413, and HCDR3 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO:414;and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 417, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 418, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 419; (z) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 422, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 423, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 424; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 427, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 428, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 429. LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 429; (aa) HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 432, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 433, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 434; and LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 437, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 438, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 439; (ab) HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 442, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 443, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 439. HCDR3 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 444; and LCVR comprising: LCDR1 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 447, LCDR2 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 448, and LCDR3 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 449;(ac) HCVR, comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 452, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 453, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 454; and LCVR, comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 457, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 458, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 459; (ad) HCVR, comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 462, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 463, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 454. HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 464; and LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 467, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 468, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 469; (ae) HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 472, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 473, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 474; and LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 477, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 478, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 474. LCDR3 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 479; and / or (af) HCVR comprising: HCDR1 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 482, HCDR2 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 483, and HCDR3 containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 484;and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 487, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 488, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 489.

[0010] Among some of these multidomain therapeutic proteins, anti-TfR antigen-binding proteins comprise: (a) HCVR, which comprises: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 392, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 393, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 394; and LCVR, which comprises: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 397, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 398, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 399; or (b) HCVR, which comprises: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 412, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 413, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 413. 414 lists HCDR3 with an amino acid sequence (or a variant thereof); and LCVR comprising: LCDR1 containing an amino acid sequence (or a variant thereof) listed in SEQ ID NO: 417, LCDR2 containing an amino acid sequence (or a variant thereof) listed in SEQ ID NO: 418, and LCDR3 containing an amino acid sequence (or a variant thereof) listed in SEQ ID NO: 419. Among some of these multidomain therapeutic proteins, anti-TfR antigen-binding proteins include: HCVR, which comprises: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 392, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 393, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 394; and LCVR, which comprises: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 397, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 398, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 399.

[0011] Among some of these multidomain therapeutic proteins, anti-TfR antigen-binding proteins comprise: (i) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 171; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 176; (ii) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 181; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 186; (iii) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 191; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 196; (iv) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 201; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 206; and (v) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 176; (vi) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 211; and LCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 221; and LCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 226; (vii) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 231; and LCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 236; (viii) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 241; and LCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 246; (ix) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 251; and LCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 251; The amino acid sequence listed in SEQ ID NO: 256 (or a variant thereof); (x) HCVR, which contains the amino acid sequence listed in SEQ ID NO: 261 (or a variant thereof); and LCVR, which contains the amino acid sequence listed in SEQ ID NO: 266 (or a variant thereof); (xi) HCVR, which contains the amino acid sequence listed in SEQ ID NO: 271 (or a variant thereof).(xii) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 276; (xii) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 281; (xiii) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 291; (xiv) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 296; (xiv) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 301; (xv) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 306; (xv) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 311; and LCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 276. The amino acid sequence listed in SEQ ID NO: 316 (or a variant thereof); (xvi) HCVR, which contains the amino acid sequence listed in SEQ ID NO: 321 (or a variant thereof); and LCVR, which contains the amino acid sequence listed in SEQ ID NO: 326 (or a variant thereof); (xvii) HCVR, which contains the amino acid sequence listed in SEQ ID NO: 331 (or a variant thereof); and LCVR, which contains the amino acid sequence listed in SEQ ID NO: 336 (or a variant thereof); (xviii) HCVR, which contains the amino acid sequence listed in SEQ ID NO: 341 (or a variant thereof); and LCVR, which contains the amino acid sequence listed in SEQ ID NO: 346 (or a variant thereof); (xix) HCVR, which contains the amino acid sequence listed in SEQ ID NO: 351 (or a variant thereof); and LCVR, which contains the amino acid sequence listed in SEQ ID NO: 356 (or a variant thereof); (xx) HCVR, which contains the amino acid sequence listed in SEQ ID NO: 326 (or a variant thereof); The amino acid sequence (or a variant thereof) listed in SEQ ID NO: 361; and LCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 366; (xxi) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 371; and LCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 376; (xxii) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 381.(xxiii) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 386; (xxiv) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 391; (xxv) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 411; (xxvi) HCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 421; and LCVR, which contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 396. The amino acid sequences (or variations thereof) listed in SEQ ID NO: 426; (xxvii) HCVR, which comprises the amino acid sequence (or variations thereof) listed in SEQ ID NO: 431; and LCVR, which comprises the amino acid sequence (or variations thereof) listed in SEQ ID NO: 436; (xxviii) HCVR, which comprises the amino acid sequence (or variations thereof) listed in SEQ ID NO: 441; and LCVR, which comprises the amino acid sequence (or variations thereof) listed in SEQ ID NO: 446; (xxix) HCVR, which comprises the amino acid sequence (or variations thereof) listed in SEQ ID NO: 451; and LCVR, which comprises the amino acid sequence (or variations thereof) listed in SEQ ID NO: 456; (xxx) HCVR, which comprises the amino acid sequence (or variations thereof) listed in SEQ ID NO: 461; and LCVR, which comprises the amino acid sequence (or variations thereof) listed in SEQ ID NO: 466; (xxxi) HCVR, which comprises the amino acid sequence (or variations thereof) listed in SEQ ID NO: 466; The amino acid sequence listed in SEQ ID NO: 471 (or a variant thereof); and the LCVR containing the amino acid sequence listed in SEQ ID NO: 476 (or a variant thereof); and / or (xxxii) HCVR containing the amino acid sequence listed in SEQ ID NO: 481 (or a variant thereof); and the LCVR containing the amino acid sequence listed in SEQ ID NO: 486 (or a variant thereof).

[0012] Among some of these multidomain therapeutic proteins, the anti-TfR antigen-binding protein comprises: (i) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 391; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 396; or (ii) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 411; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 416. Among some of these multidomain therapeutic proteins, the anti-TfR antigen-binding protein comprises: HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 391; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 396.

[0013] In some of these multi-domain therapeutic proteins, the TfR-binding delivery domain comprises an anti-TfR antibody, an antibody fragment, or a single-chain variable fragment (scFv). In some of these multi-domain therapeutic proteins, the TfR-binding delivery domain is a single-chain variable fragment (scFv), optionally wherein the multi-domain therapeutic protein comprises domains arranged in the following orientation: N'-heavy chain variable region-light chain variable region-acid sphingomyelinase polypeptide-C' or N'-light chain variable region-heavy chain variable region-acid sphingomyelinase polypeptide-C', optionally wherein the scFv and the acid sphingomyelinase polypeptide are linked by a peptide linker, and optionally wherein the peptide linker is -(GGGGS). m - (SEQ ID NO: 537); where m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, optionally wherein the scFv variable region is linked by a peptide linker, and optionally wherein the peptide linker is -(GGGGS). m -(SEQ ID NO: 537); where m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some of these multi-domain therapeutic proteins, the multi-domain therapeutic protein includes a heavy chain variable region (V... H ) and light chain variable region (V L ) and acid sphingomyelinase polypeptide, of which V H V L The polypeptide arrangement of acid sphingomyelinase is as follows: (i) V L -V H - Acidic sphingomyelinase polypeptide; (ii) V H -V L - Acidic sphingomyelinase polypeptide; (iii) V L -[(GGGGS)3(SEQ ID NO: 616)]-V H-[(GGGGS)2(SEQ ID NO: 617)]-acidic sphingomyelinase polypeptide; or (iv)V H -[(GGGGS)3(SEQ ID NO: 616)]-V L -[(GGGGS)2(SEQ ID NO: 617)]- acid sphingomyelinase polypeptide. In some of these multi-domain therapeutic proteins, the scFv comprises, is substantially composed of, or is composed of the following sequences: sequences listed in any one of SEQ ID NO: 494, 503, 505, and 508, optionally wherein the scFv comprises, is substantially composed of, or is composed of the following sequences: sequences listed in SEQ ID NO: 505 or 508, optionally wherein the scFv comprises, is substantially composed of, or is composed of the following sequences: sequences listed in SEQ ID NO: 508. In some of these multi-domain therapeutic proteins, the multi-domain therapeutic protein comprises, is substantially composed of, or is composed of the following sequences: sequences listed in SEQ ID NO: 737 or 739. Some of these multi-domain therapeutic proteins comprise, consist substantially of, or consist of the sequences listed in SEQ ID NO: 837, 839, 841, 737, or 739. (This text is repeated four times in the original.) In some of these multi-domain therapeutic proteins, the multi-domain therapeutic protein comprises, is substantially composed of, or is composed of the sequence listed in SEQ ID NO: 839. In some of these multi-domain therapeutic proteins, the multi-domain therapeutic protein comprises, is substantially composed of, or is composed of the sequence listed in SEQ ID NO: 841. In some of these multi-domain therapeutic proteins, the scFv comprises, is substantially composed of, or is composed of the sequence listed in SEQ ID NO: 813.

[0014] In some of these multi-domain therapeutic proteins, the TfR-binding delivery domain is a Fab protein comprising a complete light chain, a heavy chain variable region, and a heavy chain constant region CH1 domain, optionally with the C-terminus of the CH1 domain linked to the N-terminus of the light chain, or the C-terminus of the light chain linked to the N-terminus of the heavy chain variable region, optionally wherein the Fab protein comprises the amino acid sequences (or variations thereof) listed in SEQ ID NO: 584 and 635, or the amino acid sequences (or variations thereof) listed in SEQ ID NO: 588 and 636, and optionally wherein the C-terminus of the CH1 domain is linked to the N-terminus of an acid sphingomyelinase polypeptide, or optionally wherein the C-terminus of the light chain is linked to the N-terminus of an acid sphingomyelinase polypeptide. In some of these multi-domain therapeutic proteins, the Fab protein comprises the amino acid sequences (or variations thereof) listed in SEQ ID NO: 584 and 635, optionally wherein the Fab protein comprises, is substantially composed of, or is composed of the sequences listed in SEQ ID NO: 815 or 817. Some of these multi-domain therapeutic proteins comprise, consist substantially of, or consist of the sequence listed in SEQ ID NO: 833 or 835. Some of these multi-domain therapeutic proteins comprise, consist substantially of, or consist of the sequence listed in SEQ ID NO: 833. Some of these multi-domain therapeutic proteins comprise, consist substantially of, or consist of the sequence listed in SEQ ID NO: 835.

[0015] In some of these multi-domain therapeutic proteins, the TfR binding delivery domain is an antigen-binding protein that binds to one or more hTfR epitopes selected from the following: (a) an epitope containing the sequence LLNE (SEQ ID NO: 752) and / or an epitope containing the sequence TYKEL (SEQ ID NO: 706); (b) an epitope containing the sequence DSTDFTGT (SEQ ID NO: 753) and / or an epitope containing the sequence VKHPVTGQF (SEQ ID NO: 754) and / or an epitope containing the sequence IERIPEL (SEQ ID NO: 755); (c) an epitope containing the sequence LNENSYVPREAGSQKDEN (SEQ ID NO: 756); (d) an epitope containing the sequence FEDL (SEQ ID NO: 718); (e) an epitope containing the sequence IVDKNGRL (SEQ ID NO: 757); (f) an epitope containing the sequence IVDKNGRLVY (SEQ ID NO: 718). Epitopes containing: (g) the sequence DQTKF (SEQ ID NO: 759); (h) the sequence LVENPGGY (SEQ ID NO: 760) and / or the sequence PIVNAELSF (SEQ ID NO: 761) and / or the sequence PYLGTTMDT (SEQ ID NO: 762); (i) the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or the sequence TYKEL (SEQ ID NO: 706); (j) the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 707) and / or the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 758). (k) Epitopes containing the sequence LIERIPELNKVARAAAE (SEQ ID NO: 709); (k) Epitopes containing the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 710); (l) Epitopes containing the sequence GTKKDFEDL (SEQ ID NO: 711); (m) Epitopes containing the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 712);(n) Epitopes containing the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or epitopes containing the sequence DQTKFPIVNAEL (SEQ ID NO: 714) and / or epitopes containing the sequence TYKELIERIPELNK (SEQ ID NO: 715); (o) Epitopes containing the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or epitopes containing the sequence TYKELIERIPELNK (SEQ ID NO: 715); (p) Epitopes containing the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 716); (q) Epitopes containing the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or epitopes containing the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 716). (r) Epitopes containing the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or epitopes containing the sequence LVENPGYVAYSKAATVTGKL (SEQ ID NO: 719) and / or epitopes containing the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 720) and / or epitopes containing the sequence ISRAAAEKL (SEQ ID NO: 721) and / or epitopes containing the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 722) and / or epitopes containing the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 723); (s) Epitopes contained within or overlapping with the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or epitopes contained within the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 717). (705) Epitopes contained in or overlapping with the sequence and / or included in or overlapping with the sequence TYKEL (SEQ ID NO: 706); (t) Epitopes contained in or overlapping with the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 707) and / or included in or overlapping with the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 708) and / or included in or overlapping with the sequence LIERIPELNKVARAAAE (SEQ ID NO: 709); (u) Epitopes contained in or overlapping with the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 710);(v) Epitopes contained in or overlapping with the sequence GTKKDFEDL (SEQ ID NO: 711); (w) Epitopes contained in or overlapping with the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 712); (x) Epitopes contained in or overlapping with the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or Epitopes contained in or overlapping with the sequence DQTKFPIVNAEL (SEQ ID NO: 714) and / or Epitopes contained in or overlapping with the sequence TYKELIERIPELNK (SEQ ID NO: 715); (y) Epitopes contained in or overlapping with the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or Epitopes contained in or overlapping with the sequence TYKELIERIPELNK (SEQ ID NO: 715). (z) Epitopes contained in or overlapping with the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 716); (aa) Epitopes contained in or overlapping with the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or Epitopes contained in or overlapping with the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 717); and (ab) Epitopes contained in or overlapping with the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or Epitopes contained in or overlapping with the sequence LVENPGYVAYSKAATVTGKL (SEQ ID NO: 719) and / or Epitopes contained in or overlapping with the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 716). Epitopes contained in or overlapping with the sequence ISRAAAEKL (SEQ ID NO: 721) and / or contained in or overlapping with the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 722) and / or contained in or overlapping with the sequence FCETDYPYLGTTMDT (SEQ ID NO: 723).

[0016] In some of these multi-domain therapeutic proteins, the TfR binding delivery domain comprises an antibody or an antigen-binding fragment thereof that binds to one or more hTfR epitopes selected from: (a) an epitope consisting of the sequence LLNE (SEQ ID NO: 752) and / or an epitope consisting of the sequence TYKEL (SEQ ID NO: 706); (b) an epitope consisting of the sequence DSTDFTGT (SEQ ID NO: 753) and / or an epitope consisting of the sequence VKHPVTGQF (SEQ ID NO: 754) and / or an epitope consisting of the sequence IERIPEL (SEQ ID NO: 755); (c) an epitope consisting of the sequence LNENSYVPREAGSQKDEN (SEQ ID NO: 756); (d) an epitope consisting of the sequence FEDL (SEQ ID NO: 718); (e) an epitope consisting of the sequence IVDKNGRL (SEQ ID NO: 718). (f) Epitope composed of the sequence IVDKNGRLVY (SEQ ID NO: 758); (g) Epitope composed of the sequence DQTKF (SEQ ID NO: 759); (h) Epitope composed of the sequence LVENPGGY (SEQ ID NO: 760) and / or Epitope composed of the sequence PIVNAELSF (SEQ ID NO: 761) and / or Epitope composed of the sequence PYLGTTMDT (SEQ ID NO: 762); (i) Epitope composed of the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or Epitope composed of the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or Epitope composed of the sequence TYKEL (SEQ ID NO: 706); (j) Epitope composed of the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 757) and / or Epitope composed of the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 758) and / or Epitope composed of the sequence DQTKF (SEQ ID NO: 759); (h) Epitope composed of the sequence LVENPGGY (SEQ ID NO: 760) and / or Epitope composed of the sequence PIVNAELSF (SEQ ID NO: 761) and / or Epitope composed of the sequence PYLGTTMDT (SEQ ID NO: 762); (i) Epitope composed of the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or Epitope composed of the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or Epitope composed of the sequence TYKEL (SEQ ID NO: 706); (j) Epitope composed of the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 757) and / or Epitope composed (707) a tabletop consisting of the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 708) and / or the sequence LIERIPELNKVARAAAE (SEQ ID NO: 709); (k) a tabletop consisting of the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 710); (l) a tabletop consisting of the sequence GTKKDFEDL (SEQ ID NO: 711); (m) a tabletop consisting of the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 712);(n) Epitopes consisting of the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or the sequence DQTKFPIVNAEL (SEQ ID NO: 714) and / or the sequence TYKELIERIPELNK (SEQ ID NO: 715); (o) Epitopes consisting of the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or the sequence TYKELIERIPELNK (SEQ ID NO: 715); (p) Epitopes consisting of the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 716); (q) Epitopes consisting of the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 705). Epitopes consisting of (717); and (r) epitopes consisting of the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or the sequence LVENPGYVAYSKAATVTGKL (SEQ ID NO: 719) and / or the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 720) and / or the sequence ISRAAAEKL (SEQ ID NO: 721) and / or the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 722) and / or the sequence FCETDYPYLGTTMDT (SEQ ID NO: 723).

[0017] On the other hand, compositions are provided comprising nucleic acid constructs containing the coding sequences of any of the aforementioned multi-domain therapeutic proteins. In some such compositions, the coding sequence for the TfR-binding delivery domain is codon-optimized or CpG-depleted, the coding sequence for the acid sphingomyelinase peptide is codon-optimized or CpG-depleted, or the coding sequence for the multi-domain therapeutic protein is codon-optimized or CpG-depleted. In some such compositions, the coding sequence for the TfR-binding delivery domain is codon-optimized and CpG-depleted, the coding sequence for the acid sphingomyelinase peptide is codon-optimized and CpG-depleted, or the coding sequence for the multi-domain therapeutic protein is codon-optimized and CpG-depleted.

[0018] In some such compositions, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NO: 524-536, and encodes an scFv containing any of SEQ ID NO: 494, 503, 505, or 508; optionally, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NO: 530-532, and encodes an scFv containing SEQ ID NO: 508; or optionally, the scFv encoding sequence is identical to any of SEQ ID NO: 530-532. Any one of 527-529 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and encodes an scFv containing SEQ ID NO: 505; optionally, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 530, and encodes an scFv containing SEQ ID NO: 508; optionally, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 532, and encodes an scFv containing SEQ ID NO: 508; or .... 527 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the encoding contains scFv of SEQ ID NO: 505.In some such compositions, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NO: 524-536, is codon-optimized and CpG-depleted, and encodes an scFv comprising any of SEQ ID NO: 494, 503, 505, or 508; optionally, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NO: 530-532, is codon-optimized and CpG-depleted, and encodes an scFv comprising SEQ ID NO: 508; or optionally, the scFv encoding sequence is identical to any of SEQ ID NO: 530-532. Any one of 527-529 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, is codon-optimized and CpG-depleted, and encodes an scFv containing SEQ ID NO: 505; optionally, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 530, the scFv encoding sequence is codon-optimized and CpG-depleted, and encodes an scFv containing SEQ ID NO: 508 ... 532 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 508, wherein the scFv encoding sequence is codon-optimized and CpG-depleted, and encodes the scFv containing SEQ ID NO: 508; or optionally wherein the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 527, wherein the scFv encoding sequence is codon-optimized and CpG-depleted, and encodes the scFv containing SEQ ID NO: 505.In some such compositions, the scFv encoding sequence comprises, is substantially composed of, or consists of any of the sequences listed in SEQ ID NO: 524-536; optionally, the scFv encoding sequence comprises, is substantially composed of, or consists of any of the sequences listed in SEQ ID NO: 530-532; or optionally, the scFv encoding sequence comprises, is substantially composed of, or consists of any of the sequences listed in SEQ ID NO: 527-529; optionally, the scFv encoding sequence comprises, is substantially composed of, or consists of any of the sequences listed in SEQ ID NO: 530; optionally, the scFv encoding sequence comprises, is substantially composed of, or consists of any of the sequences listed in SEQ ID NO: 532; or optionally, the scFv encoding sequence comprises, is substantially composed of, or consists of any of the sequences listed in SEQ ID NO: 530-532. The sequence listed in 527.

[0019] In some such compositions, the nucleic acid construct includes a splice acceptor upstream of the coding sequence of a multidomain therapeutic protein, and the nucleic acid construct includes a polyadenylation signal or sequence downstream of the coding sequence of the multidomain therapeutic protein; or the nucleic acid construct includes a splice acceptor upstream of the coding sequence of the multidomain therapeutic protein and a polyadenylation signal or sequence downstream of the coding sequence of the multidomain therapeutic protein. In some such compositions, the nucleic acid construct does not contain a homologous arm. In some such compositions, the nucleic acid construct from 5' to 3' includes: a splice acceptor, a coding sequence of a multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the nucleic acid construct does not contain a promoter driving the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not contain a homologous arm. In some such compositions, the nucleic acid construct contains a homologous arm. In some such compositions, the nucleic acid construct does not contain a promoter driving the expression of the multidomain therapeutic protein. In some such compositions, the coding sequence of a multi-domain therapeutic protein is operatively linked to a promoter, optionally wherein the promoter is a liver-specific promoter.

[0020] In some of these compositions, the nucleic acid construct is contained within a nucleic acid carrier or lipid nanoparticles. In some of these compositions, the nucleic acid construct is contained within a nucleic acid carrier, optionally wherein the nucleic acid carrier is a viral vector. In some of these compositions, the nucleic acid carrier is an adeno-associated virus (AAV) vector, optionally wherein the nucleic acid construct has an inverted terminal repeat (ITR) sequence attached to each end, optionally wherein at least one ITR comprises, is substantially composed of, or is composed of SEQ ID NO: 160, and optionally wherein each ITR comprises, is substantially composed of, or is composed of SEQ ID NO: 160. In some of these compositions, the AAV vector is a single-stranded AAV (ssAAV) vector. In some of these compositions, the AAV vector is a recombinant AAV8 (rAAV8) vector, optionally wherein the AAV vector is a single-stranded rAAV8 vector.

[0021] In some such compositions, the composition is used in combination with a nuclease agent that targets a nuclease target site in a target genomic locus. In some such compositions, the target genomic locus is an albumin gene, optionally wherein the albumin gene is a human albumin gene. In some such compositions, the nuclease target site is located in intron 1 of the albumin gene. In some such compositions, the nuclease agent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein or nucleic acid encoding a Cas protein; and (ii) a guide RNA or one or more DNA sequences encoding a guide RNA, wherein the guide RNA comprises a DNA targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such compositions, the nuclease agent comprises: (a) a Cas protein or a nucleic acid encoding the Cas protein; and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA targeting segment that targets the guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

[0022] In some of these compositions, the guide RNA target sequence is located in intron 1 of the albumin gene. In some of these compositions, the DNA targeting segment has any one of SEQ ID NO: 30-61, optionally including any one of SEQ ID NO: 36, 30, 33, and 41, or wherein the DNA targeting segment is composed of any one of SEQ ID NO: 30-61, optionally including any one of SEQ ID NO: 36, 30, 33, and 41. In some of these compositions, the guide RNA includes any one of SEQ ID NO: 62-125, optionally including any one of SEQ ID NO: 68, 100, 62, 94, 65, 97, 73, and 105. In some of these compositions, the DNA targeting segment includes or is composed of SEQ ID NO: 36. In some of these compositions, the guide RNA includes SEQ ID NO: 68 or 100. In some such compositions, the composition comprises guide RNA in the form of RNA. In some such compositions, the guide RNA comprises at least one modification. In some such compositions, the at least one modification comprises: (i) a phosphate thioester bond between the first four nucleotides at the 5' end of the guide RNA; (ii) a phosphate thioester bond between the last four nucleotides at the 3' end of the guide RNA; (iii) a 2'-O-methyl modified nucleotide at the first three nucleotides at the 5' end of the guide RNA; and (iv) a 2'-O-methyl modified nucleotide at the last three nucleotides at the 3' end of the guide RNA. In some such compositions, the composition comprises a guide RNA in the form of SEQ ID NO: 100, and the guide RNA comprises: (i) a phosphate thioester bond between the first four nucleotides at the 5' end of the guide RNA; (ii) a phosphate thioester bond between the last four nucleotides at the 3' end of the guide RNA; (iii) a 2'-O-methyl modified nucleotide at the first three nucleotides at the 5' end of the guide RNA; and (iv) a 2'-O-methyl modified nucleotide at the last three nucleotides at the 3' end of the guide RNA.

[0023] In some such compositions, the Cas protein is the Cas9 protein, optionally wherein the Cas protein is derived from Streptococcus pyogenes (Streptococcus pyogenes). Streptococcus pyogenesCas9 protein. In some such compositions, the Cas protein comprises the sequence listed in SEQ ID NO:11. In some such compositions, the composition comprises a nucleic acid encoding the Cas protein, wherein the nucleic acid comprises mRNA encoding the Cas protein. In some such compositions, the mRNA encoding the Cas protein comprises at least one modification. In some such compositions, the mRNA encoding the Cas protein is completely substituted with N1-methyl-pseuuridine. In some such compositions, the mRNA encoding the Cas protein comprises the sequence listed in SEQ ID NO:1 or 2. In some such compositions, the composition comprises a nucleic acid encoding the Cas protein, wherein the nucleic acid comprises mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence listed in SEQ ID NO:1 or 2, and the mRNA encoding the Cas protein is completely substituted with N1-methyl-pseuuridine, comprises a 5' cap, and comprises a poly(A) tail.

[0024] In some such compositions, the composition comprises a guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 68 or 100, and wherein the composition comprises an administration of a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises mRNA encoding a Cas protein, and the mRNA encoding a Cas protein comprises the sequence listed in SEQ ID NO: 1 or 2. In some such compositions, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 100, and the guide RNA comprising: (i) a phosphate thioester bond between the first four nucleotides at the 5' end of the guide RNA; (ii) a phosphate thioester bond between the last four nucleotides at the 3' end of the guide RNA; (iii) a 2'-O-methyl modified nucleotide at the first three nucleotides at the 5' end of the guide RNA; and (iv) a 2'-O-methyl modified nucleotide at the last three nucleotides at the 3' end of the guide RNA, and wherein the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises mRNA encoding a Cas protein, the mRNA encoding a Cas protein comprising the sequence listed in SEQ ID NO: 1 or 2, and the mRNA encoding a Cas protein is completely replaced by N1-methyl-pseuuridine, comprises a 5' cap, and comprises a poly(A) tail.

[0025] In some such compositions, the Cas protein or nucleic acid encoding the Cas protein and guide RNA or one or more DNAs encoding the guide RNA are associated with lipid nanoparticles. In some such compositions, the lipid nanoparticles comprise cationic lipids, neutral lipids, helper lipids, and stealth lipids. In some such compositions, the cationic lipid is lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadec-9,12-dienoate), and / or the neutral lipid is distearylphosphatidylcholine or 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), and / or the helper lipid is cholesterol, and / or the stealth lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000. In some of these compositions, the cationic lipid is lipid A, the neutral lipid is DSPC, the accessory lipid is cholesterol, and the occult lipid is PEG2k-DMG. In some of these compositions, the lipid nanoparticles comprise four lipids in the following molar ratios: approximately 50 mol% lipid A, approximately 9 mol% DSPC, approximately 38 mol% cholesterol, and approximately 3 mol% PEG2k-DMG.

[0026] On the other hand, cells are provided that contain any of the aforementioned multidomain therapeutic proteins or compositions. In some such cells, the coding sequence of the nucleic acid construct or multidomain therapeutic protein is integrated into a target genomic locus, and the multidomain therapeutic protein is expressed from the target genomic locus; or the coding sequence of the nucleic acid construct or multidomain therapeutic protein is integrated into intron 1 of an endogenous albumin locus, and the multidomain therapeutic protein is expressed from the endogenous albumin locus. In some such cells, the cells are liver cells or hepatocytes. In some such cells, the cells are human cells.

[0027] In another aspect, a method is provided, comprising administering any of the aforementioned multidomain therapeutic proteins to cells or cell populations. In another aspect, a method is provided for inserting a nucleic acid encoding a multidomain therapeutic protein into a target genomic locus in a cell or cell population, the multidomain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide, the method comprising administering any of the aforementioned compositions to cells or cell populations, wherein a nuclease agent cleaves a nuclease target site in the target genomic locus, and the nucleic acid construct or nucleic acid encoding the multidomain therapeutic protein is inserted into the target genomic locus. In another aspect, a method is provided for expressing a multidomain therapeutic protein in cells or cell populations, the multidomain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide, the method comprising administering any of the aforementioned compositions to cells or cell populations, wherein the coding sequence of the multidomain therapeutic protein is operatively linked to a promoter in a nucleic acid construct and expressed in the cells or cell populations. On the other hand, a method is provided for expressing a multidomain therapeutic protein from a target genomic locus in a cell or cell population, the multidomain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide, the method comprising administering any of the above-described compositions to the cell or cell population, optionally wherein the nucleic acid construct is administered simultaneously with a nuclease agent or one or more nucleic acids encoding the nuclease agent, or the nucleic acid construct is administered before or after the nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent cleaves a nuclease target site in the target genomic locus, the coding sequence of the nucleic acid construct or the multidomain therapeutic protein is inserted into the target genomic locus to produce a modified target genomic locus, and the multidomain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide is expressed from the modified target genomic locus.

[0028] In some of these methods, the cells are liver cells, or the cell population is a population of liver cells; optionally, the cells are hepatocytes, or the cell population is a population of hepatocytes. In some of these methods, the cells are human cells, or the cell population is a population of human cells. In some of these methods, the cells are neonatal cells, or the cell population is a population of neonatal cells. In some of these methods, the cells are in vitro or ex vivo, or the cell population is in vitro or ex vivo. In some of these methods, the cells are in the body of the subject, or the cell population is in the body of the subject.

[0029] In another aspect, a method is provided, comprising administering any of the aforementioned multidomain therapeutic proteins to a subject. In another aspect, a method is provided to insert a nucleic acid encoding a multidomain therapeutic protein into a target genomic locus in cells of a subject, the multidomain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide, the method comprising administering any of the aforementioned compositions to a subject, wherein a nuclease agent cleaves a nuclease target site in the target genomic locus, and the coding sequence of the nucleic acid construct or the multidomain therapeutic protein is inserted into the target genomic locus. In another aspect, a method is provided to express a multidomain therapeutic protein in cells of a subject, the multidomain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide, the method comprising administering any of the aforementioned compositions to a subject, wherein the coding sequence of the multidomain therapeutic protein is operatively linked to a promoter in a nucleic acid construct and expressed in cells. On the other hand, a method is provided for expressing a multi-domain therapeutic protein from a target genomic locus in cells of a subject, the multi-domain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide protein. The method comprises administering any of the above-described compositions to a subject, optionally wherein the nucleic acid construct is administered simultaneously with a nuclease agent or one or more nucleic acids encoding the nuclease agent, or the nucleic acid construct is administered before or after the nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent cleaves a nuclease target site in the target genomic locus, the coding sequence of the nucleic acid construct or the multi-domain therapeutic protein is inserted into the target genomic locus to produce a modified target genomic locus, and the multi-domain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide is expressed from the modified target genomic locus.

[0030] In some methods, the expressed multi-domain therapeutic protein is delivered to and internalized by a subject's central nervous system tissue. In some such methods, the cells are liver cells, optionally hepatocytes. In some such methods, the cells are human cells. In some such methods, the cells are neonatal cells.

[0031] In another aspect, a method for treating acid sphingomyelinase deficiency in a subject in need is provided, the method comprising administering to the subject any of the aforementioned multidomain therapeutic proteins. In another aspect, a method for treating acid sphingomyelinase deficiency in a subject in need is provided, the method comprising administering to the subject any of the aforementioned compositions, wherein the coding sequence of the multidomain therapeutic protein is operatively linked to a promoter in a nucleic acid construct and expressed in the subject. In another aspect, a method for treating acid sphingomyelinase deficiency in a subject in need is provided, the method comprising administering to the subject any of the aforementioned compositions, optionally wherein the nucleic acid construct is administered simultaneously with a nuclease agent or one or more nucleic acids encoding a nuclease agent, or the nucleic acid construct is administered before or after the nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent cleaves a nuclease target site in a target genomic locus, the coding sequence of the nucleic acid construct or the multidomain therapeutic protein is inserted into the target genomic locus to produce a modified target genomic locus, and the multidomain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide is expressed from the modified target genomic locus. In another aspect, a method is provided for preventing or alleviating the onset of signs or symptoms of acid sphingomyelinase deficiency in a subject in need, the method comprising administering to the subject any of the aforementioned multi-domain therapeutic proteins, thereby preventing or alleviating the onset of signs or symptoms of acid sphingomyelinase deficiency in the subject. In another aspect, a method is provided for preventing or alleviating the onset of signs or symptoms of acid sphingomyelinase deficiency in a subject in need, the method comprising administering to the subject any of the aforementioned compositions, wherein the coding sequence of the multi-domain therapeutic protein is operatively linked to a promoter in a nucleic acid construct and expressed in the subject, thereby preventing or alleviating the onset of signs or symptoms of acid sphingomyelinase deficiency in the subject. On the other hand, a method is provided for preventing or alleviating the onset of signs or symptoms of acid sphingomyelinase deficiency in a subject in need, the method comprising administering any of the above-described compositions to the subject, optionally wherein the nucleic acid construct is administered simultaneously with a nuclease agent or one or more nucleic acids encoding a nuclease agent, or the nucleic acid construct is administered before or after the nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent cleaves a nuclease target site, the coding sequence of the nucleic acid construct or a multi-domain therapeutic protein is inserted into a target genomic locus to produce a modified target genomic locus, and a multi-domain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide is expressed from the modified target genomic locus, thereby preventing or alleviating the onset of signs or symptoms of acid sphingomyelinase deficiency in the subject.

[0032] In some of these methods, acid sphingomyelinase deficiency is Niemann-Pick disease type A. In some of these methods, acid sphingomyelinase deficiency is Niemann-Pick disease type B. In some of these methods, the subject is a human subject. In some of these methods, the subject is a neonatal subject. In some of these methods, the method results in a serum level of the multidomain therapeutic protein in the subject of at least about 1 μg / mL, at least about 2 μg / mL, at least about 3 μg / mL, at least about 4 μg / mL, at least about 5 μg / mL, at least about 6 μg / mL, at least about 7 μg / mL, at least about 8 μg / mL, at least about 9 μg / mL, or at least about 10 μg / mL. In some of these methods, the method results in a serum level of the multidomain therapeutic protein in the subject of at least about 2 μg / mL or at least about 5 μg / mL. In some such methods, the method results in serum levels of the multidomain therapeutic protein in subjects that are between about 2 μg / mL and about 30 μg / mL, or between about 2 μg / mL and about 20 μg / mL. In some such methods, the method results in serum levels of the multidomain therapeutic protein in subjects that are between about 5 μg / mL and about 30 μg / mL, or between about 5 μg / mL and about 20 μg / mL. In some such methods, the method achieves acid sphingomyelinase activity levels of at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% of the normal value.

[0033] In some of these methods, the method further includes assessing the subject's pre-existing AAV immunity prior to administration of the nucleic acid construct. In some of these methods, the pre-existing AAV immunity is pre-existing AAV8 immunity. In some of these methods, assessing pre-existing AAV immunity includes evaluating immunogenicity using a total antibody immunoassay or a neutralizing antibody assay. Attached Figure Description

[0034] Figure 1 The amino acid sequences of various anti-human transferrin receptor scFv molecules are shown in V format. k -3xG4S(SEQ ID NO: 616)-V H .

[0035] Figures 2A to 2C This demonstrates the delivery of GAA to Tfrc by an anti-human TFRC scFv antibody clone. hum Mouse brains. Anti-human TfR:GAA molecules 69261, 69329, 12839, 12841, 12843, and 12845 were tested. Figure 2A), 69348, 12795, 12799, 12801, 12850 and 12798 ( Figure 2B ); and 12802, 69340, 12847, 12848, 69307 and 69323 ( Figure 2C Each lane = 1 mouse. Delivered via HDD.

[0036] Figure 3 This image shows a subset of anti-hTFRC antibodies (12798, 12850, 69323, 12841, 12843, 12845, 12847, 12848, 12799, 69307, and 12839) delivering mature GAA to the brain parenchyma in scfv:GAA format (via HDD delivery). Lane E corresponds to the endothelium, and lane P corresponds to the parenchyma. The affinity ratio of mfTfR to human TfR is indicated below the image (mf refers to cynomolgus monkey). Macaca fascicularis monkey).

[0037] Figure 4 This illustrates the delivery of mature GAA to the brain parenchyma via anti-hTFRC antibodies (12799, 12843, 12847, and 12839) in the scfv:GAA format (AAV8 episome liver reservoir gene therapy). Lane E corresponds to the endothelium, and lane P corresponds to the parenchyma.

[0038] Figure 5 This demonstrates how the anti-hTFRC scfv:GAA antibody delivers GAA protein to the liver reservoir of AAV8 episomes. - / - / Tfrc hum The mouse's central nervous system (cerebellum, cerebrum, spinal cord), heart, and muscles (quadriceps).

[0039] Figure 6 The liver reservoir of AAV8 episomes was rescued by anti-hTFRC scfv:GAA antibodies (12839, 12843, and 12847). Gaa - / - / Tfrc hum Glycogen storage in the central nervous system (CNS) (cerebellum, cerebrum, spinal cord), heart, and muscles (quadriceps) of mice.

[0040] Figures 7A to 7D The liver reservoir of AAV8 episomes was rescued by anti-hTFRC scfv:GAA antibodies (12847, 12843, and 12799). Gaa - / - / Tfrc hum Mouse brain (thalamus) Figure 7A ), cerebral cortex ( Figure 7B ), Haima CA1 area ( Figure 7C )) and muscles (quadriceps ( Figure 7D Glycogen storage in ))

[0041] Figure 8 The albumin insertion of anti-hTFRC 12847scfv:GAA demonstrates the delivery of mature GAA protein to the central nervous system and muscles of a Pompe disease model mouse.

[0042] Figure 9 Albumin insertion of anti-hTFRC 12847scfv:GAA was shown to rescue glycogen stores in the central nervous system and muscles of Pompe disease model mice. Univariate ANOVA (*p<0.01; **p<0.001; ***p<0.0001).

[0043] Figure 10 The image shows GAA activity in serum after insertion of anti-TfR1:GAA or anti-CD63:GAA into the cynomolgus macaque albumin locus via Cas9-mediated AAV delivery. The mediators alone were used as negative controls. One unit of GAA activity was defined as the amount of enzyme producing 1.0 µmol 4-MU per minute at pH 4.5 and 37 °C. Error bars are from SEM. Mediator group N=1; all other groups N=2–4.

[0044] Figure 11 The albumin insertion of anti-hTFRC 12847scfv:GAA demonstrates the delivery of mature GAA protein to the central nervous system and muscles of cynomolgus monkeys. For the bar chart, mature GAA was quantified by Western blot of tissue lysates; error bars are SD.

[0045] Figure 12 The Machupo mammalian sand virus (MDV) is shown as a symmetrical unit superimposed on two TfR molecules. Mammarenavirus machupoense GP1 protein (PDB 3KAS), human ferritin (PDB 6GSR), Plasmodium vivax ( Plasmodium vivax Interactions of Sal-1 PvRBP2b protein (PDB 6D04), human HFE protein (PDB 1DE4), and human transferrin (PDB 1SUV) molecules. For Machupo mammalian sandvirus GP1 protein and human ferritin, only one copy of the symmetric unit is shown for clarity to reduce the complexity of the figure.

[0046] Figure 13 The hydrogen-deuterium exchange mass spectrometry (HDX) protection of the antibody tested in HDX-MS experiments was depicted to be allocated to 5 regions in TfR (PDB 1SUV).

[0047] Figure 14The TfR regions protected by REGN17513 are shown, where REGN17513 is representative of antibodies that induce HDX protection in the TfR apical domain. These TfR regions overlap with the binding sites of Machupo mammalian isovirus GP1 protein, human ferritin, and Plasmodium vivax PvRBP2b protein.

[0048] Figure 15 This demonstrates the TfR region protected by REGN17510, where REGN17510 is a non-volatile component within the top structural domain of the TfR. Figure 15 Representative of other TfR-binding partner-shared HDX-protected antibodies shown.

[0049] Figure 16 The TfR region protected by REGN17515 is shown, representing antibodies with HDX protection at the TfR apical domain. These antibodies are effective against human ferritin and Plasmodium vivax. Sal-1 PvRBP2b proteins share a binding site.

[0050] Figure 17 This displays the TfR region protected by REGN17514, where REGN17514 is a TfR protease-like domain that interacts with Plasmodium vivax. Sal-1 A representative of HDX-protected antibodies that share a common binding site with the PvRBP2b protein.

[0051] Figure 18 This image shows the TfR region protected by REGN17508, where REGN17508 is representative of antibodies with HDX protection within the TfR protease-like domain. This region is not... Figure 18 Other TfR interacting molecules are shown.

[0052] Figure 19A and Figure 19B The study demonstrates the GAA enzyme activity in culture medium after various anti-TfR:GAA insert templates (CpG depleted and native) were inserted into the albumin locus of primary human hepatocytes following delivery via rAAV2.

[0053] Figure 20A Western blots were shown demonstrating the delivery of GAA to intravenously administered LNP-g666 (3 mg / kg) by anti-human TfR antibody clones (0 CpG and native) and various recombinant AAV8 anti-TfR:GAA or AAV8 anti-CD63:GAA insert templates in 3-month-old infants. Gaa - / - / Tfrc hum The mouse's brain. Each lane = 1 mouse.

[0054] Figure 20B The results showed that albumin insertion of anti-hTfR:GAA rescued intravenously administered LNP-g666 (3 mg / kg) and various recombinant AAV8 anti-TfR:GAA or AAV8 anti-CD63:GAA insertion templates. Gaa - / - / Tfrc hum Glycogen storage in the brain, quadriceps femoris muscle, diaphragm, and heart of mice. Glycogen levels were measured 3 weeks after administration. Wt Untreated mice served as a positive control, and Gaa - / - Untreated mice served as a negative control.

[0055] Figure 21 A schematic diagram of LNP-g9860 and recombinant AAV8 (rAAV8) capsid is shown; the former contains a target human albumin (rAAV8). ALB The lipid nanoparticles containing intron 1 Cas9 mRNA and sgRNA 9860, the latter packaged with an anti-TfR:ASM insert template.

[0056] Figure 22 This demonstrates CRISPR / Cas9-mediated... ALB A schematic diagram of the insertion template for anti-TfR:ASM at the gene locus. It depicts the human... ALB Loci, where Cas9 cleavage sites are indicated by scissors. The cleavage acceptor sites for the TfR:ASM transgene lateralized in the inserted template are depicted. In endogenous... ALB Following promoter-driven insertion and transcription, in ALB Splicing occurs between exon 1 and the inserted anti-TfR:ASMDNA template, as shown by the dashed line, producing a heterozygous strain. ALB - Anti-TfR:ASM mRNA. The ALB signal peptide promotes the secretion of anti-TfR:ASM and is removed during protein maturation to produce anti-TfR:ASM in plasma.

[0057] Figure 23 This demonstrates that the AAV plasmid construct expresses stable anti-TfR:ASM in vitro.

[0058] Figure 24 This demonstrates that the anti-TfR:ASM fusion protein retains sphingomyelinase activity in vitro.

[0059] Figure 25 The fluid dynamics delivery demonstrated that the anti-TfR:ASM fusion protein was effective in [the following context is missing from the original text] Tfrc hum Expression, secretion, and blood-brain barrier crossing in mice.

[0060] Figure 26The experimental setup for testing the rAAV8 episome liver reservoir for TfR-targeting ASM in ASMD mice is shown.

[0061] Figure 27 The expression of hepatic hSMPD1 DNA and ASM protein is shown after ASM: anti-TfR is delivered via the rAAV8 appendage.

[0062] Figures 28A to 28E The ASM: TfR-resistant rAAV8 appender delivery was shown to be reduced. Smpd1 - / - mouse cerebellum ( Figure 28A ),brain( Figure 28B ),liver( Figure 28C ),spleen( Figure 28D ) and lungs ( Figure 28E Sphingomyelin accumulation in ).

[0063] Figure 29 This shows the redesigned TfR-targeting ASM plasmid used for testing. Tfrc hum Experimental setup for hydrodynamic delivery in mice.

[0064] Figure 30 The fluid dynamics delivery demonstrated that the anti-TfR:ASM fusion protein was effective in [the following context is missing from the original text] Tfrc hum Expression and secretion in mice.

[0065] Figure 31 The AAV plasmid construct was shown to express stable anti-TfR:ASM in Huh-7 cells and retain normal ASM activity in vitro.

[0066] Figure 32 Showing the use of in Tfrc hum / hum ; Smpd1 - / - Experimental setup for testing albumin insertion of anti-TfR:ASM template in mice.

[0067] Figure 33 Show all TfR:ASM formats in Tfrc hum / hum ; Smpd1 - / - In mice, albumin insertion resulted in uniform transcript delivery and expression. TaqMan assays showed no significant differences in DNA or RNA levels.

[0068] Figures 34A to 34D The study showed that albumin insertion of anti-TfR:ASM was reduced. Smpd1 - / - Sphingomyelin in mouse target tissues, including the cerebellum ( Figure 34A),lung( Figure 34B ),spleen( Figure 34C ) and heart ( Figure 34D Significance was determined using the Kruskal-Wallis test; only significant differences between saline and treated samples are shown.

[0069] Figure 35 The albumin insertion of anti-TfR:ASM was not observed in... Tfrc hum / hum ; Smpd1 - / - Splenomegaly was induced in mice. Statistical analysis: One-way ANOVA (Kruskal-Wallis test). Lack of statistical significance indicated no significant difference; p-values ​​were almost all ≥0.9999.

[0070] definition The terms “protein,” “polypeptide,” and “peptide,” used interchangeably herein, encompass amino acids of any length in polymeric form, including coding and non-coding amino acids, as well as amino acids modified or derived chemically or biochemically. These terms also include modified polymers, such as polypeptides having a modified peptide backbone. The term “domain” refers to any portion of a protein or polypeptide that has a specific function or structure.

[0071] Proteins are considered to have an "N-terminus" and a "C-terminus". The term "N-terminus" refers to the starting point of a protein or polypeptide that terminates at an amino acid with a free amine group (-NH2). The term "C-terminus" refers to one end of an amino acid chain (protein or polypeptide) that terminates at a free carboxyl group (-COOH).

[0072] The terms “nucleic acid” and “polynucleotide”, used interchangeably herein, encompass nucleotides in polymeric form of any length, including ribonucleotides, deoxyribonucleotides, or their analogues or modified forms. These terms include single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derived nucleotide bases.

[0073] Nucleic acids are considered to have both a "5' end" and a "3' end" because mononucleotides react to form oligonucleotides in such a way that the 5' phosphate of one mononucleotide's pentose ring attaches in one direction to the 3' oxygen of its adjacent mononucleotide's pentose ring via a phosphodiester bond. If the 5' phosphate of the oligonucleotide is not attached to the 3' oxygen of the mononucleotide's pentose ring, then the end of the oligonucleotide is called the "5' end." If the 3' oxygen of the oligonucleotide is not attached to the 5' phosphate of another mononucleotide's pentose ring, then the end of the oligonucleotide is called the "3' end." Even if a nucleic acid sequence is inside a larger oligonucleotide, the nucleic acid sequence can be considered to have both a 5' end and a 3' end. In linear or circular DNA molecules, discrete elements are referred to as "downstream" or "upstream" of 3' elements or 5' elements.

[0074] The term "genome-integrated" refers to nucleic acids that have been introduced into cells so that their nucleotide sequences are integrated into the cell's genome. Nucleic acids can be stably incorporated into the cell's genome using any method.

[0075] The term "viral vector" refers to a recombinant nucleic acid containing at least one viral element and containing elements sufficient or permissible for packaging into a viral vector particle. Vectors and / or particles can be used for the purpose of transferring DNA, RNA, or other nucleic acids into cells, in vitro, ex vivo, or in vivo. Many forms of viral vectors are known.

[0076] The term "isolated" in relation to cells, tissues (e.g., liver samples), proteins, and nucleic acids includes cells, tissues (e.g., liver samples), proteins, and nucleic acids that are relatively purified relative to other bacteria, viruses, cells, or other components that may normally be present in situ, up to and including substantially pure formulations of cells, tissues (e.g., liver samples), proteins, and nucleic acids. The term "isolated" also includes cells, tissues (e.g., liver samples), proteins, and nucleic acids that do not have naturally occurring counterparts, have been chemically synthesized, and are therefore substantially uncontaminated by other cells, tissues (e.g., liver samples), proteins, and nucleic acids, or have been isolated or purified from most of their naturally occurring accompanying components (e.g., cellular components) (e.g., other cellular proteins, polynucleotides, or cellular components).

[0077] The term "wildtype" includes entities that have the structure and / or activity found in normal states or conditions (compared to mutated, diseased, altered, etc.). Wildtype genes and polypeptides often exist in many different forms (e.g., alleles).

[0078] The term "endogenous sequence" refers to nucleic acid sequences that naturally exist within cells or animals. For example, in humans... ALB Sequence refers to what naturally exists in humans ALB Natural at the gene locus ALB sequence.

[0079] "Exogenous" molecules or sequences include molecules or sequences that are not normally present in cells in the form described. Normal presence includes presence with respect to a specific developmental stage of the cell and environmental conditions. For example, exogenous molecules or sequences may include mutant forms of corresponding endogenous sequences within the cell (such as humanized forms of endogenous sequences), or sequences that correspond to endogenous sequences within the cell but are in a different form (i.e., not within chromosomes). In contrast, endogenous molecules or sequences include molecules or sequences that are normally present in that form in a specific cell at a specific developmental stage under specific environmental conditions.

[0080] When used in the context of nucleic acids or proteins, the term "heterologous" indicates that a nucleic acid or protein contains at least two segments that are not naturally found together in the same molecule. For example, when used with respect to nucleic acid segments or protein segments, the term "heterologous" indicates that a nucleic acid or protein contains two or more subsequences that are not found in nature to be in the same relationship (e.g., linked together). As an example, a "heterologous" region of a nucleic acid vector is a nucleic acid segment within or attached to another nucleic acid molecule that is not found in nature to associate with other molecules. For example, a heterologous region of a nucleic acid vector may contain a coding sequence flanked by a sequence that is not found in nature to associate with a coding sequence. Similarly, a "heterologous" region of a protein is an amino acid segment within or attached to another peptide molecule (e.g., a fusion protein or a tagged protein) that is not found in nature to associate with other peptide molecules. Similarly, nucleic acids or proteins may contain heterologous tags or heterologous secretion or localization sequences.

[0081] “Codon optimization” (i.e., “codon-optimized” sequences) takes advantage of the degeneracy of codons, as demonstrated by the diversity of tribase pair codon combinations for a given amino acid, and typically involves modifying nucleic acid sequences to enhance expression in a specific host cell by replacing at least one codon of the natural sequence with a codon that is more frequently or most frequently used in the host cell’s gene while maintaining the natural amino acid sequence. For example, a nucleic acid encoding a polypeptide of interest can be modified to replace a codon that has a higher frequency of use in a given prokaryotic or eukaryotic cell, including bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, or any other host cell, compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, at “Codon Usage Databases.” These tables can be modified in various ways. See Nakamura et al. (2000). Nucleic Acids Res. 28(1):292, which is incorporated herein by reference in its entirety for all purposes. Computer algorithms for codon optimization of specific sequences expressed in a particular host are also available (see, for example, Gene Forge).

[0082] The term "locus" refers to a specific location on a chromosome within an organism's genome, representing a gene (or significant sequence), DNA sequence, or polypeptide coding sequence. For example, " ALB "Locus" can refer to ALB Gene, ALB A specific location in a DNA sequence, an albumin-coding sequence, or on a chromosome in an organism's genome. ALB The ALB location has been identified as the site where this type of sequence resides. ALB "Locus" may include ALB Regulatory elements of a gene include, for example, enhancers, promoters, 5' and / or 3' untranslated regions (UTRs), or combinations thereof.

[0083] The term "gene" refers to a DNA sequence in a chromosome that, if naturally present, may contain at least one coding region and at least one non-coding region. The DNA sequence of a chromosome encoding a product (e.g., but not limited to RNA products and / or polypeptide products) may contain a coding region interrupted by non-coding introns and a sequence located adjacent to the coding region at both the 5' and 3' ends such that the gene corresponds to a full-length mRNA sequence (containing the 5' and 3' untranslated sequences). Additionally, other non-coding sequences, including regulatory sequences (e.g., but not limited to promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulating sequences, and matrix attachment regions, may be present in a gene. These sequences may be located near the coding region of the gene (e.g., but not limited to within 10 kb) or at distant sites, and these sequences may influence the level or rate of transcription and translation of the gene.

[0084] The term "allele" refers to a variant form of a gene. Some genes have multiple different forms, located at the same location on a chromosome, or at a genetic locus. Diploid organisms have two alleles at each locus. Each pair of alleles represents the genotype at a specific locus. If there are two identical alleles at a particular locus, the genotype is described as homozygous, and if the two alleles are different, the genotype is described as heterozygous.

[0085] A promoter is a regulatory region of DNA that typically contains a TATA box that guides RNA polymerase II to initiate RNA synthesis at the appropriate transcription start site of a specific polynucleotide sequence. Promoters may additionally contain other regions that influence the rate of transcription initiation. The promoter sequences disclosed herein regulate the transcription of operatively linked polynucleotides. Promoters may be active in one or more cell types disclosed herein (e.g., mouse cells, rat cells, pluripotent cells, single-cell stage embryos, differentiated cells, or combinations thereof). Promoters may be, for example, constitutively active promoters, conditional promoters, inducible promoters, time-restricted promoters (e.g., developmentally regulated promoters), or spatially restricted promoters (e.g., cell-specific or tissue-specific promoters). Examples of promoters can be found, for example, in WO 2013 / 176772, which is incorporated herein by reference in its entirety for all purposes.

[0086] "Operationally linked" or "operably coupled" refers to juxtaposing two or more components (e.g., a promoter and another sequence element) such that both components function normally and allow at least one component to mediate a function imposed on at least one other component. For example, if a promoter controls the transcriptional level of a coding sequence in response to the presence or absence of one or more transcriptional regulators, then a promoter can be operationally coupled to a coding sequence. Operationally coupled connections may include sequences that are adjacent to each other or act in a trans-regulatory manner (e.g., regulatory sequences can act at a distance to control the transcription of a coding sequence).

[0087] The methods and compositions provided herein employ a variety of different components. Some components throughout the specification may have active variants and fragments. The term "functional" refers to the innate ability of a protein or nucleic acid (or a fragment or variant thereof) to exhibit biological activity or function. The biological function of a functional fragment or variant may be the same as or may actually be altered (e.g., regarding its specificity or selectivity or efficacy) but retains the essential biological function of the molecule compared to the original molecule.

[0088] The term "variant" refers to a nucleotide sequence that differs from the most common sequence in the population (e.g., by one nucleotide) or a protein sequence that differs from the most common sequence in the population (e.g., by one amino acid).

[0089] When referring to proteins, the term "fragment" means a protein that is shorter than a full-length protein or has fewer amino acids. When referring to nucleic acids, the term "fragment" means a nucleic acid that is shorter than a full-length nucleic acid or has fewer nucleotides. When referring to protein fragments, a fragment can be, for example, an N-terminal fragment (i.e., a portion of the C-terminus of a protein has been removed), a C-terminal fragment (i.e., a portion of the N-terminus of a protein has been removed), or an internal fragment (i.e., a portion of both the N-terminus and C-terminus of a protein has been removed). When referring to nucleic acid fragments, a fragment can be, for example, a 5' fragment (i.e., a portion of the 3' end of a nucleic acid has been removed), a 3' fragment (i.e., a portion of the 5' end of a nucleic acid has been removed), or an internal fragment (i.e., a portion of both the 5' end and 3' end of a nucleic acid has been removed).

[0090] In the context of two polynucleotide or polypeptide sequences, "sequence identity" or "identity" refers to the same residues in two sequences when compared for maximum correspondence within a specified comparison window. When using a percentage of sequence identity relative to a protein, dissimilar residue positions are often distinguished by conserved amino acid substitutions, where an amino acid residue is replaced by another amino acid residue with similar chemical properties (e.g., charge or hydrophobicity) and therefore does not alter the molecule's functional properties. When the conserved substitutions of sequences differ, the percentage of sequence identity can be adjusted upwards to correct for the conservatism of the substitution. Sequences that differ due to such conserved substitutions are considered to have "sequence similarity" or "similarity." The means of making this adjustment are well known. Typically, this involves counting conserved substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, a score for a conserved substitution is between zero and 1 when the score for an identical amino acid is 1 and the score for a non-conservative substitution is zero. For example, the score for a conserved substitution is calculated, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0091] The "Sequence Identity Percentage" is a value determined by comparing two optimally aligned sequences within a comparison window (the maximum number of perfectly matched residues). The portion of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., vacancies) compared to the reference sequence (excluding additions or deletions) to achieve optimal alignment. This percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Unless otherwise specified (e.g., the shorter sequence contains linked heterologous sequences), the comparison window is the full length of the shorter of the two compared sequences.

[0092] Unless otherwise stated, sequence identity / similarity values ​​include those obtained using GAP version 10 with the following parameters: nucleotide sequence identity % and similarity using a GAP weight of 50 and a length weight of 3 and an nwsgapdna.cmp scoring matrix; amino acid sequence identity % and similarity using a GAP weight of 8 and a length weight of 2 and a BLOSUM62 scoring matrix; or any equivalent procedure thereof. "Equivalent procedure" includes any sequence comparison procedure that generates alignments with the same nucleotide or amino acid residue match and the same percentage of sequence identity for any two sequences in question when compared to corresponding alignments generated by GAP version 10.

[0093] The term "conservative amino acid substitution" refers to replacing a normally present amino acid in a sequence with a different amino acid having similar size, charge, or polarity. Examples of conservative substitution include replacing another nonpolar residue with a nonpolar (hydrophobic) residue (such as isoleucine, valine, or leucine). Similarly, examples of conservative substitution include replacing another polar residue with a polar (hydrophilic) residue, such as a polar residue between arginine and lysine, a polar residue between glutamine and asparagine, or a polar residue between glycine and serine. Additionally, replacing another basic residue with a basic residue (such as lysine, arginine, or histidine) or replacing another acidic residue with an acidic residue (such as aspartic acid or glutamic acid) are further examples of conservative substitution. Examples of nonconservative substitution include replacing a polar (hydrophilic) residue (such as cysteine, glutamine, glutamic acid, or lysine) with a nonpolar (hydrophobic) amino acid residue (such as isoleucine, valine, leucine, alanine, or methionine) and / or replacing a nonpolar residue with a polar residue. The typical amino acid classifications are summarized below.

[0094] Table 1. Amino acid classification

[0095] "Homologous" sequences (e.g., nucleic acid sequences) include sequences that are identical or substantially similar to a known reference sequence, such that they are, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence. Homologous sequences can include, for example, orthologous and paralogous sequences. For example, homologous genes typically arise from a common ancestral DNA sequence through speciation events (orthologous genes) or genetic duplication events (paralogous genes). "Orthologous" genes include genes from different species that evolved from a common ancestor through speciation. Orthologs typically retain the same function during evolution. "Paralleloid" genes include genes associated with duplication within the genome. Paralogs may evolve new functions during evolution.

[0096] The term "in vitro" includes an artificial environment and processes or reactions that occur within that artificial environment (e.g., test tubes or isolated cells or cell lines). The term "in vivo" includes a natural environment (e.g., cells, organisms, or the body) and processes or reactions that occur within that natural environment. The term "ex vivo" includes cells that have been removed from an individual and processes or reactions that occur within such cells.

[0097] As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains, and two light (L) chains linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR, VL, or VK) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs can be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs can be abbreviated as LCDR1, LCDR2, and LCDR3). The term "high affinity" antibody refers to an antibody with at least 10... -9 M, at least 10 -10 M; at least 10 -11 M; or at least 10 -12 Those antibodies that bind to M with affinity, such as those that do so via surface plasmon resonance, for example, BIACORE. ™Or it can be measured by solution affinity ELISA. The term "antibody" can cover any type of antibody, such as monoclonal or polyclonal antibodies. Furthermore, antibodies can be of any origin, such as mammalian or non-mammal. In one embodiment, the antibody can be mammalian or avian. In another embodiment, the antibody can be human or human, and may also be a human monoclonal antibody.

[0098] The phrase "bispecific antibody" refers to an antibody capable of selectively binding to two or more epitopes. Bispecific antibodies typically comprise two distinct heavy chains, each specifically binding to different epitopes on two different molecules (e.g., antigens) or on the same molecule (e.g., the same antigen). If a bispecific antibody is capable of selectively binding to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will typically be at least one to two, three, or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa. The epitopes recognized by a bispecific antibody can be located on the same or different targets (e.g., on the same or different proteins). Bispecific antibodies can be prepared, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, a nucleic acid sequence encoding a variable sequence of a heavy chain recognizing different epitopes of the same antigen can be fused with a nucleic acid sequence encoding a constant region of a different heavy chain, and such sequences can be expressed in cells expressing immunoglobulin light chains. A typical bispecific antibody has two heavy chains and one immunoglobulin light chain. Each heavy chain has three heavy chain CDRs, followed by a CH1 domain (N-terminus to C-terminus), a hinge, a CH2 domain, and a CH3 domain. The immunoglobulin light chain does not confer antigen-binding specificity but can associate with each heavy chain, or can associate with each heavy chain and bind one or more epitopes bound by the heavy chain antigen-binding region, or can associate with each heavy chain and enable one or two heavy chains to bind one or two epitopes.

[0099] The phrase "heavy chain" or "immunoglobulin heavy chain" contains a constant region sequence of an immunoglobulin heavy chain from any organism and, unless otherwise stated, contains a heavy chain variable domain. Unless otherwise stated, the heavy chain variable domain contains three heavy chain CDRs and four FR regions. Fragments of the heavy chain contain CDRs, CDRs, and FRs, and combinations thereof. A typical heavy chain has a CH1 domain, a hinge, a CH2 domain, and a CH3 domain following the variable domain (from the N-terminus to the C-terminus). Functional fragments of the heavy chain contain fragments capable of specifically recognizing antigens (e.g., recognizing antigens with KD in the micromolar, nanomolar, or picomolar range), capable of being expressed and secreted by cells, and including at least one CDR.

[0100] The phrase “light chain” encompasses the constant region sequence of an immunoglobulin light chain from any organism and, unless otherwise specified, includes human κ and λ light chains. Unless otherwise specified, the light chain variable (VL) domain typically comprises three light chain CDRs and four frame (FR) regions. Generally, a full-length light chain contains a VL domain and a light chain constant domain from the amino terminus to the carboxyl terminus, the VL domain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Light chains that may be used herein include, for example, those that do not selectively bind to the first or second antigen selectively bound by an antigen-binding protein. Suitable light chains include those that can be identified by screening the most commonly used light chains in existing antibody libraries (wet libraries or computer simulations), where the light chain substantially does not interfere with the affinity and / or selectivity of the antigen-binding domain of the antigen-binding protein. Suitable light chains include those that can bind one or both epitopes bound by the antigen-binding region of an antigen-binding protein.

[0101] The phrase “variable domain” includes an amino acid sequence (modified as needed) of the immunoglobulin light or heavy chain containing the following amino acid regions ordered from the N-terminus to the C-terminus (unless otherwise specified): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. “Variable domain” includes an amino acid sequence capable of folding into a canonical domain (VH or VL) with a double β-sheet structure, wherein these β-sheets are linked by disulfide bonds between residues of the first and second β-sheets.

[0102] The phrase "complementarity-determining region" or the term "CDR" contains an amino acid sequence encoded by the nucleic acid sequence of an organism's immunoglobulin gene. This amino acid sequence typically (i.e., in wild-type animals) occurs between two framework regions within the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T-cell receptor). The CDR can be encoded by, for example, germline sequences, rearranged sequences, or unrearranged sequences, and is encoded, for example, by naive or mature B cells or T cells. In some cases (e.g., for CDR3), the CDR can be encoded by two or more sequences (e.g., germline sequences) that are discontinuous (e.g., in an unrearranged nucleic acid sequence) but are continuous in the B-cell nucleic acid sequence, for example, due to splicing or linking sequences (e.g., VDJ recombination to form the heavy chain CDR3).

[0103] The term “antibody fragment” refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term “antibody fragment” include (i) the Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) the F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by disulfide bonds at the hinge region; (iii) the Fd fragment consisting of VH and CH1 domains; (iv) the Fv fragment consisting of the VL and VH domains of a single arm of the antibody; (v) the dAb fragment (Ward et al., (1989) Nature 241:544-546), which consists of the VH domain; (vi) the isolated CDR; and (vii) the scFv fragment consisting of the two domains VL and VH of the Fv fragment linked by a synthetic linker to form a single protein chain, wherein the VL and VH regions pair to form a monovalent molecule. Other forms of single-chain antibodies, such as dimers, are also covered under the term "antibody" (see, for example, Holliger et al., (1993)). Proc.Natl.Acad.Sci. USA90:6444-6448; Poljak et al., (1994) Structure 2:1121-1123).

[0104] The phrase "Fc-containing protein" includes antibodies, bispecific antibodies, immunoadhesins, and other binding proteins that contain at least the functional portions of the immunoglobulin CH2 and CH3 regions. "Functional portion" refers to the CH2 and CH3 regions that can bind to Fc receptors (e.g., FcyR; or FcRn, i.e., the neonatal Fc receptor) and / or participate in complement activation. If the CH2 and CH3 regions contain deletions, substitutions, and / or insertions or other modifications that prevent them from binding to any Fc receptors and from activating complement, then the CH2 and CH3 regions are inactive.

[0105] Fc-containing proteins may contain modifications in their immunoglobulin domains, including modifications that affect one or more effector functions of the bound protein (e.g., modifications affecting FcyR binding, FcRn binding, thereby affecting half-life and / or CDC activity). Such modifications include, but are not limited to, the following modifications and combinations thereof, referring to the EU numbers for the immunoglobulin constant region: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 311, 312, 315. 318, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375, 376, 378, 380, 382, ​​383, 384, 386, 388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and 439.

[0106] For example, but not as a limitation, the binding protein is an Fc-containing protein that exhibits a prolonged serum half-life (compared to the same Fc-containing protein without the said modification) and has the following modifications: modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., L / R / SI / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In another example, the modifications may include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 2591 (e.g., V259I) and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); 307 and / or 308 modifications (e.g., 308F or 308P).

[0107] As used herein, the term "antigen-binding protein" refers to a polypeptide or protein (one or more polypeptides complexed in a functional unit) that specifically recognizes epitopes on antigens, such as the cell-specific antigens and / or target antigens provided herein. Antigen-binding proteins can be multispecific. The term "multispecific" with respect to antigen-binding proteins means that the protein recognizes different epitopes on the same antigen or on different antigens. The multispecific antigen-binding proteins provided herein can be a single multifunctional polypeptide or a polymeric complex formed by two or more polypeptides covalently or non-covalently associated with each other. The term "antigen-binding protein" includes antibodies or fragments thereof provided herein that can be linked to or co-expressed with another functional molecule (e.g., another peptide or protein). For example, antibodies or fragments thereof can be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent binding, or other means) to one or more other molecular entities (such as proteins or fragments thereof) to produce bispecific or multispecific antigen-binding molecules with a second binding specificity.

[0108] As used herein, the term "epitope" refers to an antigenic moiety recognized by a multispecific antigen-binding polypeptide. A single antigen (such as an antigenic polypeptide) may have more than one epitope. Epitopes can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and are defined as those residues that directly promote the affinity between the antigen-binding polypeptide and the antigen. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In some embodiments, epitopes may comprise determinants of chemically active surface groups (such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups) as molecules, and in some embodiments, may have specific three-dimensional structural features and / or specific charge features. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed from tertiary folds are generally lost upon treatment with denaturing solvents.

[0109] The term "domain" refers to any part of a protein or polypeptide that has a specific function or structure. Preferably, the domains provided herein bind to cell-specific antigens or target antigens. As used herein, cell-specific antigen-binding domains or target antigen-binding domains include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to antigens.

[0110] The interchangeable terms "half-body" or "half-antibody" refer to half of an antibody that essentially contains one heavy chain and one light chain. Antibody heavy chains can form dimers, so the heavy chain of one half can associate with a different molecule (e.g., another half) or another heavy chain containing an Fc polypeptide. Two slightly different Fc domains can undergo "heterodimerization," as in the formation of bispecific antibodies or other heterodimers, heterotrimers, heterotetramers, etc. See Vincent and Murini (2012). Biotechnol.J. 7(12):1444-1450; and Shimamoto et al., (2012) MAbs 4(5):586-91. In one embodiment, the hemisomal variable domain specifically recognizes internalization effectors, and the hemisomal Fc domain dimers with Fc fusion proteins that include alternative enzymes (e.g., peptide bodies).

[0111] The term "single-chain variable fragment" or "scFv" refers to a single-chain fusion polypeptide containing both the variable region (VH) of the immunoglobulin heavy chain and the variable region (VL) of the immunoglobulin light chain. In some embodiments, the VH and VL are linked by a linker sequence of 10 to 25 amino acids. scFv polypeptides may also contain other amino acid sequences, such as CL or CH1 regions. scFv molecules can be prepared by phage display or by direct subcloning of the heavy and light chains from hybridoma or B cells. See Ahmad et al., (2012). Clin.Dev.Immunol. 2012:980250, this document is incorporated herein by reference in its entirety for all purposes.

[0112] As used herein, in the context of humans, the term "newborn" encompasses human subjects who are at most or less than 1 year old (52 weeks), preferably at most or less than 24 weeks, more preferably at most or less than 12 weeks, more preferably at most or less than 8 weeks, and even more preferably at most or less than 4 weeks old. In some embodiments, the newborn human subject is at most 4 weeks old. In some embodiments, the newborn human subject is at most 8 weeks old. In another embodiment, the newborn human subject is within 3 weeks of birth. In another embodiment, the newborn human subject is within 2 weeks of birth. In another embodiment, the newborn human subject is within 1 week of birth. In another embodiment, the newborn human subject is within 7 days of birth. In another embodiment, the newborn human subject is within 6 days of birth. In another embodiment, the newborn human subject is within 5 days of birth. In another embodiment, the newborn human subject is within 4 days of birth. In another embodiment, the newborn human subject is within 3 days of birth. In another embodiment, the newborn human subject is within 2 days of birth. In another embodiment, the newborn human subject is within 1 day of birth. The time windows disclosed above apply to human subjects and are also intended to cover corresponding developmental time windows in other animals. As used herein, “newborn cells” refer to cells from newborn subjects, and a newborn cell population refers to a population of cells from newborn subjects.

[0113] As used herein, a “control” in a control sample or control subject is a comparison used for a measurement (e.g., a diagnostic measurement of a sign or symptom of a disease). In some embodiments, a control may be a subject sample from an earlier time point (e.g., prior to a treatment intervention) of the same subject. In some embodiments, a control may be a measurement from a normal subject (i.e., a subject who does not have the disease of the treated subject) to provide a normal control, such as enzyme concentration or activity in a subject sample. In some embodiments, a normal control may be a population control, i.e., the mean of subjects in a general population. In some embodiments, a control may be an untreated subject with the same disease. In some embodiments, a control may be a subject treated with a different therapy (e.g., standard of care). In some embodiments, a control may be a subject or group of subjects from a natural history study of subjects with the disease of the subject being compared. In some embodiments, a control is matched to the subject being tested for certain factors, such as age and sex. In some embodiments, a control may be a control level from a specific laboratory (e.g., a clinical laboratory). The selection of an appropriate control is within the competence of those skilled in the art.

[0114] A composition or method that “comprising” or “contains” one or more of the listed elements may include other elements not specifically listed. For example, a composition that “comprising” or “contains” a protein may contain a protein alone or in combination with other ingredients. The transitional phrase “consistently made of” means that the scope of the claims should be interpreted to cover the specified elements listed in the claims as well as those elements that do not substantially affect the essential and novel characteristics of the claimed invention. Therefore, when used in the claims of this invention, the term “consistently made of” should not be interpreted as equivalent to “comprising”.

[0115] "Optional" or "optionally" means that the event or situation described below may or may not occur, and this description includes instances in which the event or situation occurs as well as instances in which the event or situation does not occur.

[0116] The specification of a numerical range includes all integers within that range or defining that range, as well as all subranges defined by the integers within that range. For example, 5-10 nucleotides is understood as 5, 6, 7, 8, 9, or 10 nucleotides, while 5%-10% is understood as including 5% and all possible values ​​up to 10%.

[0117] At least 17 nucleotides in a 20-nucleotide sequence are understood to include 17, 18, 19, or 20 nucleotides in the provided sequence, thus providing an upper limit, even if no upper limit is explicitly provided as would be clearly understood. Similarly, at most 3 nucleotides are understood to cover 0, 1, 2, or 3 nucleotides, thus providing a lower limit, even if no lower limit is explicitly provided. When “at least,” “at most,” or other similar language modifies a number, it is understood to modify each number in the series.

[0118] As used herein, “no more than” or “less than” is understood to be the value adjacent to the phrase and a logically lower value or integer to zero that is reasonable from the context. For example, the double-stranded region of “no more than 2 nucleotide base pairs” has 2, 1, or 0 nucleotide base pairs. When “no more than” or “less than” precedes a series of numbers or ranges, it should be understood that each number in the series or range is modified.

[0119] As used herein, “detection of analyte” should be understood as a determination in which the analyte (if present) can be detected, wherein the analyte is present in an amount higher than the detection level of the determination.

[0120] As used herein, “loss of function” should be understood as the absence of activity for any reason, such as the absence of enzyme activity. In some embodiments, the absence of activity may be due to the absence of a functional protein, for example, the protein is not transcribed or translated, the protein is translated but unstable, or it cannot be properly transported intracellularly or systemically. In some embodiments, the absence of activity may be due to the presence of mutations, such as point mutations, truncations, or aberrant splicing, that result in the presence of a protein without function. Loss of function can be partial or complete. In some embodiments, it is known that varying degrees of loss of function can lead to a variety of conditions, disease severity, or age of onset. As used herein, loss of function is preferably not a temporary loss of function, for example, due to a stress response or other response that results in a temporary loss of function of the protein. Therapeutic interventions to correct protein loss of function may include compensating for the loss of function with the deficient protein, or compensating for the loss of function with a protein that compensates for the loss of function but has a different sequence or structure than the protein with the loss of function. It should be understood that the loss of function of a protein can be compensated for by providing or altering the activity of another protein in the same biological pathway. In some implementations, compensating for loss-of-function proteins includes one or more of truncated, mutated, or non-natural sequences to guide protein transport within cells or throughout the body, thereby overcoming the loss of protein function. Therapeutic interventions may or may not correct loss of protein function in all cell types or tissues. Therapeutic interventions may include expressing proteins to compensate for loss of function at sites distant from where the lacking protein is normally expressed, such as at sites where the deficiency leads to cellular or organ dysfunction. Therapeutic interventions may include expressing proteins in the liver to compensate for loss of function at sites distant from the liver. In humans and other species, many genetic mutations are associated with specific loss-of-function mutations.

[0121] As used herein, “enzyme deficiency” should be understood as insufficient enzyme activity due to loss of protein function. Enzyme deficiency can be partial or complete and may result in variations in the timing of onset or the severity of signs or symptoms, depending on the extent and location of the loss of function. As used herein, enzyme deficiency is preferably not a temporary deficiency caused by stress or other factors. In humans and other species, many genetic mutations are associated with enzyme deficiency. In some embodiments, enzyme deficiency leads to congenital metabolic defects. In some embodiments, enzyme deficiency leads to lysosomal storage diseases. In some embodiments, enzyme deficiency leads to galactosemia. In some embodiments, enzyme deficiency leads to hemorrhagic diseases.

[0122] As used herein, it should be understood that when the maximum value is expressed as 100% (e.g., 100% inhibition or 100% encapsulation), the value is limited by the detection method. For example, 100% inhibition is understood as inhibition to a level below the detection level to be determined, and 100% encapsulation is understood as the material intended for encapsulation not being detected outside the vesicle.

[0123] Unless the context otherwise requires, the term "about" covers values ​​±5% of the stated value. In some embodiments, the term "about" should be understood to cover variations or errors tolerable in the art, such as two standard deviations from the average or the sensitivity of the method used to make the measurement, or percentages of values ​​tolerable in the art, such as variations with age. When "about" precedes the first value in a series, it can be understood to modify each value in that series.

[0124] The term “and / or” means and covers any and all possible combinations of one or more of the listed items, as well as the absence of such combinations when interpreted in the alternative (“or”) sense.

[0125] The term "or" refers to any one member of a particular list, and also includes any combination of members of that list.

[0126] Unless the context clearly indicates otherwise, the singular forms “an,” “a,” and “the” used herein include plural referents. For example, the term “protein” or “at least one protein” can include a variety of proteins, or mixtures thereof.

[0127] Statistically significant means p≤0.05.

[0128] In the event of a conflict between the sequence in this application and the specified login number or position within the login number, the sequence in this application shall prevail. Detailed Implementation

[0129] I. Overview It provides multi-domain therapeutic proteins containing a TfR-binding delivery domain fused to an acid sphingomyelinase (ASM) peptide; and allows insertion of multi-domain therapeutic protein coding sequences into target genomic loci (such as endogenous). ALB The multidomain therapeutic protein and / or nucleic acid constructs and compositions encoding the multidomain therapeutic protein locus. The multidomain therapeutic protein, along with the nucleic acid constructs and compositions, can be applied to cells, cell populations, or subjects, and can be used for methods of integrating the multidomain therapeutic protein nucleic acid into a target genomic locus, methods of expressing the multidomain therapeutic protein in cells, methods of treating ASM deficiency (ASMD) in subjects, and methods of preventing or alleviating the onset of ASMD signs or symptoms in subjects.

[0130] Compositions, combinations, or kits are also provided, comprising: a nucleic acid construct containing a coding sequence of a multi-domain therapeutic protein, and a nuclease agent or one or more nucleic acids encoding a nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus. As used herein, the term “combined with” means that an additional component may be applied before, after, or simultaneously with the application of the nucleic acid construct. The different components of the combination may be formulated as a single composition, for example for simultaneous delivery, or individually formulated as two or more compositions (e.g., a kit comprising each component, for example in which the additional agent is in a separate formulation).

[0131] More specifically, this document describes, in some embodiments, a CRISPR / Cas9 gene-editing-based therapeutic product optionally contained in a lipid nanoparticle (LNP) delivery system, associated with a multi-domain therapeutic protein DNA gene insertion template optionally contained in recombinant adeno-associated virus serotype 8 (rAAV8). The CRISPR / Cas9 component has been programmed to target and cleave target loci (e.g., safe harbor loci, such as those in hepatocytes). ALB The gene locus is a double-stranded DNA, allowing a multi-domain therapeutic protein DNA template to be inserted into the genome at the target genomic locus. This transgene insertion provides a functional multi-domain therapeutic protein gene encoding a missing or defective genome found in ASMD patients. SMPD1 .

[0132] Some of the multi-domain therapeutic protein coding sequences in the constructs disclosed herein have been optimized for expression. For example, the coding sequences in the constructs disclosed herein may contain one or more modifications, such as codon optimization (e.g., codon optimization of human codons), CpG dinucleotide depletion, mutations in cryptic splicing sites, or any combination thereof.

[0133] II. Nucleic acid constructs for inserting multi-domain therapeutic proteins into cells and / Or express multiple structures Multi-domain therapeutic proteins and compositions It provides multi-domain therapeutic proteins containing a TfR-binding delivery domain fused to an acid sphingomyelinase (ASM) peptide; and allows insertion of multi-domain therapeutic protein coding sequences into target genomic loci (such as endogenous). ALBThe multidomain therapeutic protein and / or nucleic acid constructs and compositions encoding the multidomain therapeutic protein locus. The multidomain therapeutic protein, along with the nucleic acid constructs and compositions, can be applied to cells, cell populations, or subjects, and can be used for methods of integrating the multidomain therapeutic protein nucleic acid into a target genomic locus, methods of expressing the multidomain therapeutic protein in cells, methods of treating ASM deficiency (ASMD) in subjects, and methods of preventing or alleviating the onset of ASMD signs or symptoms in subjects.

[0134] This article provides a multi-domain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase (ASM) peptide. The multi-domain therapeutic protein and composition can be used for methods of introducing the multi-domain therapeutic protein into cells or cell populations or subjects, methods of treating ASMD in subjects, and methods of preventing or alleviating the onset of ASMD signs or symptoms in subjects.

[0135] This article provides a method for inserting multi-domain therapeutic protein coding sequences into target genomic loci (such as endogenous albumin). ALB This document provides nucleic acid constructs and compositions for expressing multi-domain therapeutic proteins (e.g., episome expression vectors) at a specific locus. These constructs and compositions can be used for introducing nucleic acid constructs containing multi-domain therapeutic protein coding sequences into cells or cell populations or subjects; for integrating multi-domain therapeutic protein nucleic acids into target genomic loci; for expressing multi-domain therapeutic proteins in cells; for treating ASMD in subjects; and for preventing or alleviating the onset of ASMD signs or symptoms in subjects. Nuclease agents (e.g., targeting endogenous nucleases) are also provided. ALB (A locus) or nucleic acid encoding a nuclease agent to promote the integration of nucleic acid constructs into target genomic loci such as endogenous loci. ALB In the locus.

[0136] A. Multi-domain therapeutic proteins and nucleic acid constructs encoding multi-domain therapeutic proteins The compositions and methods described herein include the use of a multidomain therapeutic protein comprising an acid sphingomyelinase (ASM) polypeptide (ASM or its bioactive portion thereof, for providing alternative ASM enzyme activity) linked to or fused to a TfR-binding delivery domain. The compositions and methods described herein also include the use of a nucleic acid construct comprising a coding sequence of a multidomain therapeutic protein. The compositions and methods described herein may further include the use of a nucleic acid construct comprising a coding sequence of a multidomain therapeutic protein or an inverse complementary sequence thereof. Such nucleic acid constructs can be used to express multidomain therapeutic proteins in cells. Such nucleic acid constructs can be used for insertion into target genomic loci or into cleavage sites generated by nuclease agents or CRISPR / Cas systems as disclosed elsewhere herein. The term cleavage site includes a DNA sequence in which a nick or double-strand break is formed by a nuclease agent (e.g., a Cas9 protein complexed with a guide RNA). In some embodiments, the double-strand break is generated by a Cas9 protein complexed with a guide RNA, such as a SpyCas9 protein complexed with SpyCas9 guide RNA.

[0137] The length of the nucleic acid constructs disclosed herein can vary. The constructs can be, for example, from about 1 kb to about 5 kb, such as from about 1 kb to about 4.5 kb or from about 1 kb to about 4 kb. Exemplary nucleic acid constructs are between about 1 kb and about 5 kb or between about 1 kb and about 4 kb. Alternatively, the length of the nucleic acid constructs can be between about 1 kb and about 1.5 kb, about 1.5 kb and about 2 kb, about 2 kb and about 2.5 kb, about 2.5 kb and about 3 kb, about 3 kb and about 3.5 kb, about 3.5 kb and about 4 kb, about 4 kb and about 4.5 kb or about 4.5 kb and about 5 kb. Alternatively, the length of the nucleic acid constructs can be, for example, no more than 5 kb, no more than 4.5 kb, no more than 4 kb, no more than 3.5 kb, no more than 3 kb, or no more than 2.5 kb.

[0138] The construct may contain deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and may be single-stranded, double-stranded, or partially single-stranded and partially double-stranded, and may be introduced into the host cell in linear or circular (e.g., microloop) form. See, for example, US 2010 / 0047805, US 2011 / 0281361, and US 2011 / 0207221, each of which is incorporated herein by reference in its entirety for all purposes. If introduced in a linear form, the ends of the construct may be protected by known methods (e.g., from exonuclease degradation). For example, one or more dideoxynucleotide residues may be added to the 3' end of a linear molecule and / or self-complementary oligonucleotides may be attached to one or both ends. See, for example, Chang et al. (1987). Proc.Natl.Acad.Sci.USA84:4959-4963 and Nehls et al. (1996) Science 272:886-889, each of these references is incorporated herein by reference in its entirety for all purposes. Other methods for protecting exogenous polynucleotides from degradation include, but are not limited to, adding terminal amino groups and using modified internucleotide bonds, such as, for example, thiophosphates, aminophosphates, and O-methylribose or deoxyribose residues. The construct can be introduced into cells as part of a vector molecule having additional sequences, such as origin of replication, promoter, and genes encoding antibiotic resistance. Viral elements can be omitted from the construct. Furthermore, the construct can be introduced as naked nucleic acid, as a nucleic acid complexed with agents such as liposomes or poloxamer, or delivered via viruses (e.g., adenovirus, adeno-associated virus (AAV), herpesvirus, retrovirus, or lentivirus).

[0139] The constructs disclosed herein can be modified at one or both ends to incorporate one or more suitable structural features and / or impart one or more functional benefits as needed. For example, structural modifications can vary depending on the method used to deliver the constructed constructs disclosed herein to host cells (e.g., delivery using a viral vector or packaging into lipid nanoparticles for delivery). Such modifications include, for example, terminal structures such as inverted terminal repeats (ITRs), hairpins, loops, and other structures such as toroidal surfaces. For example, the constructs disclosed herein may contain one, two, or three ITRs, or may contain no more than two ITRs. Various methods of structural modification are known.

[0140] Some constructs can be inserted such that their expression is driven by an endogenous promoter at the insertion site (e.g., when the construct is integrated into the host cell). ALB Endogenous in the locus ALB (Promoter). Such constructs may not contain a promoter that drives the expression of multi-domain therapeutic proteins. For example, the expression of multi-domain therapeutic proteins may be driven by a host cell promoter (e.g., when a transgene is integrated into a host cell). ALB Endogenous in the locus ALB(Promoter). In this case, the construct may lack control elements driving its expression (e.g., promoters and / or enhancers) (e.g., promoterless construct). In other cases, the construct may contain promoters and / or enhancers, such as constitutive promoters, inducible promoters, or tissue-specific (e.g., liver or platelet-specific) promoters that drive the expression of multi-domain therapeutic proteins in the episome or upon integration. For example, the construct may be a construct for expression but not for insertion (e.g., episome construct). In some embodiments, the construct is not used for insertion. Non-limiting examples of constitutive promoters include the cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor α (EF1a) promoter, ubiquitin promoter, actin promoter, microtubule promoter, immunoglobulin promoter, functional fragments thereof, or any combination of the foregoing. For example, the promoter can be a CMV promoter or a truncated CMV promoter. Another example is the EF1a promoter. Non-limiting exemplary inducible promoters include those induced by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohols. Inducible promoters can be promoters with low basal (non-inducible) expression levels, such as Tet-On. ®Promoters (Clontech). Although not essential for expression, constructs may contain transcriptional or translational regulatory sequences, such as promoters, enhancers, isolators, internal ribosome entry sites, additional sequences encoding peptides, and / or polyadenylation signals. The construct may contain a sequence encoding a multi-domain therapeutic protein, located downstream of and operatively linked to a signal sequence encoding a signal peptide. In some examples, the nucleic acid construct works in a homology-independent manner by inserting a nucleic acid encoding a multi-domain therapeutic protein. Such nucleic acid constructs can function in, for example, non-dividing cells (e.g., cells where non-homologous end joining (NHEJ) and non-homologous recombination (HR) are the primary mechanisms for repairing double-strand DNA breaks) or dividing cells (e.g., actively dividing cells). Such constructs can be, for example, homology-independent donor constructs. In a preferred embodiment, the promoter and other regulatory sequences are adapted for human use, for example, recognized by regulatory factors in human cells (e.g., human liver cells), and are acceptable to regulatory agencies for human use. Examples of liver-specific promoters include TTR promoters, such as human or mouse TTR promoters. In one example, the construct may contain a TTR promoter, such as a mouse TTR promoter or a human TTR promoter (e.g., the coding sequence of a multi-domain therapeutic protein is operatively linked to the TTR promoter). In one example, the construct may contain a SERPINA1 enhancer, such as a mouse SERPINA1 enhancer or a human SERPINA1 enhancer (e.g., the coding sequence of a multi-domain therapeutic protein is operatively linked to the SERPINA1 enhancer). In one example, the construct may contain both a TTR promoter and a SERPINA1 enhancer, such as a human SERPINA1 enhancer and a mouse TTR promoter (e.g., the coding sequence of a multi-domain therapeutic protein is operatively linked to both the SERPINA1 enhancer and the TTR promoter).

[0141] The constructs disclosed herein can be modified to include or exclude any suitable structural features required for any particular purpose and / or to impart one or more desired functions. For example, some constructs disclosed herein do not contain homologous arms. Some constructs disclosed herein are capable of insertion into the cleavage site of a target genomic locus or target DNA sequence of a nuclease agent via non-homologous end joining (e.g., capable of inserting into safe harbor genes such as...). ALB (In the locus). For example, such constructs can be inserted into blunt-ended double-strand breaks after being cleaved with nuclease agents as disclosed herein (e.g., CRISPR / Cas systems, such as the SpyCas9 CRISPR / Cas system). In a specific example, the construct can be delivered via AAV and can be inserted by non-homologous end joining (e.g., the nucleic acid construct does not contain homologous arms).

[0142] In one specific example, the construct can be inserted via homology-independent targeted integration. For example, a multi-domain therapeutic protein-coding sequence in the construct can be flanked at a target site with a nuclease agent on each side (e.g., the same target site as the target DNA sequence used for targeted insertion, e.g., in a safe harbor gene, and the same nuclease agent is used to cleave the target DNA sequence for targeted insertion). The nuclease agent can then cleave the target site flanked by the multi-domain therapeutic protein. In one specific example, the construct is delivered via AAV-mediated delivery, and cleaving the target site flanked by the multi-domain therapeutic protein-coding sequence can remove the inverted terminal repeat (ITR) of the AAV. In some cases, if the multi-domain therapeutic protein-coding sequence is inserted into the cleavage site or target DNA sequence in the correct orientation, the target DNA sequence used for targeted insertion (e.g., the target DNA sequence in a safe harbor locus, such as a gRNA target sequence containing a motif flanked by a prototype spacer region) will no longer be present; however, if the multi-domain therapeutic protein-coding sequence is inserted into the cleavage site or target DNA sequence in the opposite orientation, the target DNA sequence will be reformed. This helps ensure that multi-domain therapeutic protein coding sequences are inserted with the correct expression orientation.

[0143] The constructs disclosed herein may contain polyadenylated sequences or polyadenylated tail sequences (e.g., downstream or at the 3' end of a multi-domain therapeutic protein-coding sequence). Methods for designing suitable polyadenylated tail sequences are well known. The polyadenylated tail sequence may encode, for example, a continuous “poly-A” sequence downstream of a multi-domain therapeutic protein-coding sequence. The poly-A tail may contain, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenine nucleotides, and optionally up to 300 adenine nucleotides. In a specific example, the poly-A tail may contain 95, 96, 97, 98, 99, or 100 adenine nucleotides. Methods for designing suitable polyadenylated tail sequences and / or polyadenylated signal sequences are well known. For example, the polyadenylated signal sequence AAUAAA is commonly used in mammalian systems, but variants such as UAUAAA or AU / GUAAA have been identified. See, for example, Proudfoot (2011). Genes & Dev.25(17):1770-82, which is incorporated herein by reference in its entirety for all purposes. The term polyadenylation signal sequence refers to any sequence that directs transcription termination and the addition of a poly-A tail to an mRNA transcript. In eukaryotes, transcription terminators are recognized by protein factors, and polyadenylation follows termination; it is the process of adding a poly(A) tail to an mRNA transcript in the presence of poly(A) polymerase. Mammalian poly(A) signals typically consist of a core sequence of about 45 nucleotides in length, which may be side-mounted with various auxiliary sequences to enhance cleavage and polyadenylation efficiency. The core sequence consists of a highly conserved upstream element (AATAAA or AAUAAA) in the mRNA, referred to as the poly A recognition motif or poly A recognition sequence, which is recognized by the cleavage and polyadenylation specific factor (CPSF); and an ill-defined downstream region constrained by the cleavage stimulating factor (CstF) (enriched in U or G and U). Examples of usable transcription terminators include, for example, human growth hormone (HGH) polyadenylation signals, simian virus 40 (SV40) late polyadenylation signals, rabbit β-globin polyadenylation signals, bovine growth hormone (BGH) polyadenylation signals, phosphoglycerate kinase (PGK) polyadenylation signals, AOX1 transcription termination sequences, CYC1 transcription termination sequences, or any transcription termination sequence known to be suitable for regulating gene expression in eukaryotic cells. In one example, the polyadenylation signal is the simian virus 40 (SV40) late polyadenylation signal. For example, a polyadenylation signal may comprise, consist essentially of, or consist of the following sequences: SEQ ID NO: 615, 169, or 161. For example, a polyadenylation signal may comprise, consist essentially of, or consist of SEQ ID NO: 169 or 161. For example, a polyadenylation signal may comprise, consist essentially of, or consist of SEQ ID NO: 169. For example, a polyadenylation signal may include, consist substantially of, or consist of SEQ ID NO: 615. In another example, the polyadenylation signal is a bovine growth hormone (BGH) polyadenylation signal or a CpG-depleted BGH polyadenylation signal. For example, a polyadenylation signal may include, consist substantially of, or consist of SEQ ID NO: 162.

[0144] In one example, the polyadenylation signal may include a BGH polyadenylation signal. For example, the BGH polyadenylation signal may include, consist substantially of, or consist of the following sequence: SEQ ID NO: 797. In another example, the polyadenylation signal may include an SV40 polyadenylation signal. For example, the SV40 polyadenylation signal may be a unidirectional SV40 late polyadenylation signal. For example, the transcription terminator sequence present in the “early” reverse orientation of SV40 may be mutated (e.g., by mutating the reverse strand AAUAAA sequence to AAUCAA). SV40 polyA is bidirectional, but “late” orientation polyadenylation is more efficient than “early” orientation polyadenylation. For example, a unidirectional SV40 late polyadenylation signal may include, consist substantially of, or consist of the following sequence: SEQ ID NO: 798. In another example, a synthetic polyadenylation signal may be used. For example, a synthetic polyadenylation signal may comprise, consist substantially of, or consist of the following sequence: SEQ ID NO: 799. In another example, two or more polyadenylation signals may be used in combination. For example, a polyadenylation signal may comprise a combination of a BGH polyadenylation signal and an SV40 polyadenylation signal (e.g., a late SV40 polyadenylation signal, such as a unidirectional late SV40 polyadenylation signal). For example, a polyadenylation signal may comprise a combination of a BGH polyadenylation signal and a unidirectional late SV40 polyadenylation signal. For example, a BGH polyadenylation signal may comprise, consist substantially of, or consist of the following sequence: SEQ ID NO: 797, and a unidirectional late SV40 polyadenylation signal may comprise, consist substantially of, or consist of the following sequence: SEQ ID NO: 798. In one specific example, the BGH polyadenylation signal may be upstream (5' end) of the SV40 polyadenylation signal (e.g., a unidirectional SV40 late polyadenylation signal). For example, the combined polyadenylation signal may comprise the sequence listed in SEQ ID NO: 800. In another example, the polyadenylation signal may comprise a combination of the BGH polyadenylation signal and a synthetic polyadenylation signal. For example, the BGH polyadenylation signal may comprise, consist substantially of, or consist of the sequence SEQ ID NO: 797, and the synthetic polyadenylation signal may comprise, consist substantially of, or consist of the sequence SEQ ID NO: 799. In some embodiments, the nucleic acid construct is a unidirectional construct.

[0145] In some implementations, a filler sequence can be used to increase the time between RNA polymerase transcription of polyA and its transcription of the next splice acceptor. For example, the filler sequence can be used between two different polyadenylation signals (e.g., between a BGH polyadenylation signal and a synthetic polyadenylation signal). For example, the filler sequence can comprise, consist substantially of, or consist of the following sequence: SEQ ID NO: 801.

[0146] In some embodiments, the MAZ element that causes polymerase pausing is used in combination with a polyadenylation signal (e.g., a BGH polyadenylation signal or an SV40 polyadenylation signal). For example, one or more (e.g., at least 1, at least 2, at least 3, at least 4, or about 1 to about 4, about 2 to about 4, about 3 to about 4, or 1, 2, 3, or 4) MAZ elements may be used in combination with a polyadenylation signal. For example, the MAZ element may comprise, consist substantially of, or consist of the following sequence: SEQ ID NO: 802.

[0147] In some embodiments, a unidirectional SV40 late polyadenylation signal is used. SV40 polyA is bidirectional, but polyadenylation in the "late" orientation is more effective than polyadenylation in the "early" orientation. The unidirectional SV40 late polyadenylation signal described herein is placed in the "late" orientation, while polyadenylation signals present in the "early" orientation are mutated or inactivated. In some embodiments, each AATAAA sequence instance in the reverse strand of the unidirectional SV40 late polyadenylation signal is mutated. For example, the two conserved AATAAA poly(A) signals present in the SV40 "early" poly(A) are mutated to AATCACAA. In some embodiments, the unidirectional SV40 late polyadenylation signal is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence listed in SEQ ID NO:798. In some implementations, the unidirectional SV40 late polyadenylation signal comprises, is substantially composed of, or is composed of the sequence listed in SEQ ID NO: 798.

[0148] The unidirectional SV40 late polyadenylation signal can be used in combination (e.g., in tandem) with one or more additional polyadenylation signals. Examples of usable transcription terminators include, for example, human growth hormone (HGH) polyadenylation signals, simian virus 40 (SV40) late polyadenylation signals, rabbit β-globin polyadenylation signals, bovine growth hormone (BGH) polyadenylation signals, phosphoglycerate kinase (PGK) polyadenylation signals, AOX1 transcription termination sequences, CYC1 transcription termination sequences, or any transcription termination sequence known to be suitable for regulating gene expression in eukaryotic cells. For example, the unidirectional SV40 late polyadenylation signal can be used in combination (e.g., in tandem) with a bovine growth hormone (BGH) polyadenylation signal, optionally wherein the BGH polyadenylation signal is upstream (5' end) of the unidirectional SV40 late polyadenylation signal. In some embodiments, the BGH polyadenylation signal is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence listed in SEQ ID NO: 797. In some embodiments, the BGH polyadenylation signal comprises, is substantially composed of, or is composed of the sequence listed in SEQ ID NO: 797. In some embodiments, the combination of the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence listed in SEQ ID NO: 800. In some embodiments, the combination of the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises, is substantially composed of, or is composed of the sequence listed in SEQ ID NO: 800.

[0149] In some implementations, a filler sequence can be used to increase the time between RNA polymerase transcription of polyA and its transcription of the next splice acceptor. For example, the filler sequence can be used between two different polyadenylation signals (e.g., between a BGH polyadenylation signal and a synthetic polyadenylation signal). For example, the filler sequence can comprise, consist substantially of, or consist of the following sequence: SEQ ID NO: 801.

[0150] In some embodiments, the MAZ element that causes polymerase pausing is used in combination with a polyadenylation signal (e.g., a BGH polyadenylation signal or an SV40 polyadenylation signal). For example, one or more (e.g., at least 1, at least 2, at least 3, at least 4, or about 1 to about 4, about 2 to about 4, about 3 to about 4, or 1, 2, 3, or 4) MAZ elements may be used in combination with a polyadenylation signal. For example, the MAZ element may comprise, consist substantially of, or consist of the following sequence: SEQ ID NO: 802.

[0151] The constructs disclosed herein may also include a splice acceptor site (e.g., operatively linked to a multi-domain therapeutic protein-coding sequence, such as located upstream or at the 5' end of such a sequence). The splice acceptor site may, for example, contain or consist of NAGs. In a specific example, the splice acceptor is... ALB Scissor acceptor (e.g., used to cut) ALB Exons 1 and 2 are spliced ​​together. ALB Scissor acceptor (i.e., ALB Exon 2 scissor acceptor). For example, this scissor acceptor can be derived from humans. ALB Gene. In another example, the splice acceptor can be derived from mice. Alb Genes (e.g., used to modify mice) Alb Exons 1 and 2 are spliced ​​together. ALB Scission receptor (i.e., mouse) Alb Exon 2 splice acceptor). In another example, the splice acceptor is a splice acceptor from a gene encoding a polypeptide of interest (e.g., SMPD1 (Scipector receptors). For example, such scissor receptors can originate from humans. SMPD1 Gene. Alternatively, this splice acceptor can be derived from mice. SMPD1 Genes. Other suitable splice acceptor sites (including artificial splice acceptors) that can be used in eukaryotes are well known. See, for example, Shapiro et al., (1987). Nucleic Acids Res .15:7155-7174 and Burset et al., (2001) Nucleic Acids Res Each of these references (.29:255-259) is incorporated herein by reference in its entirety for all purposes. In a specific example, the scissor acceptor is the mouse. Alb Exon 2 splicing acceptor. In a specific example, the splicing acceptor may contain, consist substantially of, or consist of SEQ ID NO: 163.

[0152] In some examples, the nucleic acid constructs disclosed herein may be bidirectional constructs, which are described in more detail below. In some examples, the nucleic acid constructs disclosed herein may be unidirectional constructs, which are described in more detail below. Similarly, in some examples, the nucleic acid constructs disclosed herein may be contained in vectors (e.g., viral vectors, such as AAV or rAAV8) and / or lipid nanoparticles, as described in more detail elsewhere herein.

[0153] (1) Multi-domain therapeutic proteins Multidomain therapeutic proteins, as described herein, include acid sphingomyelinase (ASM) polypeptides (ASM or its biologically active portion, used to provide alternative ASM enzyme activity) linked to or fused to a TfR-binding delivery domain. The TfR-binding domain and ASM are described in more detail below. The TfR-binding domain provides binding to the internalizing factor TfR. Multidomain therapeutic proteins produced by the liver target muscle and the CNS by targeting TfR, which is expressed in muscle and on brain endothelial cells. Transcytosis of TfR in these cells enables crossing the blood-brain barrier. In some multidomain therapeutic proteins, the TfR-binding delivery domain is covalently linked to ASM. The covalent bond can be of any type (i.e., any bond involving shared electrons). In some cases, the covalent bond is a peptide bond between two amino acids, such that the ASM and the TfR-binding delivery domain, in whole or in part, form a continuous polypeptide chain, as in fusion proteins. In some cases, the ASM portion and the TfR-binding delivery domain portion are directly linked. In other cases, a linker such as a peptide linker is used to connect the two parts. Any suitable linker can be used. See Chen et al., “Fusion protein linkers: property, design and functionality,” 65(10) Adv Drug Deliv Rev. 1357-69 (2013). In some cases, a cleavable linker is used. For example, a cathepsin-cleavable linker can be inserted between the TfR-binding delivery domain and the ASM to facilitate the removal of the TfR-binding delivery domain from the lysosome. In another example, the linker may contain an amino acid sequence, for example, about 10 amino acids in length, such as 1, 2, 3, 4, 5, 6, 7, 8, 8 or 10 Gly4Ser (SEQ ID NO: 537) repeat sequences. In one example, the linker contains, is substantially composed of or consists of three such repeat sequences (SEQ ID NO: 616). For example, the encoding sequence of the connector may contain, consist substantially of, or consist of any of the following sequences: SEQ ID NO: 618-622 and 803. In another example, the connector contains, consists substantially of, or consists of two such repeating sequences (SEQ ID NO: 617). For example, the encoding sequence of the connector may contain, consist substantially of, or consist of any of the following sequences: SEQ ID NO: 623-629. In another example, the connector contains, consists substantially of, or consists of one such repeating sequence (SEQ ID NO: 537).For example, the coded sequence of the connector may contain, consist substantially of, or consist of the following sequence: SEQ ID NO: 630 or 804. In another example, a rigid connector, such as a 2XH4 connector, may be used. In one example, the connector contains, consists substantially of, or consists of the following sequence: AEAAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO: 808). For example, the coded sequence of the connector may contain, consist substantially of, or consist of the following sequence: SEQ ID NO: 807.

[0154] In a specific multidomain therapeutic protein, the ASM (e.g., N-terminus) is covalently linked to the C-terminus of the heavy chain or light chain of the anti-TfR antibody (i.e., the format of the multidomain therapeutic protein is anti-TfR:ASM from N-terminus to C-terminus). In another specific multidomain therapeutic protein, the ASM is covalently linked to the N-terminus of the heavy chain or light chain of the anti-TfR antibody (i.e., the format of the multidomain therapeutic protein is ASM:anti-TfR from N-terminus to C-terminus). In yet another specific embodiment, the ASM (e.g., N-terminus) is linked to the C-terminus of the anti-TfR scFv domain (i.e., the format of the multidomain therapeutic protein is anti-TfR-scFv:ASM, such as anti-TfR-scFv(V) from N-terminus to C-terminus). L V H In another specific embodiment, the ASM (e.g., N-terminus) is attached to the C-terminus of the anti-TfR Fab heavy chain (i.e., the format of the multi-domain therapeutic protein is anti-TfR-Fab (light chain heavy chain):ASM from the N-terminus to the C-terminus). In another specific embodiment, the ASM (e.g., N-terminus) is attached to the C-terminus of the anti-TfR Fab light chain (i.e., the format of the multi-domain therapeutic protein is anti-TfR-Fab (heavy chain light chain):ASM from the N-terminus to the C-terminus).

[0155] (a) Acidic sphingomyelinase (ASM) Acidic sphingomyelinase (ASM; sphingomyelin phosphodiesterase; aSMase; ASM; SMPD1) is composed of SMPD1 ( ASM The gene ASM is encoded by the lysosomal acid sphingomyelinase that converts sphingomyelin into ceramides. It also possesses phospholipase C activity. Defects in this gene are the cause of acid sphingomyelinase deficiency (ASMD), such as Niemann-Pick disease type A (NPA) and Niemann-Pick disease type B (NPB).

[0156] The ASM expressed by the compositions and methods disclosed herein can be any wild-type or variant ASM. In one example, the ASM is the human ASM protein. SMPD1 The gene (NCBI GeneID 6609) is located in the 11p15.4 region on chromosome 11 (genome version: GRCh38.p14 (GCF_000001405.40); location: NC_000011.10 (6390474..6394996)). Human ASM is designated as UniProt reference number P17405. An exemplary amino acid sequence of human ASM is designated as NCBI accession number NP_000534.3 and is listed as SEQ ID NO: 728 (signal peptide: positions 1-46; mature ASM: positions 47-631). Exemplary human ASM The mRNA (cDNA) sequence is designated as NCBI accession number NM_000543.5 and is listed as SEQ ID NO: 729. Exemplary human ASM The coding sequence is designated as CCDS ID CCDS44531.1 and is listed as SEQ ID NO: 730 (the stop codon has been removed). An exemplary mature human ASM (ASM(47-631)) amino acid sequence (i.e., the human ASM sequence after removing the signal peptide) is listed as SEQ ID NO: 731. An exemplary mature human ASM (ASM(47-631)) coding sequence is listed as SEQ ID NO: 734. Another exemplary mature human ASM (ASM(47-631)) coding sequence is listed as SEQ ID NO: 735. An exemplary amino acid sequence (ASM(62-631)) of a processed, secreted, intermediate form of human ASM (sASM) lacking the first 61 amino acids is listed as SEQ ID NO: 733. An exemplary coding sequence of human ASM (ASM(62-631)) lacking the first 61 amino acids is listed as SEQ ID NO: 732.

[0157] In some examples, ASM (e.g., human ASM) is a wild-type ASM (e.g., wild-type human ASM) sequence, or its biologically active portion or fragment. For example, ASM may lack the ASM signal peptide. For example, ASM may be a fragment containing the mature ASM amino acid sequence (i.e., the ASM sequence after removing the signal peptide).

[0158] In one specific example, the ASM may contain SEQ ID NO: 728, or may be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 728. In another specific example, the ASM may consist substantially of SEQ ID NO: 728. In yet another specific example, the ASM may consist of SEQ ID NO: 728.

[0159] In one specific example, the ASM may contain SEQ ID NO: 731, or may be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 731. In another specific example, the ASM may consist substantially of SEQ ID NO: 731. In yet another specific example, the ASM may consist of SEQ ID NO: 731.

[0160] In one specific example, the ASM may contain SEQ ID NO: 733, or may be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 733. In another specific example, the ASM may consist substantially of SEQ ID NO: 733. In yet another specific example, the ASM may consist of SEQ ID NO: 733.

[0161] The ASM coding sequence in the constructs disclosed herein may contain one or more modifications, such as codon optimization (e.g., codon optimization of human codons), CpG dinucleotide depletion, mutation of cryptic splice sites, addition of one or more glycosylation sites, or any combination thereof. CpG dinucleotides in the constructs can limit the therapeutic efficacy of the construct. First, unmethylated CpG dinucleotides can interact with the host toll-like receptor 9 (TLR-9) to stimulate an innate, pro-inflammatory immune response. Second, once CpG dinucleotides are methylated, they can lead to inhibition of transgene expression coordinated by methyl-CpG binding proteins. Cryptic splice sites are sequences in premessenger RNA that are not normally used as splice sites but can be activated, for example, by inactivating typical splice sites or by mutations that form splice sites that were not previously present. Accurate splice site selection is crucial for successful gene expression, and removal of cryptic splice sites can facilitate the use of normal or intended splice sites.

[0162] In one example, the ASM coding sequence in the construct disclosed herein has been mutated or one or more hidden splicing sites have been removed. In another example, the ASM coding sequence in the construct disclosed herein has been mutated or all identified hidden splicing sites have been removed. In another example, the ASM coding sequence in the construct disclosed herein has one or more CpG dinucleotides removed (i.e., it is CpG depleted). In another example, the ASM coding sequence in the construct disclosed herein has all CpG dinucleotides removed (i.e., it is completely CpG depleted). In another example, the ASM coding sequence in the construct disclosed herein is codon-optimized (e.g., codon-optimized for expression in humans or mammals). In one specific example, the ASM coding sequence in the construct disclosed herein has one or more CpG dinucleotides removed (i.e., it is CpG depleted) and one or more hidden splicing sites have been mutated or removed. In another specific example, the ASM coding sequence in the construct disclosed herein has all CpG dinucleotides removed and one or more or all identified hidden splicing sites have been mutated or removed. In another specific example, the ASM coding sequence in the construct disclosed herein has had one or more CpG dinucleotides removed (i.e., is CpG depleted) and is codon-optimized (e.g., codon-optimized for expression in humans or mammals). In yet another specific example, the ASM coding sequence in the construct disclosed herein has all CpG dinucleotides removed (i.e., is completely CpG depleted) and is codon-optimized (e.g., codon-optimized for expression in humans or mammals).

[0163] Various ASM coding sequences are provided. In one example, the ASM coding sequence is (or comprises) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 730. In another example, the ASM coding sequence is (or comprises) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 730. In another example, the ASM coding sequence is (or comprises) at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 730. In another example, the ASM coding sequence consists essentially of the sequence in SEQ ID NO: 730. In another example, the ASM coding sequence consists of the sequence listed in SEQ ID NO: 730. Optionally, the ASM coding sequence encodes an ASM protein (or an ASM protein comprising the sequence described below), which or the sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 728 (and, for example, retains the activity of native ASM). Optionally, the ASM coding sequence encodes an ASM protein (or an ASM protein comprising the sequence described below), which or the sequence is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 728 (and, for example, retains the activity of native ASM). Optionally, the ASM-coding sequence in the above examples encodes an ASM protein (or an ASM protein comprising the sequence listed below), which is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 728 (and, for example, retains the activity of native ASM). Optionally, the ASM-coding sequence in the above examples encodes an ASM protein comprising the sequence listed in SEQ ID NO: 728. Optionally, the ASM-coding sequence in the above examples encodes an ASM protein substantially composed of the sequence listed in SEQ ID NO: 728. Optionally, the ASM-coding sequence in the above examples encodes an ASM protein composed of the sequence listed in SEQ ID NO: 728.

[0164] In one example, the ASM coding sequence is (or comprises) a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 730. In another example, the ASM coding sequence is (or comprises) a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 730, and encodes an ASM protein (or an ASM protein comprising the sequence described below), which or the sequence is at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 728. In another example, the ASM coding sequence is (or comprises) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 730, and encodes an ASM protein comprising the sequence listed in SEQ ID NO: 728. In another example, the ASM coding sequence is (or comprises) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 730. In another example, the ASM coding sequence is (or comprises) a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 730, and encodes an ASM protein (or an ASM protein comprising the sequence listed in SEQ ID NO: 728), which or the sequence is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 728. In another example, the ASM coding sequence is (or comprises) a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 730, and encodes an ASM protein comprising the sequence listed in SEQ ID NO: 728. In another example, the ASM coding sequence is (or comprises) a sequence that is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 730. In another example, the ASM coding sequence is (or contains) a sequence that is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 730, and encodes an ASM protein (or an ASM protein containing the sequence described below), which or the sequence is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 728.In another example, the ASM coding sequence is (or comprises) at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 730, and encodes an ASM protein comprising the sequence listed in SEQ ID NO: 728. In another example, the ASM coding sequence comprises the sequence listed in SEQ ID NO: 730. In another example, the ASM coding sequence consists substantially of the sequence listed in SEQ ID NO: 730. In another example, the ASM coding sequence consists of the sequence listed in SEQ ID NO: 730. The ASM coding sequence may be, for example, CpG depleted (e.g., fully CpG depleted) and / or codon-optimized. For example, the ASM coding sequence may be CpG depleted (e.g., fully CpG depleted) and codon-optimized. Optionally, the ASM coding sequence encodes an ASM protein (or an ASM protein comprising the sequence described below), which or the sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 728 (and, for example, retains the activity of native ASM). Optionally, the ASM-coding sequence in the above examples encodes an ASM protein (or an ASM protein comprising the sequence listed below), which is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 728 (and, for example, retains the activity of native ASM). Optionally, the ASM-coding sequence in the above examples encodes an ASM protein comprising the sequence listed in SEQ ID NO: 728. Optionally, the ASM-coding sequence in the above examples encodes an ASM protein substantially composed of the sequence listed in SEQ ID NO: 728. Optionally, the ASM-coding sequence in the above examples encodes an ASM protein composed of the sequence listed in SEQ ID NO: 728.

[0165] Various ASM-coded sequences are provided. In one example, the ASM-coded sequence is (or comprises) a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 732. In another example, the ASM-coded sequence is (or comprises) a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 732. In another example, the ASM-coded sequence is (or comprises) a sequence that is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 732. In another example, the ASM-coded sequence comprises the sequence listed in SEQ ID NO: 732. In yet another example, the ASM-coded sequence consists essentially of the sequence listed in SEQ ID NO: 732. In another example, the ASM coding sequence consists of the sequence listed in SEQ ID NO: 732. Optionally, the ASM coding sequence encodes an ASM protein (or an ASM protein comprising the sequence below), which or the sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 733 (and, for example, retains the activity of native ASM). Optionally, the ASM coding sequence encodes an ASM protein (or an ASM protein comprising the sequence below), which or the sequence is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 733 (and, for example, retains the activity of native ASM). Optionally, the ASM-coding sequence in the above examples encodes an ASM protein (or an ASM protein comprising the sequence listed below), which is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 733 (and, for example, retains the activity of native ASM). Optionally, the ASM-coding sequence in the above examples encodes an ASM protein comprising the sequence listed in SEQ ID NO: 733. Optionally, the ASM-coding sequence in the above examples encodes an ASM protein substantially composed of the sequence listed in SEQ ID NO: 733. Optionally, the ASM-coding sequence in the above examples encodes an ASM protein composed of the sequence listed in SEQ ID NO: 733.

[0166] In one example, the ASM coding sequence is (or comprises) a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 732. In another example, the ASM coding sequence is (or comprises) a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 733, and encodes an ASM protein (or an ASM protein comprising the sequence described below), which or the sequence is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 733. In another example, the ASM coding sequence is (or comprises) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 732, and encodes an ASM protein comprising the sequence listed in SEQ ID NO: 733. In another example, the ASM coding sequence is (or comprises) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 732. In another example, the ASM coding sequence is (or comprises) a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 732, and encodes an ASM protein (or an ASM protein comprising the sequence listed in SEQ ID NO: 733), which or the sequence is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 733. In another example, the ASM coding sequence is (or comprises) a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 732, and encodes an ASM protein comprising the sequence listed in SEQ ID NO: 733. In another example, the ASM coding sequence is (or comprises) a sequence that is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 732. In another example, the ASM coding sequence is (or contains) a sequence that is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 732, and encodes an ASM protein (or an ASM protein containing the sequence described below), which or the sequence is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 733.In another example, the ASM coding sequence is (or comprises) at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 732, and encodes an ASM protein comprising the sequence listed in SEQ ID NO: 733. In another example, the ASM coding sequence comprises the sequence listed in SEQ ID NO: 732. In another example, the ASM coding sequence consists substantially of the sequence listed in SEQ ID NO: 732. In another example, the ASM coding sequence consists of the sequence listed in SEQ ID NO: 732. The ASM coding sequence may be, for example, CpG depleted (e.g., fully CpG depleted) and / or codon-optimized. For example, the ASM coding sequence may be CpG depleted (e.g., fully CpG depleted) and codon-optimized. Optionally, the ASM coding sequence encodes an ASM protein (or an ASM protein comprising the sequence described below), which or the sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 733 (and, for example, retains the activity of native ASM). Optionally, the ASM-coding sequence in the above examples encodes an ASM protein (or an ASM protein comprising the sequence listed below), which is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 733 (and, for example, retains the activity of native ASM). Optionally, the ASM-coding sequence in the above examples encodes an ASM protein comprising the sequence listed in SEQ ID NO: 733. Optionally, the ASM-coding sequence in the above examples encodes an ASM protein substantially composed of the sequence listed in SEQ ID NO: 733. Optionally, the ASM-coding sequence in the above examples encodes an ASM protein composed of the sequence listed in SEQ ID NO: 733.

[0167] Various ASM coding sequences are provided. In one example, the ASM coding sequence is (or comprises) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 734 or 735. In another example, the ASM coding sequence is (or comprises) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 734 or 735. In yet another example, the ASM coding sequence is (or comprises) at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 734 or 735. In yet another example, the ASM coding sequence consists essentially of the sequence in SEQ ID NO: 734 or 735. In another example, the ASM coding sequence consists of the sequence listed in SEQ ID NO: 734 or 735. Optionally, the ASM coding sequence encodes an ASM protein (or an ASM protein comprising the sequence below), which or the sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 731 (and, for example, retains the activity of native ASM). Optionally, the ASM coding sequence encodes an ASM protein (or an ASM protein comprising the sequence below), which or the sequence is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 731 (and, for example, retains the activity of native ASM). Optionally, the ASM-coding sequence in the above examples encodes an ASM protein (or an ASM protein comprising the sequence listed below), which is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 731 (and, for example, retains the activity of native ASM). Optionally, the ASM-coding sequence in the above examples encodes an ASM protein comprising the sequence listed in SEQ ID NO: 731. Optionally, the ASM-coding sequence in the above examples encodes an ASM protein substantially composed of the sequence listed in SEQ ID NO: 731. Optionally, the ASM-coding sequence in the above examples encodes an ASM protein composed of the sequence listed in SEQ ID NO: 731.

[0168] In one example, the ASM coding sequence is (or comprises) a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 734 or 735. In another example, the ASM coding sequence is (or comprises) a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 731, and encodes an ASM protein (or an ASM protein comprising the sequence described below), which is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 731. In another example, the ASM coding sequence is (or comprises) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 734 or 735, and encodes an ASM protein comprising the sequence listed in SEQ ID NO: 731. In another example, the ASM coding sequence is (or comprises) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the sequence in SEQ ID NO: 734 or 735. In another example, the ASM coding sequence is (or comprises) a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 734 or 735, and encodes an ASM protein (or an ASM protein comprising the sequence listed in SEQ ID NO: 731), which or the sequence is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 731. In another example, the ASM coding sequence is (or comprises) a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 734 or 735, and encodes an ASM protein comprising the sequence listed in SEQ ID NO: 731. In another example, the ASM coding sequence is (or comprises) a sequence that is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 734 or 735.In another example, the ASM coding sequence is (or comprises) a sequence that is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 734 or 735, and encodes an ASM protein (or an ASM protein comprising the sequence listed below), which or the sequence is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 731. In another example, the ASM coding sequence is (or comprises) a sequence that is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 734 or 735, and encodes an ASM protein comprising the sequence listed in SEQ ID NO: 731. In another example, the ASM coding sequence comprises the sequence listed in SEQ ID NO: 734 or 735. In another example, the ASM coding sequence consists substantially of the sequence listed in SEQ ID NO: 734 or 735. In another example, the ASM coding sequence consists of the sequence listed in SEQ ID NO: 734 or 735. The ASM coding sequence may be, for example, CpG-depleted (e.g., completely CpG-depleted) and / or codon-optimized. For example, the ASM coding sequence may be CpG-depleted (e.g., completely CpG-depleted) and codon-optimized. Optionally, the ASM coding sequence encodes an ASM protein (or an ASM protein comprising the sequence described below), which or the sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 733 (and, for example, retaining the activity of native ASM). Optionally, the ASM coding sequence encodes an ASM protein (or an ASM protein comprising the sequence described below), which or the sequence is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 731 (and, for example, retaining the activity of native ASM). Optionally, the ASM-coding sequence in the above examples encodes an ASM protein (or an ASM protein comprising the sequence listed below), which is at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 731 (and, for example, retains the activity of native ASM). Optionally, the ASM-coding sequence in the above examples encodes an ASM protein comprising the sequence listed in SEQ ID NO: 731. Optionally, the ASM-coding sequence in the above examples encodes an ASM protein substantially composed of the sequence listed in SEQ ID NO: 731. Optionally, the ASM-coding sequence in the above examples encodes an ASM protein composed of the sequence listed in SEQ ID NO: 731.

[0169] When a specific ASM or multidomain therapeutic protein nucleic acid construct sequence is disclosed herein, it means that the disclosed sequence or its reverse complement is covered. For example, if the ASM or multidomain therapeutic protein nucleic acid construct disclosed herein consists of the hypothetical sequence 5'-CTGGACCGA-3', it also means that the reverse complement of that sequence (5'-TCGGTCCAG-3') is covered. Similarly, when construct elements are disclosed herein in a specific 5' to 3' order, it also means that the reverse complement of those element orders is covered. One reason for this is that in many embodiments disclosed herein, the ASM or multidomain therapeutic protein nucleic acid construct is part of a single-stranded recombinant AAV vector. The single-stranded AAV genome is packaged as sense (positive strand) or antisense (negative strand) genomes, and the + and - polar single-stranded AAV genomes are packaged into mature rAAV virions at equal frequencies. See, for example, LING et al., (2015). J. Mol.Genet.Med. 9(3):175, Zhou et al., (2008) Mol.Ther. 16(3):494-499, and Samulski et al., (1987) J. Virol. Each of these references (61:3096-3101) is incorporated herein by reference in its entirety for all purposes.

[0170] (b) TfR combined with delivery domain The multi-domain therapeutic proteins disclosed herein may include a TfR-binding delivery domain fused to an ASM peptide. The TfR-binding domain provides binding to the internalizing factor transferrin receptor protein 1 (TfR; UniProt Ref. P02786). TfR (also known as TR, TfR1, and Trfr) is composed of… TFRC Genetically encoded. TfR is expressed in muscle and on brain endothelial cells. Transcytosis of TfR in these cells enables crossing the blood-brain barrier. In some embodiments, multi-domain therapeutic proteins containing a TfR-binding delivery domain fused to ASM (e.g., scFv) do not alter transferrin uptake. In some embodiments, multi-domain therapeutic proteins containing a TfR-binding delivery domain fused to ASM (e.g., scFv) do not alter iron homeostasis. In some embodiments, multi-domain therapeutic proteins containing a TfR-binding delivery domain fused to ASM (e.g., scFv) do not alter transferrin uptake or iron homeostasis.

[0171] Transferrin receptor 1 (TfR) is a membrane receptor involved in controlling iron supply to cells via binding to transferrin (the main siderophore protein). Transferrin receptor 1 is composed of… TFRCGene expression. Transferrin receptor 1 may be referred to herein as TFRC. This receptor plays a crucial role in the control of cell proliferation because iron is essential for maintaining the activity of ribonucleotide reductase, and is the only enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides. Preferably, the TfR is human TfR (hTfR). See, for example, accession numbers NP_001121620.1; BAD92491.1 and NP_001300894.1; and e!Ensembl entry: ENSG00000072274. Human transferrin receptor 1 is expressed in several tissues, including but not limited to: cerebral cortex; cerebellum; hippocampus; caudate nucleus; parathyroid gland; adrenal gland; bronchus; lung; oral mucosa; esophagus; stomach; duodenum; small intestine; colon; rectum; liver; gallbladder; pancreas; kidney; bladder; testis; epididymis; prostate; vagina; ovary; fallopian tube; endometrium; cervix; placenta; breast; myocardium; smooth muscle; soft tissue; skin; appendix; lymph nodes; tonsils; and bone marrow. The associated transferrin receptor is transferrin receptor 2 (TfR2). Human transferrin receptor 2 shares approximately 45% sequence identity with human transferrin receptor 1. "Trinder & Baker, Transferrin receptor 2: a new molecule iniron metabolism." Int J Biochem Cell Biol. 2003, March; 35(3):292-6. Unless otherwise stated, the transferrin receptor as used herein generally refers to transferrin receptor 1 (e.g., human transferrin receptor 1).

[0172] Human transferrin (Tf) is a single-chain 80 kDa member of the anion-binding protein superfamily. Transferrin is a 698-amino acid precursor, broken down into a 19-amino acid signal sequence followed by a 679-amino acid mature segment typically containing 19 intrachain disulfide bonds. N-terminal and C-terminal sidebands (or domains) bind ferric iron via interactions with specific anions (e.g., bicarbonate) and four amino acids (His, Asp, and two Tyr). Iron-deferrotransferrin initially binds an iron atom to its C-terminus, followed by subsequent iron binding through the N-terminus to form iron-saturated transferrin (diferrotransferrin, iron-saturated Tf). Through its C-terminal iron-binding domain, iron-saturated transferrin interacts with TfR on the cell surface, where it is internalized into acidified endosomes. Iron dissociates from the Tf molecules in these endosomes and is transported as ferrous iron into the cytosol. In addition to TfR, transferrin has also been reported to bind to cupulin, IGFBP3, microbial iron-binding protein, and liver-specific TfR2.

[0173] The blood-brain barrier (BBB) ​​is located within the brain's microvascular system and regulates the pathways through which molecules from the blood enter the brain. (Burkhart et al., Accessing targeted nanoparticles to the brain: the vascularroute. Curr Med Chem. 2014; 21(36):4092-9.) Transcellular transport across brain capillary endothelial cells can occur via the following pathways: 1) leukocyte entry into the cell; 2) carrier-mediated inward transport, such as glucose transporter 1 (GLUT-1) inward transport of glucose, amino acid transporter 1 (LAT-1) inward transport of amino acids, and small peptide transporter peptide B (OATP-B) inward transport of small anion transporters; 3) paracellular channels of small hydrophobic molecules; 4) adsorption-mediated transcytosis, such as transcytosis of albumin and cationized molecules; 5) passive diffusion of lipid-soluble nonpolar solutes (including CO2 and O2); and 6) receptor-mediated transcytosis, such as transcytosis of insulin by the insulin receptor and transcytosis of Tf by the TfR. (Johnsen et al., Targeting the transferrin receptor for brain drug delivery, Prog Neurobiol. 2019 Oct; 181:101665.)

[0174] For example, an anti-TfR:ASM fusion protein was presented, exhibiting high affinity for the transferrin receptor and excellent blood-brain barrier crossing. Surprisingly, the fusion exhibiting high binding affinity for TfR crossed the blood-brain barrier more efficiently than low-affinity binders. We found that the high-affinity antibody, in the anti-hTFRscfv:payload format, showed optimal delivery to the central nervous system and muscles. This contrasts with previous findings using monovalent and bivalent anti-TFR antibodies, in which low-affinity antibodies crossed the blood-brain barrier more efficiently. The fusion protein presented in this paper enables efficient delivery of ASM to the brain, thus potentially offering effective treatment for diseases such as ASM deficiency (ASMD).

[0175] This document provides antigen-binding proteins that specifically bind to transferrin receptors, preferably human transferrin receptor 1 (anti-hTfR), such as antibodies, and their antigen-binding fragments, such as Fab and scFv. For example, in one embodiment, anti-hTfR is in the form of a fusion protein. The fusion protein comprises an anti-hTfR antigen-binding protein fused to an ASM. Anti-hTfR effectively crosses the blood-brain barrier (BBB) ​​and thereby delivers the fused ASM to the brain.

[0176] Antigen-binding proteins that specifically bind to transferrin receptors and their fusion proteins (e.g., fused with tags such as His6 and / or myc) (e.g., human transferrin receptors (e.g., REGN2431) or monkey transferrin receptors (e.g., REGN2054)) at approximately 25 °C (e.g., in surface plasmon resonance assays) at approximately 20 nM K. D Or bind with a higher affinity. This antigen-binding protein may be referred to as "anti-TfR". In some embodiments, the antigen-binding protein binds at approximately 0.41 nM K. D Or it may bind with a stronger affinity to the human transferrin receptor. In some embodiments, the antigen-binding protein binds at approximately 3 nM K. D Or it may bind with a stronger affinity to the human transferrin receptor. In some embodiments, the antigen-binding protein binds at a K+ level of approximately 0.45 nM to 3 nM. D It binds to the human transferrin receptor. In some embodiments, Fab with HCVR and LCVR binds at approximately 0.65 nM K. D Or it may bind with a stronger affinity to the human transferrin receptor. In some embodiments, the fusion protein disclosed herein binds at approximately 1 × 10⁻⁶. -7 M of K D Or it may bind to the human transferrin receptor with a stronger affinity.

[0177] In one embodiment, the anti-hTfR scFv:ASM fusion protein comprises a scFv containing a variable region arrangement of LCVR-HCVR or HCVR-LCVR, wherein HCVR and LCVR are optionally linked by a linker and the scFv is optionally linked to ASM via a linker (e.g., LCVR-(Gly4Ser)3(SEQ ID NO: 616)-HCVR-(Gly4Ser)2(SEQ ID NO: 617))-ASM; or LCVR-(Gly4Ser)3(SEQ ID NO: 616)-HCVR-(Gly4Ser)2(SEQ ID NO: 617))-ASM (Gly4Ser = SEQ ID NO: 537)). In one example, the scFv contains a variable region arrangement of LCVR-HCVR. In another example, the scFv contains a variable region arrangement of HCVR-LCVR. In one example, the connector between the HCVR and LCVR comprises, is substantially composed of, or consists of three such repeating sequences (SEQ ID NO: 616). For example, the coding sequence of this connector may comprise, is substantially composed of, or consists of any of SEQ ID NO: 618-622 and 803. In another example, the connector between the HCVR and LCVR comprises, is substantially composed of, or consists of two such repeating sequences (SEQ ID NO: 617). For example, the coding sequence of this connector may comprise, is substantially composed of, or consists of any of SEQ ID NO: 623-629. In yet another example, the connector between the HCVR and LCVR comprises, is substantially composed of, or consists of one such repeating sequence (SEQ ID NO: 537). For example, the coding sequence of this connector may comprise, is substantially composed of, or consists of SEQ ID NO: 630 or 804. In one example, the connector between the scFv and the ASM comprises, is substantially composed of, or consists of three such repeating sequences (SEQ ID NO: 616). For example, the encoding sequence of this connector may comprise, is substantially composed of, or consists of any of SEQ ID NO: 618-622 and 803. In another example, the connector between the scFv and the ASM comprises, is substantially composed of, or consists of two such repeating sequences (SEQ ID NO: 617).For example, the coded sequence of the connector may contain, consist substantially of, or consist of any of the following sequences: SEQ ID NO: 623-629. In another example, the connector between the scFv and the ASM contains, consists substantially of, or consists of a repeating sequence (SEQ ID NO: 537). For example, the coded sequence of the connector may contain, consist substantially of, or consist of the following sequences: SEQ ID NO: 630 or 804. In another example, a rigid connector, such as a 2XH4 connector, may be used. In one example, the connector contains, consists substantially of, or consists of the following sequences: AEAAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO: 808). For example, the coded sequence of the connector may contain, consists substantially of, or consist of the following sequences: SEQ ID NO: 807.

[0178] Anti-hTfR:ASM optionally includes a signal peptide linked to an antigen-binding protein that specifically binds to a transferrin receptor (TfR), preferably, (optionally via a linker) a human transferrin receptor (hTfR) fused to ASM. In one embodiment, the signal peptide is an mROR signal sequence (e.g., mROR signal sequence -LCVR-(Gly4Ser)3(SEQ ID NO: 616)-HCVR-(Gly4Ser)2(SEQ ID NO: 617))-ASM; or LCVR-(Gly4Ser)3(SEQ ID NO: 616)-HCVR-(Gly4Ser)2(SEQ ID NO: 617))-ASM (Gly4Ser = SEQ ID NO: 537)). The terms "fused" or "tethered" in relation to fusion peptides refer to peptides that are directly or indirectly linked (e.g., via a linker or other peptides).

[0179] In one implementation, amino acids are assigned to each frame or CDR domain in the immunoglobulin according to the definitions in the following literature: Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md., 5th ed., NIH Publication No. 91-3242 (1991); Kabat (1978) Adv.Prot.Chem. 32:1-75; Kabat et al., (1977) J. Biol.Chem. 252:6609-6616; Chothia et al., (1987) J Mol.Biol. 196:901-917; or Chothia et al., (1989) Nature 342: 878-883. Therefore, antibody and antigen-binding fragments containing V are included. H CDR and V L The CDR, the V H and V L It contains amino acid sequences as listed herein (see, for example, the sequences in Table 2, or variants thereof), wherein the CDR is as defined according to Kabat and / or Chothia.

[0180] In some multi-domain therapeutic proteins, the TfR-binding delivery domain is an antibody, antibody fragment, or other antigen-binding protein. Examples of antigen-binding proteins include, for instance, receptor-fusion molecules, capture molecules, receptor-Fc fusion molecules, antibodies, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-chain Fv (scFv) molecules, dAb fragments, separated complementarity-determining regions (CDRs), CDR3 peptides, restricted FR3-CDR3-FR4 peptides, domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, nanobodies, monovalent nanobodies, divalent nanobodies, small modular immunopharmaceuticals (SMIPs), camelid antibodies (VHH heavy chain homodimer antibodies), and shark variable IgNAR domains.

[0181] This document provides antibodies that specifically bind to human transferrin receptor 1. As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains interconnected by disulfide bonds: two heavy chains (HC) and two light chains (LC). In one embodiment, each antibody heavy chain (HC) contains a heavy chain variable region ("HCVR" or "V"). H(e.g., containing SEQ ID NO: 171, 680, 181, 681, 191, 682, 201, 211, 221, 685, 231, 687, 241, 689, 251, 261, 691, 271, 281, 692, 291, 301, 311, 694, 321, 331, 696, 341, 351, 697, 361, 699, 371, 700, 381, 391, 401, 411, 421, 701, 431, 441, 451, 461, 471, 702 and / or 481, or variants thereof) and a heavy chain constant region (e.g., human IgG, human IgG1 or human IgG4); and each antibody light chain (LC) contains a light chain variable region (“LCVR” or “V”). L (e.g., SEQ ID NO: 176, 186, 196, 206, 683, 216, 684, 226, 686, 236, 688, 246, 690, 256, 266, 276, 286, 693, 296, 306, 316, 695, 326, 336, 346, 356, 698, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 632, 486 and / or 703, or variants thereof) and a light chain constant region (e.g., human κ or human λ). In one embodiment, each antibody heavy chain (HC) includes a heavy chain variable region (“HCVR” or “V”). H (e.g., containing SEQ ID NO: 391 or 411, or a variant thereof) and a heavy chain constant region (e.g., human IgG, human IgG1, or human IgG4); and each antibody light chain (LC) contains a light chain variable region (“LCVR” or “V”). L (e.g., SEQ ID NO: 396 or 416, or variants thereof) and a light chain constant region (e.g., human κ or human λ). In one embodiment, each antibody heavy chain (HC) includes a heavy chain variable region (“HCVR” or “V”). H (e.g., containing SEQ ID NO: 391, or a variant thereof) and a heavy chain constant region (e.g., human IgG, human IgG1, or human IgG4); and each antibody light chain (LC) contains a light chain variable region (“LCVR” or “V”). L (e.g., SEQ ID NO: 396, or a variant thereof) and a light chain constant region (e.g., human κ or human λ). In one embodiment, each antibody heavy chain (HC) contains a heavy chain variable region (“HCVR” or “V”). H(e.g., containing SEQ ID NO: 411, or a variant thereof) and a heavy chain constant region (e.g., human IgG, human IgG1, or human IgG4); and each antibody light chain (LC) contains a light chain variable region (“LCVR” or “V”). L (e.g., SEQ ID NO: 416, or a variant thereof) and light chain constant regions (e.g., human κ or human λ). V H District and V L The region can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conservative regions known as framing regions (FRs). Each V H and V L It contains three CDRs and four FRs. The anti-TfR antibody disclosed in this paper can also be fused with ASM.

[0182] The anti-TfR antigen-binding proteins described herein can be antigen-binding fragments of antibodies capable of tethering to ASM. As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to an immunoglobulin molecule that binds an antigen but does not contain the complete antibody (preferably, the complete antibody is IgG) of its entire sequence. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; and (vi) dAb fragments; consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., a separated complementarity-determining region (CDR), such as a CDR3 peptide) or a bound FR3-CDR3-FR4 peptide. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-grafted antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, and small modular immunopharmaceuticals (SMIPs) are also covered under the term "antigen-binding fragment" as used herein.

[0183] Anti-TfR antigen-binding protein can be scFv, which can tether to ASM. scFv (single-chain variable fragment) has a weight (V) H ) and light (V) LThe variable regions of the V domain (in any order) are preferably linked together by a flexible linker (e.g., a peptide linker). The length of the flexible linker used to connect the two V regions may be important for the proper folding of the polypeptide chain. It has been previously estimated that the peptide linker must span 3.5 nm (35 Å) between the carboxyl terminus of the variable domain and the amino terminus of the other domain without affecting the domain's ability to fold and form a complete antigen-binding site (Huston et al., Protein engineering of single-chain Fv analogs and fusion proteins. Methods in Enzymology. 1991; 203:46-88). In one embodiment, the linker contains an amino acid sequence of such length that it separates the variable domains by approximately 3.5 nm.

[0184] In some embodiments, the anti-TfR antigen-binding proteins described herein comprise monovalent or “single-arm” antibodies. As used herein, a monovalent or “single-arm” antibody refers to an immunoglobulin containing a single variable domain. For example, the single-arm antibody may contain a single variable domain within a Fab, wherein the Fab is linked to at least one Fc fragment. In some embodiments, the single-arm antibody comprises: (i) a heavy chain containing a heavy chain constant region and a heavy chain variable region, (ii) a light chain containing a light chain constant region and a light chain variable region, and (iii) a polypeptide containing an Fc fragment or a truncated heavy chain. In some embodiments, the Fc fragment or truncated heavy chain contained in a separate polypeptide is a “virtual Fc,” which refers to an Fc fragment not linked to an antigen-binding domain. The single-arm antibodies of this disclosure may have any HCVR / LCVR pair or CDR amino acid sequence as listed herein in Table 2. Single-arm antibodies comprising a full-length heavy chain, a full-length light chain, and an additional Fe domain polypeptide can be constructed using standard methods (see, for example, WO2010151792, the full text of which is incorporated herein by reference), wherein the heavy chain constant region differs from the Fc domain polypeptide by at least two amino acids (e.g., H95R and Y96F according to the IMGT exon numbering system; or H435R and Y436F according to the EU numbering system). Such modifications can be used to purify monovalent antibodies (see WO2010151792).

[0185] In one embodiment, the antigen-binding fragment of the antibody will contain at least one variable domain. The variable domain can be of any size or amino acid composition and typically contains at least one CDR, which is adjacent to or within one or more frame sequences. L V of domain association H In the antigen-binding fragment of the domain, V H and V LDomains can be positioned relative to each other in any suitable arrangement. For example, variable regions can be dimers and contain V. H -V H V H -V L or V L -V L Dimer. Alternatively, the antigen-binding fragment of the antibody may contain monomer V. H or V L Structural domain.

[0186] In some embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of the variable and constant domains that may be found within the antigen-binding fragment of the antibody described herein include: (i) V H -CH1、(ii)V H -CH2、(iii)V H -CH3、(iv)V H -CH1-CH2、(v)V H -CH1-CH2-CH3、(vi)V H -CH2-CH3、(vii)V H -CL、(viii)V L -CH1、(ix)V L -CH2、(x)V L -CH3、(xi)V L -CH1-CH2, (xii)VL-CH1-CH2-CH3, (xiii)V L -CH2-CH3 and (xiv)V L -CL. In any configuration of the variable and constant domains (including any of the exemplary configurations listed above), the variable and constant domains may be directly connected to each other or connected via complete or partial hinge regions or linker regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, forming a flexible or semi-flexible linker between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody described herein may comprise a homodimer or heterodimer (or other multimer) having any of the variable and constant domain configurations listed above, non-covalently associated with each other and / or with one or more monomers V H or V L Domains (e.g., via disulfide bonds) are non-covalently associated. This disclosure includes antigen-binding fragments of antigen-binding proteins, such as antibodies listed herein.

[0187] Antigen-binding proteins (e.g., antibodies and antigen-binding fragments) can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding proteins are discussed further herein. This disclosure includes both monospecific and multispecific (e.g., bispecific) antigen-binding fragments containing one or more variable domains from antigen-binding proteins specifically described herein.

[0188] The term "specific binding" refers to the ability of an antigen (such as human TfR protein, mouse TfR protein, or monkey TfR protein) to bind specifically to it. D At least about 10 -9 Those antigen-binding proteins (e.g., antibodies or their antigen-binding fragments) with binding affinity of M (e.g., 0.01 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, or 1.0 nM), such as those determined by real-time, label-free biolayer interferometry, for example at 25°C or 37°C (e.g., Octet). ® Measurements can be made using HTX biosensors or through surface plasmon resonance (e.g., BIACORE). ™ The measurement can be performed by means of solution affinity ELISA. This disclosure includes antigen-binding proteins that specifically bind to TfR proteins. "Anti-TfR" refers to an antigen-binding protein (or other molecule) that specifically binds to TfR, such as an antibody or its antigen-binding fragment.

[0189] "Isolated" antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof), polypeptides, polynucleotides, and carriers are at least partially free of other biomolecules from the cells or cell cultures in which they are produced. Such biomolecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other materials such as cell debris and growth media. Isolated antigen-binding proteins may also be at least partially free of expression system components, such as biomolecules from host cells or their growth media. Generally, the term "isolated" is not intended to mean the complete absence of such biomolecules (e.g., small or insignificant amounts of impurities may be retained), or the absence of water, buffers, or salts, or a component of a pharmaceutical formulation containing an antigen-binding protein (e.g., an antibody or antigen-binding fragment).

[0190] This disclosure includes antigen-binding proteins, such as antibodies or antigen-binding fragments, that bind to the same epitopes as the antigen-binding proteins described herein. In some embodiments, an antigen-binding protein is provided that specifically binds to the transferrin receptor or an antigenic fragment thereof or a variant thereof, the antigen-binding protein binding to one or more hTfR epitopes selected from: (a) an epitope containing the sequence LLNE (SEQ ID NO: 752) and / or an epitope containing the sequence TYKEL (SEQ ID NO: 706); (b) an epitope containing the sequence DSTDFTGT (SEQ ID NO: 753) and / or an epitope containing the sequence VKHPVTGQF (SEQ ID NO: 754) and / or an epitope containing the sequence IERIPEL (SEQ ID NO: 755); (c) an epitope containing the sequence LNENSYVPREAGSQKDEN (SEQ ID NO: 756); (d) an epitope containing the sequence FEDL (SEQ ID NO: 718); (e) an epitope containing the sequence IVDKNGRL (SEQ ID NO: 757); (f) an epitope containing the sequence IVDKNGRLVY (SEQ ID NO: 718). Epitopes containing: (g) the sequence DQTKF (SEQ ID NO: 759); (h) the sequence LVENPGGY (SEQ ID NO: 760) and / or the sequence PIVNAELSF (SEQ ID NO: 761) and / or the sequence PYLGTTMDT (SEQ ID NO: 762); (i) the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or the sequence TYKEL (SEQ ID NO: 706); (j) the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 707) and / or the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 758). (k) Epitopes containing the sequence LIERIPELNKVARAAAE (SEQ ID NO: 709); (l) Epitopes containing the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 710); (m) Epitopes containing the sequence GTKKDFEDL (SEQ ID NO: 711); (v) Epitopes containing the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 712);(n) Epitopes containing the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or epitopes containing the sequence DQTKFPIVNAEL (SEQ ID NO: 714) and / or epitopes containing the sequence TYKELIERIPELNK (SEQ ID NO: 715); (o) Epitopes containing the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or epitopes containing the sequence TYKELIERIPELNK (SEQ ID NO: 715); (p) Epitopes containing the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 716); (q) Epitopes containing the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or epitopes containing the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 716). Epitopes of (717); (r) epitopes containing the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or epitopes containing the sequence LVENPGYVAYSKAATVTGKL (SEQ ID NO: 719) and / or epitopes containing the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 720) and / or epitopes containing the sequence ISRAAAEKL (SEQ ID NO: 721) and / or epitopes containing the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 722) and / or epitopes containing the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 723); (s) epitopes contained within or overlapping with the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or epitopes contained within the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 717). (705) Epitopes contained in or overlapping with the sequence and / or included in or overlapping with the sequence TYKEL (SEQ ID NO: 706); (t) Epitopes contained in or overlapping with the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 707) and / or included in or overlapping with the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 708) and / or included in or overlapping with the sequence LIERIPELNKVARAAAE (SEQ ID NO: 709); (u) Epitopes contained in or overlapping with the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 710);(v) Epitopes contained in or overlapping with the sequence GTKKDFEDL (SEQ ID NO: 711); (w) Epitopes contained in or overlapping with the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 712); (x) Epitopes contained in or overlapping with the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or Epitopes contained in or overlapping with the sequence DQTKFPIVNAEL (SEQ ID NO: 714) and / or Epitopes contained in or overlapping with the sequence TYKELIERIPELNK (SEQ ID NO: 715); (y) Epitopes contained in or overlapping with the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or Epitopes contained in or overlapping with the sequence TYKELIERIPELNK (SEQ ID NO: 715). (z) Epitopes contained in or overlapping with the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 716); (aa) Epitopes contained in or overlapping with the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or Epitopes contained in or overlapping with the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 717); and (bb) Epitopes contained in or overlapping with the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or Epitopes contained in or overlapping with the sequence LVENPGYVAYSKAATVTGKL (SEQ ID NO: 719) and / or Epitopes contained in or overlapping with the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 716). Epitopes contained in or overlapping with the sequence ISRAAAEKL (SEQ ID NO: 721) and / or contained in or overlapping with the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 722) and / or contained in or overlapping with the sequence FCETDYPYLGTTMDT (SEQ ID NO: 723). In some embodiments, an antigen-binding protein is provided, wherein the antigen-binding protein comprises an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof binding to one or more hTfR epitopes selected from: (a) an epitope consisting of the sequence LLNE (SEQ ID NO: 752) and / or an epitope consisting of the sequence TYKEL (SEQ ID NO: 706);(b) Epitopes consisting of the sequence DSTDFTGT (SEQ ID NO: 753) and / or the sequence VKHPVTGQF (SEQ ID NO: 754) and / or the sequence IERIPEL (SEQ ID NO: 755); (c) Epitopes consisting of the sequence LNENSYVPREAGSQKDEN (SEQ ID NO: 756); (d) Epitopes consisting of the sequence FEDL (SEQ ID NO: 718); (e) Epitopes consisting of the sequence IVDKNGRL (SEQ ID NO: 757); (f) Epitopes consisting of the sequence IVDKNGRLVY (SEQ ID NO: 758); (g) Epitopes consisting of the sequence DQTKF (SEQ ID NO: 759); (h) Epitopes consisting of the sequence LVENPGGY (SEQ ID NO: 760) and / or the sequence PIVNAELSF (SEQ ID NO: 759). (i) Epitopes consisting of the sequence PYLGTTMDT (SEQ ID NO: 762); (ii) Epitopes consisting of the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or the sequence TYKEL (SEQ ID NO: 706); (j) Epitopes consisting of the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 707) and / or the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 708) and / or the sequence LIERIPELNKVARAAAE (SEQ ID NO: 709); (k) Epitopes consisting of the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 710); (l) Epitopes consisting of the sequence GTKKDFEDL (SEQ ID NO: 762). Epitopes consisting of (n) the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 712); (m) the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or the sequence DQTKFPIVNAEL (SEQ ID NO: 714) and / or the sequence TYKELIERIPELNK (SEQ ID NO: 715);(o) an epitope consisting of the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or an epitope consisting of the sequence TYKELIERIPELNK (SEQ ID NO: 715); (p) an epitope consisting of the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 716); (q) an epitope consisting of the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or an epitope consisting of the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 717); and (r) an epitope consisting of the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or an epitope consisting of the sequence LVENPGYVAYSKAATVTGKL (SEQ ID NO: 719) and / or an epitope consisting of the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 716). Epitopes consisting of the sequence ISRAAAEKL (SEQ ID NO: 720) and / or the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 722) and / or the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 723).

[0191] An antigen is a molecule, such as an antibody or an antigen-binding fragment thereof, to which it binds, such as a peptide (e.g., TfR or a fragment thereof (antigenic fragment)). A specific region on an antigen that an antibody recognizes and binds to is called an epitope. Antigen-binding proteins (e.g., antibodies) that specifically bind to such antigens, as described herein, are part of this disclosure.

[0192] The term "epitope" refers to an antigenic determinant (e.g., on a TfR) that interacts with a specific antigen-binding site of an antigen-binding protein, such as a variable region of an antibody, called a complementary site. A single antigen may have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and can have different biological effects. The term "epitope" can also refer to a site on an antigen to which B cells and / or T cells respond, and / or a region on an antigen to which an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can be linear or conformational, i.e., composed of non-linear amino acids. In some embodiments, an epitope may comprise a determinant of chemically active surface groups (such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups) as molecules, and in some embodiments, may have specific three-dimensional structural features and / or specific charge features. Epitopes bound by the antigen-binding proteins described herein may be contained in fragments of a TfR (e.g., its extracellular domain). The antigen-binding proteins (e.g., antibodies) that bind to such epitopes as described herein are part of this disclosure.

[0193] Methods for identifying epitopes of antigen-binding proteins (e.g., antibodies, fragments, or peptides) include alanine scanning mutation analysis, peptide blotting (Reineke (2004) Methods Mol.Biol. 248: 443-63), peptide cleavage analysis, crystallographic studies, and NMR analysis. Additionally, methods such as epitope excision, epitope extraction, and antigen chemical modification can be employed (Tomer (2000) Prot.Sci. 9: 487-496). Another method that can be used to identify the amino acids that interact with an antigen-binding protein (e.g., antibody, fragment, or peptide) within a peptide is the detection of hydrogen / deuterium exchange via mass spectrometry. See, for example, Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal.Chem. 73: 256A-265A.

[0194] This disclosure includes antigen-binding proteins that compete with the antigen-binding proteins described herein for binding to TfR epitopes as discussed herein. As used herein, the term "competition" means that an antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) binds to an antigen (e.g., a TfR) and inhibits or blocks the binding of another antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) to that antigen. Unless otherwise stated, the term also includes competition between two antigen-binding proteins (e.g., antibodies) in two orientations, i.e., a first antibody binds to the antigen and blocks the binding of a second antibody to the antigen, or vice versa. Thus, in one embodiment, the competition occurs in one such orientation. In some embodiments, a first antigen-binding protein (e.g., an antibody) and a second antigen-binding protein (e.g., an antibody) may bind to the same epitope. Alternatively, a first antigen-binding protein and a second antigen-binding protein (e.g., an antibody) may bind to different but, for example, overlapping or non-overlapping epitopes, wherein the binding of one antigen-binding protein inhibits or blocks the binding of the second antibody, for example, via steric hindrance. Competition between antigen-binding proteins (e.g., antibodies) can be measured using methods known in the art, such as real-time, label-free biolayer interferometry. Alternatively, binding competition between TfR-binding proteins (e.g., monoclonal antibodies (mAbs)) can be determined using real-time, label-free biolayer interferometry on the Octet RED384 biosensor (Pall ForteBio Corp.).

[0195] Typically, the antibody or antigen-binding fragments described herein, modified in some way, retain the ability to specifically bind to TfR, for example, retaining at least 10% of their TfR-binding activity when expressed on a molar basis (compared to the parent antibody). Preferably, the antibody or antigen-binding fragments described herein retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or higher of the parent antibody's TfR-binding affinity. It is also contemplated that the antibody or antigen-binding fragments described herein may include conserved or non-conserved amino acid substitutions (referred to as “conserved variants” or “functionally conserved variants” of the antibody) that substantially do not alter its biological activity.

[0196] The anti-TfR antigen-binding proteins described herein can be monoclonal antibodies or antigen-binding fragments of monoclonal antibodies that can be tethered to ASM. This document provides monoclonal anti-TfR antigen-binding proteins, such as antibodies and their antigen-binding fragments, as well as monoclonal compositions comprising a variety of isolated monoclonal antigen-binding proteins. As used herein, the term "monoclonal antibody" or "mAb" refers to a member of a substantially homogeneous group of antibodies, i.e., the antibody molecules constituting that group have the same amino acid sequence, differing only in possibly small amounts of potentially naturally occurring mutations. "A plurality of" such monoclonal antibodies and fragments in a composition refers to the concentration of identical (i.e., as discussed above, the amino acid sequence is identical, differing only in possibly small amounts of potentially naturally occurring mutations) antibodies and fragments that is higher than the concentrations typically found in nature (e.g., in the blood of a host organism such as a mouse or human).

[0197] In one embodiment, the anti-TfR antigen-binding protein, such as an antibody or antigen-binding fragment (which may be tethered to a payload), comprises a heavy chain constant domain of type IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, and IgG4), or IgM. In one embodiment, the antigen-binding protein (e.g., an antibody or antigen-binding fragment) comprises a light chain constant domain of type κ or λ. In one embodiment, V, as listed herein... H Linked to human heavy chain constant domains (e.g., IgG), and as listed herein, V L Linked to a human light chain constant domain (e.g., κ). This disclosure includes antigen-binding proteins containing variable domains listed herein, which are linked to, for example, heavy chain constant domains and / or light chain constant domains listed herein.

[0198] This document includes human anti-TfR antigen-binding proteins that can be tethered to ASM. As used herein, the term "human" antigen-binding protein, such as an antibody or antigen-binding fragment, includes antibodies and fragments having variable and constant regions derived from human germline immunoglobulin sequences, whether in human cells or transplanted into non-human cells such as mouse cells. See, for example, US8502018, US6596541, or US5789215. The anti-TfR human mAbs provided herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in CDRs, particularly CDR3. However, as used herein, the term "human antibody" is not intended to include mAbs in which a CDR sequence derived from another mammalian species (e.g., mouse) has been transplanted onto a human FR sequence. The term includes antibodies recombinantly generated in non-human mammals or in cells of non-human mammals. The term is not intended to include naturally occurring antibodies isolated directly from human subjects. This disclosure includes human antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof described herein).

[0199] This document also includes anti-TfR chimeric antigen-binding proteins, such as antibodies and their antigen-binding fragments (which can tether to ASM), and methods of use thereof. As used herein, a “chimeric antibody” is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein the first and second antibodies are from different species. (See, for example, US4816567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855). This disclosure includes chimeric antibodies having the variable domains and nonhuman constant domains listed herein.

[0200] The term "recombinant" anti-TfR antigen-binding protein, such as an antibody or its antigen-binding fragment (which can be tethered to ASM), refers to such molecules produced, expressed, isolated, or obtained by techniques or methods known in the art, such as recombinant DNA techniques (which include, for example, DNA splicing and transgenic expression). This term includes antibodies expressed in non-human mammals (including transgenic non-human mammals, such as transgenic mice) or cells (e.g., CHO cells) such as cell expression systems, or antibodies isolated from a library of recombinant combined human antibodies. This disclosure includes recombinant antigen-binding proteins, such as antibodies and antigen-binding fragments as described herein.

[0201] In one embodiment, the antigen-binding fragment of the antibody will contain less structure than the complete antibody but will still specifically bind to the antigen, such as a TfR, for example, including at least one variable domain. The variable domain can have any size or amino acid composition and will typically contain at least one (e.g., 3) CDRs adjacent to or within one or more frame sequences. L V of domain association H In the antigen-binding fragment of the domain, V H and V L Domains can be positioned relative to each other in any suitable arrangement. For example, variable regions can be dimers and contain V. H -V H V H -V L or V L -V L Dimer. Alternatively, the antigen-binding fragment of the antibody may contain a non-covalently bound monomer V. H and / or V L Structural domain.

[0202] In some embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of the variable and constant domains that may be found within the antigen-binding fragment of the antibody described herein include: (i) V H -CH1、(ii)V H -CH2、(iii)V H -CH3、(iv)V H -CH1-CH2、(v)V H -CH1-CH2-CH3、(vi)V H -CH2-CH3、(vii)V H -CL、(viii)V L -CH1、(ix)V L -CH2、(x)V L -CH3、(xi)V L -CH1-CH2, (xii)VL-CH1-CH2-CH3, (xiii)V L -CH2-CH3 and (xiv)V L-CL. In any configuration of the variable and constant domains (including any of the exemplary configurations listed above), the variable and constant domains may be directly connected to each other or connected via complete or partial hinge regions or linker regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, forming a flexible or semi-flexible linker between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody described herein may comprise a homodimer or heterodimer (or other multimer) having any of the variable and constant domain configurations listed above, non-covalently associated with each other and / or with one or more monomers V H or V L Domains (e.g., via disulfide bonds) are non-covalently associated. This disclosure includes antigen-binding fragments of antigen-binding proteins, such as antibodies listed herein.

[0203] Antigen-binding proteins (e.g., antibodies and antigen-binding fragments) can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding proteins are discussed further herein. This disclosure includes both monospecific and multispecific (e.g., bispecific) antigen-binding fragments containing one or more variable domains from antigen-binding proteins specifically described herein.

[0204] A “variant” of a polypeptide (such as an immunoglobulin chain) refers to a sequence containing the same amino acid sequence as the reference amino acid sequences listed herein (e.g., SEQ ID NO: 171-174, 680, 176-179, 181-184, 681, 186-189, 191-194, 682, 196-199, 201-204, 206-209, 683, 211-214, 216-219, 684, 221-224, 685, 226-229, 686, 231-234, 687, 236-239, 688, 241-244, 689, 246-249, 690, 251-254, 256-259, 261-264, 69...). 1, 266-269, 271-274, 276-279, 281-284, 692, 286-289, 693, 291-294, 296-299, 301-304, 306-309, 311-314, 694, 316-319, 695, 321-324, 326-329, 331-334, 696, 336-339, 341-344, 346-349, 351-354, 697, 356-359, 698, 361-364, 699, 366- 369, 371-374, 700, 376-379, 381-384, 386-389, 391-394, 396-399, 401-404, 406-409, 411-414, 416-419, 421-424, 701, 426-429, 431-434, 436-439, 441-444, 446-449, 451-454, 456-459, 461-464, 466-469, 471-474, 702, 476-479, 481-48 4. A polypeptide having at least about 70% to 99.9% (e.g., at least 70%, 72%, 74%, 75%, 76%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) the same or similar amino acid sequence.The algorithm parameters are selected to give the maximum match between corresponding sequences over the entire length of the corresponding reference sequence (e.g., expected threshold: 10; word length: 3; maximum match in the query range: 0; BLOSUM 62 matrix; gap penalty: 11 for existence, 1 for expansion; conditional combination score matrix adjustment); and / or peptides containing the amino acid sequence but with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mutations (e.g., point mutations, insertions, truncations, and / or deletions).

[0205] Furthermore, variants of the polypeptide may include polypeptides such as immunoglobulin chains, which may include the amino acid sequence of a reference polypeptide (the amino acid sequence of which is specifically described herein), but have one or more mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), such as one or more missense mutations (e.g., conserved substitutions), nonsense mutations, deletions, or insertions. For example, this disclosure includes TfR-binding proteins comprising: immunoglobulin light chains (or V... L Variants comprising the amino acid sequences listed in SEQ ID NO: 176, 186, 196, 206, 683, 216, 684, 226, 686, 236, 688, 246, 690, 256, 266, 276, 286, 693, 296, 306, 316, 695, 326, 336, 346, 356, 698, 366, 376, 386, 396, 406, 416, 426, 436, 446, 456, 466, 476, 632, 486, or 703, but having one or more such mutations; and / or immunoglobulin heavy chain (or V HVariants comprising the amino acid sequences listed in SEQ ID NO: 171, 680, 181, 681, 191, 682, 201, 211, 221, 685, 231, 687, 241, 689, 251, 261, 691, 271, 281, 692, 291, 301, 311, 694, 321, 331, 696, 341, 351, 697, 361, 699, 371, 700, 381, 391, 401, 411, 421, 701, 431, 441, 451, 461, 471, 702, or 481, but having one or more such mutations. In one embodiment, the TfR binding protein comprises: immunoglobulin light chain variants containing CDR-L1, CDR-L2, and CDR-L3, wherein one or more (e.g., 1, 2, or 3) of such CDRs have one or more such mutations (e.g., conserved substitutions); and / or immunoglobulin heavy chain variants containing CDR-H1, CDR-H2, and CDR-H3, wherein one or more (e.g., 1, 2, or 3) of such CDRs have one or more such mutations (e.g., conserved substitutions).

[0206] The following references relate to the BLAST algorithm commonly used in sequence analysis: BLAST algorithm: Altschul et al., (2005) FEBS J. 272(20): 5101-5109; Altschul, SF et al., (1990) J. Mol.Biol.215:403-410; Gish, W. et al., (1993) Nature Genet.3:266-272; Madden, TL et al., (1996) Meth.Enzymol.266:131-141; Altschul, SF et al., (1997) Nucleic Acids Res.25:3389-3402; Zhang, J. et al., (1997) Genome Res.7:649-656; Wootton, JC et al., (1993) Comput. Chem. 17:149-163; Hancock, JM et al., (1994) Comput. Appl. Biosci. 10:67-70; Comparison scoring system: Dayhoff, MO et al., “A model of evolutionary change in proteins.”, Atlas of Protein Sequence and Structure, (1978) Vol. 5 Supplement 3, MO Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, DC; Schwartz, RM et al., “Matrices for detecting distant relationships.”, Atlas of Protein Sequence and Structure, (1978) Vol. 5 Supplement 3, MO Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, SF, (1991) J.Mol.Biol.219:555-565; States, DJ et al., (1991) Methods 3:66-70; Henikoff, S. et al., (1992) Proc.Natl.Acad.Sci.USA 89:10915-10919; Altschul, SF et al., (1993) J.Mol.Evol.36:290-300; Comparative Statistics: Karlin, S.et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S. et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A. et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, SF., “Evaluating the statistical significance of multiple distinct local alignments.”, Theoretical and Computational Methods in Genome Research (edited by S. Suhai), (1997) pp. 1-14, Plenum, NY.

[0207] (For example, the "conserved modified variants" or "conserved substitutions" of immunoglobulin chains listed herein refer to variants in which one or more amino acids in a polypeptide are replaced by other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.). Such changes can be made frequently without significantly impairing the biological activity of the antibody or fragment. Those skilled in the art will recognize that, in general, single-amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al., (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to significantly impair biological activity. This disclosure includes TfR-binding proteins containing such conserved modified variants of immunoglobulin chains.

[0208] Examples of amino acid groups with chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Alternatively, conservative substitutions are any changes with positive values ​​in the PAM250 log-likelihood matrix disclosed in Gonnet et al., (1992) Science 256: 1443-45.

[0209] The antibody and antigen-binding fragments described herein comprise immunoglobulin chains, including the amino acid sequences (and variants thereof) specifically listed herein, as well as cellular and in vitro post-translational modifications against the antibody or fragment. For example, this disclosure includes antibodies and antigen-binding fragments thereof that specifically bind to TfRs having the heavy chain amino acid sequences and / or light chain amino acid sequences listed herein, and antibodies and fragments wherein one or more asparagine, serine, and / or threonine residues are glycosylated, one or more asparagine residues are deamidated, one or more residues (e.g., Met, Trp, and / or His) are oxidized, N-terminal glutamine is pyroglutamate (pyroE), and / or C-terminal lysine or other amino acid is missing.

[0210] In one embodiment, the anti-hTfR:loador or anti-hTfR:loador (e.g., in the format of scFv, Fab, antibody, or antigen-binding fragment thereof) (e.g., where the load is human GAA) exhibits one or more of the following characteristics: • Affinity (K0.05) for binding with human TfR in a surface plasmon resonance scheme at 25 °C D The value is approximately 41 nM or higher (e.g., approximately 1 nM or 0.1 nM, or approximately 0.18 nM to approximately 1.2 nM or higher). • Affinity (K0) for binding with monkey TfR in the surface plasmon resonance scheme at 25 °C D The value is approximately 0 nM (binding cannot be detected) or higher (e.g., approximately 20 nM or higher). • K-type TfR bound to monkey TfR / human TfR in a surface plasmon resonance scheme at 25 °C D The ratio is between 0 and 278 (e.g., about 17 or 18). • When in the Fab format (IgG1), it blocks approximately 3%, 5%, 10%, or 13% of the binding of hTfR (e.g., Hmm-hTFRC such as REGN2431) to iron-saturated Tf, for example, no more than approximately 45% blockade; • When in scFv(V) K -V H When in the format, it blocks approximately 6%, 8%, 10%, or 13% of the binding of hTfR (e.g., Hmm-hTFRC such as REGN2431) to human iron-saturated Tf, for example, no more than approximately 45% blocking; • When in scFv(V) H -V L When in the format, it blocks approximately 11%, 17%, 23%, or 26% of the binding of hTfR (e.g., Hmm-hTFRC such as REGN2431) to human iron-saturated Tf, for example, no more than approximately 45% blocking; • When in the anti-hTfR scFv:hGAA format, mice administered this molecule via HDD (e.g., Tfrc hum / hum Knock-in mice showed a ratio of mature hGAA protein in the brain (normalized to the ratio of the positive control 8D3:GAA scFv) of approximately 1 or greater; 0.67 or greater, 1.08 or greater, 0.91 or greater, 0.65 or greater, 0.55 or greater, 0.50 or greater, 0.27 or greater, 0.72 or greater, 1.05 or greater, 0.49 or greater, 0.29 or greater, 1.29 or greater, 1.72 or greater, 1.79 or greater, 3.08 or greater, 1.24 or greater; 0.59 or greater, or 0.47 or greater (or approximately 1–2 or greater); or delivery of mature human GAA protein to the human brain that had been administered the scFv:hGAA molecule; • When in the anti-hTfR scFv:hGAA format, mice administered this molecule via HDD (e.g., Tfrc) hum / hum Knock-in mice showed a ratio of mature hGAA protein in the brain parenchyma (normalized to the ratio of the positive control 8D3:GAA scFv) of approximately 0.44, 0.05, 1.13, or 0.60 (approximately 0.1–1.2); or mature human GAA protein was delivered to the brain parenchyma of humans who had been administered the scFv:hGAA molecule. • When in the anti-hTfR scFv:hGAA format, mice administered this molecule via HDD (e.g., Tfrc) hum / hum Knock-in mice showed a ratio of mature hGAA protein in the quadriceps femoris muscle (normalized to the ratio of the positive control 8D3:GAA scFv) of approximately 0.67, 1.80, 1.78, or 7.74 (approximately 1–2); or mature human GAA protein was delivered to the human quadriceps femoris muscle or other muscle tissue that had been administered the scFv:hGAA molecule. • Mice administered this molecule via the AAV8 liver reservoir while in the anti-hTfR scFv:hGAA formulation (e.g., Tfrc mice) hum Knock-in mice showed a ratio of mature hGAA protein in the brain parenchyma (normalized to the ratio of the positive control 8D3:GAA scFv) of approximately 0.94, 0.49, 0.61, or 1.90 (approximately 0.1–1.2); or mature human GAA protein was delivered to human brain parenchyma that had been administered the scFv:hGAA molecule via a virus (e.g., AAV), a liver reservoir, or via non-enteric delivery in the scFv:hGAA fusion protein format; • When in the anti-hTfR scFv:hGAA format, mature hGAA protein is delivered to mice that have already been given the molecule by an AAV8 liver reservoir (e.g., Tfrchum Serum, liver, brain, cerebellum, spinal cord, heart, and / or quadriceps muscle of knock-in mice; or delivery of mature human GAA protein to human serum, liver, brain, cerebellum, spinal cord, heart, and / or quadriceps muscle that has been administered the scFv:hGAA molecule via a virus (e.g., AAV), liver reservoir, or non-enteric delivery in the scFv:hGAA fusion protein format. • When in the anti-hTfR scFv:hGAA format, reduce the amount of this molecule that has been administered to mice already treated with the AAV8 liver reservoir (e.g., Tfrc). hum Glycogen stored in the brain, cerebellum, spinal cord, heart, and / or quadriceps of knock-in mice; for example, reducing glycogen by at least 75% to more than 95% or more than 99%; or reducing glycogen stored in the human brain, cerebellum, spinal cord, heart, and / or quadriceps of mice that have been administered the scFv:hGAA molecule via non-enteric delivery in the scFv:hGAA fusion protein format; • Compared to unprocessed Gaa - / - / Tfrc hum Mice will be treated with liver reservoir AAV8 anti-hTFRC scfv:hGAA (e.g., 4e11vg / kg AAV8). Gaa - / - / Tfrc hum Glycogen levels in the mouse's tissues (e.g., cerebellum) were reduced by at least about 90% (e.g., about 95% or more). • Compared to unprocessed Gaa - / - / Tfrc hum Mice will be treated with liver reservoir AAV8 anti-hTFRC scfv:hGAA (e.g., 4e11vg / kg AAV8). Gaa - / - / Tfrc hum Glycogen levels in mouse tissues (e.g., quadriceps femoris) are reduced by at least about 89% (e.g., about 90% or 91% or more); or glycogen levels in human tissues (e.g., quadriceps femoris) treated with a fusion protein (e.g., via parenteral delivery of the fusion protein); • When administered (e.g., via HDD or AAV8 epitope liver bank) Tfrc humIn mice, it does not cause abnormal iron homeostasis; for example, mice maintain normal serum, cardiac, liver and / or spleen iron levels, normal total iron-binding capacity (TIBC) and / or normal hepcidin levels; or when administered to humans (e.g. via parenteral delivery of the fusion protein), it does not cause abnormal iron homeostasis. • When delivered to a subject, for example in an AAV8 vector, via chromosomal insertion (e.g., into an albumin locus) or in free form (e.g., to a human or... Gaa - / - / Tfrc hum / hum In mice, the DNA encoding the fusion variant caused mature human GAA to be expressed in serum, liver, brain, and / or quadriceps muscle; and / or • When delivered, for example, in an AAV8 vector, via chromosomal insertion (e.g., into an albumin locus) or as an episome (e.g., delivered to a human or...). Gaa - / - / Tfrc hum / hum In mice, the DNA encoding the fusion protein reduced glycogen levels in the brain and / or quadriceps muscle.

[0211] * Tfrc hum or Tfrc hum / hum It is a homozygous knock-in mouse.

[0212] The amino acid sequences of the domains in the anti-human transferrin receptor antigen-binding proteins of the fusion variants disclosed herein are summarized in Table 2 below. For example, this document discloses anti-human transferrin receptor 1 antibodies fused to ASM and their antigen-binding fragments (e.g., scFv and Fab) that contain the HCVR and LCVR of the molecules in Table 2; or contain their CDR. In one specific example, the anti-human transferrin receptor 1 antibody and its antigen-binding fragment (e.g., scFv and Fab) contain the HCVR and LCVR of #23 or #25 in Table 2, or contain the CDR of #23 or #25 in Table 2. In one specific example, the anti-human transferrin receptor 1 antibody and its antigen-binding fragment (e.g., scFv and Fab) contain the HCVR and LCVR of #23 in Table 2, or contain the CDR of #23 in Table 2. In one specific example, the anti-human transferrin receptor 1 antibody and its antigen-binding fragment (e.g., scFv and Fab) contain the HCVR and LCVR of #25 in Table 2, or contain the CDR of #25 in Table 2.

[0213] Table 2. Domains in anti-hTfR antibodies, antigen-binding fragments (e.g., Fab), or scFv molecules in fusion proteins

[0214] 31874B HCVR (V) H nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCGCCTTTAGCAGCTATGCCATGACCTGGGTCCGACAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATCAGTGGTACTGGTGGTAGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGGGGAGCAGCTCGTAGAATGGAATACTTCCAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 170) HCVR (V) H amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYAMTWVRQAPGKGLEWVSVISGTGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGAARRMEYFQYWGQGTLVTVSS (SEQ ID NO: 171) Or EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYAMTWVRQAPGKGLEWVSVISGTGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGAARRMEYFQYWGQGT T VTVSS (SEQ ID NO: 680) HCDR1: GFAFSSYA (SEQ ID NO: 172) HCDR2: ISGTGGST (SEQ ID NO: 173) HCDR3: AKGGAARRMEYFQY (SEQ ID NO: 174) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAACCTCCTTATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGATTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACAGTGCCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA(SEQ ID NO: 175) LCVR (V) L amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPNLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPLTFGGGTKVEIK(SEQ ID NO: 176) LCDR1:QGISNY(SEQ ID NO: 177) LCDR2:AAS(SEQ ID NO: 178) LCDR3:QKYNSAPLT(SEQ ID NO: 179) 31863B HCVR (V) H nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAACAGCTATGCCATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATTTATTGGTGGTAGTACTGGTAACACATACTACGCAGGCTCCGTGAAGGGCCGGTTCACCATCTCCAGCGACAATTCCAAGAAGACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGGGGAGCAGCTCGTAGAATGGAATACTTCCAGCACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA (SEQ IDNO: 180) HCVR (V) H amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMTWVRQAPGKGLEWVSFIGGSTGNTYYAGSVKGRFTISSDNSKKTLYLQMNSLRAEDTAVYYCAKGGAARRMEYFQHWGQGTLVTVSS (SEQ ID NO: 181) Or EVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMTWVRQAPGKGLEWVSFIGGSTGNTYYAGSVKGRFTISSDNSKKTLYLQMNSLRAEDTAVYYCAKGGAARRMEYFQHWGQGT T VTVSS (SEQ ID NO: 681) HCDR1: GFTFNSYA (SEQ ID NO: 182) HCDR2: IGGSTGNT (SEQ ID NO: 183) HCDR3: AKGGAARRMEYFQH (SEQ ID NO: 184) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTATAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAACAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAACCATAACAGTGTCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA(SEQ ID NO: 185) LCVR (V) L amino acid sequence DIQMTQSPSSLSASIGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQNHNSVPLTFGGGTKVEIK(SEQ ID NO: 186) LCDR1:QGISNY(SEQ ID NO: 187) LCDR2:AAS(SEQ ID NO: 188) LCDR3:QNHNSVPLT(SEQ ID NO: 189) 69348 HCVR (V) H nucleotide sequence CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCACTACCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCTGTTATATGGTATGATGGAAGTAATAAATATTATGGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACAGCCTGAGAGTCGACGACACGGCTGTTTATTACTGTACGAGAACCCATGGCTATACCAGGTCGTCGGACGGTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 190) HCVR (V) [[ID=~]] H amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFTTYGMHWVRQAPGKGLEWVAVIWYDGSNKYYGDSVKGRFTISRDNSKNTLYLQMNSLRVDDTAVYYCTRTHGYTRSSDGFDYWGQGTLVTVSS (SEQ ID NO: 191) Or E VQLVESGGGVVQPGRSLRLSCAASGFTFTTYGMHWVRQAPGKGLEWVAVIWYDGSNKYYGDSVKGRFTISRDNSKNTLYLQMNSLRVDDTAVYYCTRTHGYTRSSDGFDYWGQGT M VTVSS (SEQ ID NO: 682) HCDR1: GFTFTTYG (SEQ ID NO: 192) HCDR2: IWYDGSNK (SEQ ID NO: 193) 」 HCDR3: TRTHGYTRSSDGFDY (SEQ ID NO: 194) LCVR (V) <00~]] L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGAAATGTTTTAGGCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTCAGCGCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTACAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAATTTTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA(SEQ ID NO: 195) LCVR (V) L amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSIRNVLGWFQQKPGKAPQRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNFYPLTFGGGTKVEIK(SEQ ID NO: 196) LCDR1:QSIRNV(SEQ ID NO: 197) LCDR2:AAS(SEQ ID NO: 198) LCDR3:LQHNFYPLT(SEQ ID NO: 199) 69340 HCVR (V) H nucleotide sequence GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATAAAGCCATGCACTGGGTCCGGCAAGTTCCAGGGAAGGGCCTGGAATGGATCTCAGGTATTAGTTGGAATAGTGGTACTATAGGCTATGCGGACTCTGTGAAGGGCCGATTCATCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGCGCAAAAGATGGAGATACCAGTGGCTGGTACTGGTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQID NO: 200) HCVR (V) H amino acid sequence EVQLVESGGGLVQPGRSLRLSCAASGFTFDDKAMHWVRQVPGKGLEWISGISWNSGTIGYADSVKGRFIISRDNAKNSLYLQMNSLRAEDTALYYCAKDGDTSGWYWYGLDVWGQGTTVTVSS(SEQ ID NO: 201) HCDR1:GFTFDDKA(SEQ ID NO: 202) HCDR2:ISWNSGTI(SEQ ID NO: 203) HCDR3:AKDGDTSGWYWYGLDV(SEQ ID NO: 204) LCVR (V) L nucleotide sequence GAAATTGTGTTGACACAGTCTCCTGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCCATGATGTATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGTCTAGAGCCTGAAGATTTTGTAGTTTATTACTGTCAGCAGCGTAGCGACTGGCCCATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA (SEQ ID NO: 205) LCVR (V) L amino acid sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIHDVSNRATGIPARFSGSGSGTDFTLTISSLEPEDFVVYYCQQRSDWPITFGQGTRLEIK (SEQ ID NO: 206) Or D IV M TQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIHDVSNRATGIPARFSGSGSGTDFTLTISSLEPEDFVVYYCQQRSDWPITFGQGTRLEIK (SEQ ID NO: 683) LCDR1: QSVSSY (SEQ ID NO: 207) LCDR2: DVS (SEQ ID NO: 208) LCDR3: QQRSDWPIT (SEQ ID NO: 209) 69331 HCVR (V) H nucleotide sequence CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTATAGCCTCTGGATTCACCTTCAGTGTCTATGGCATTCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGATGGCAGTAATATCACATGATGGAAATATTAAACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATTAACAGCCTGAGAACTGAGGACACGGCTGTGTATTACTGTGCGAAAGATACCTGGAACTCCCTTGATACTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA(SEQ ID NO:210) HCVR (V) H amino acid sequence QVQLVESGGGVVQPGRSLRLSCIASGFTFSVYGIHWVRQAPGKGLEWMAVISHDGNIKHYADSVKGRFTISRDNSKNTLYLQINSLRTEDTAVYYCAKDTWNSLDTFDIWGQGTMVTVSS(SEQ ID NO: 211) HCDR1:GFTFSVYG(SEQ ID NO: 212) HCDR2:ISHDGNIK(SEQ ID NO: 213) HCDR3:AKDTWNSLDTFDI(SEQ ID NO: 214) LCVR (V) L nucleotide sequence GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCTGGGCCAGTCAGGGCATTAGCAGTTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTAATAGTTACCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO: 215) LCVR (V) L amino acid sequence DIQLTQSPSSLSASVGDRVTITCWASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPLTFGGGTKVEIK (SEQ ID NO: 216) Or DIQ M TQSPSSLSASVGDRVTITCWASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPLTFGGGTKVEIK (SEQ ID NO: 684) LCDR1: QGISSY (SEQ ID NO: 217) LCDR2: AAS (SEQ ID NO: 218) LCDR3: QQLNSYPLT (SEQ ID NO: 219) 69332 HCVR (V) H nucleotide sequence CAGGTCACCTTGAGGGAGTCTGGTCCCGCGCTGGTGAAACCCTCACAGACCCTCACACTGACCTGCACCTTCTCTGGATTCTCACTCAACACTTATGGGATGTTTGTGAGCTGGATCCGTCAGCCTCCAGGGAAGGCCCTAGAGTGGCTTGCACACATTCATTGGGATGATGATAAATACTACAGCACATCTCTGAAGACCAGGCTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACGTATTATTGTGCACGGGGGCACAATAATTTGAACTACATCATCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO:220) HCVR (V) H amino acid sequence QVTLRESGPALVKPSQTLTLTCTFSGFSLNTYGMFVSWIRQPPGKALEWLAHIHWDDDKYYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGHNNLNYIIHWGQGTLVTVSS (SEQ ID NO: 221) Or QV Q L V ESGPALVKPSQTLTLTCTFSGFSLNTYGMFVSWIRQPPGKALEWLAHIHWDDDKYYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGHNNLNYIIHWGQGTLVTVSS (SEQ ID NO: 685) HCDR1: GFSLNTYGMF (SEQ ID NO: 222) HCDR2: IHWDDDK (SEQ ID NO: 223) HCDR3: ARGHNNLNYIIH (SEQ ID NO: 224) LCVR (V) L nucleotide sequence GCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGCACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAAGATTACAATTACCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA (SEQ ID NO: 225) LCVR (V) L amino acid sequence AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPFTFGPGTKVDIK (SEQ ID NO: 226) Or D I L MTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPFTFGPGTKV E IK (SEQ ID NO: 686) LCDR1: QGIRND (SEQ ID NO: 227) LCDR2: AAS (SEQ ID NO: 228) LCDR3: LQDYNYPFT (SEQ ID NO: 229) 69326 HCVR (V) H nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGTCTCTGGATTCATCTTCAGTAGTTATGAAATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATTCTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGTGTCTGGAGTGGTCCTTTTTGATGTCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA (SEQ ID NO: 230) HCVR (V) H amino acid sequence EVQLVESGGGLVQPGGSLRLSCAVSGFIFSSYEMNWVRQAPGKGLEWVSYISSSGSTIFYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVSGVVLFDVWGQGTMVTVSS (SEQ ID NO: 231) Or Q VQLVESGGGLVQPGGSLRLSCAVSGFIFSSYEMNWVRQAPGKGLEWVSYISSSGSTIFYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVSGVVLFDVWGQGTMVTVSS (SEQ ID NO: 687) HCDR1: GFIFSSYE (SEQ ID NO: 232) HCDR2: ISSSGSTI (SEQ ID NO: 233) HCDR3: VSGVVLFDV (SEQ ID NO: 234) LCVR (V) L nucleotide sequence GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCGGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTTGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAGTGCATCCTCCAGGGCCACTGGTATCCCAGTCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATATCTGGCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO: 235) LCVR (V) L amino acid sequence EIVMTQSPATLSVSPGERATLSCRASQSVSSNFAWYQQKPGQAPRLLIYSASSRATGIPVRFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNIWPRTFGQGTKVEIK (SEQ ID NO: 236) Or D IVMTQSPATLSVSPGERATLSCRASQSVSSNFAWYQQKPGQAPRLLIYSASSRATGIPVRFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNIWPRTFGQGTKVEIK (SEQ ID NO: 688) LCDR1: QSVSSN (SEQ ID NO: 237) LCDR2: SAS (SEQ ID NO: 238) LCDR3: QQYNIWPRT (SEQ ID NO: 239) 69329 HCVR (V) H nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAACTATTGGATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGGAAGATGGAAGTGAGAAAGACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGGCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGGGAGCAGCTCGTCGATTACTACTACTACTACGTTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 240) HCVR (V) H amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMTWVRQAPGKGLEWVANIKEDGSEKDYVDSVKGRFTISRDNAKNSLYLQMNSLRGEDTAVYYCARDGEQLVDYYYYYVMDVWGQGTTVTVSS (SEQ ID NO: 241) Or Q VQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMTWVRQAPGKGLEWVANIKEDGSEKDYVDSVKGRFTISRDNAKNSLYLQMNSLRGEDTAVYYCARDGEQLVDYYYYYVMDVWGQGTTVTVSS (SEQ ID NO: 689) HCDR1: GFTFSNYW (SEQ ID NO: 242) HCDR2: IKEDGSEK (SEQ ID NO: 243) HCDR3: ARDGEQLVDYYYYYVMDV (SEQ ID NO: 244) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAAAAGGCTAACAGTTTCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 245) LCVR (V) L amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQKANSFPYTFGQGTKLEIK (SEQ ID NO: 246) Or DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQKANSFPYTFGQGTK V EIK (SEQ ID NO: 690) LCDR1: QGISSW (SEQ ID NO: 247) LCDR2: AAS (SEQ ID NO: 248) LCDR3: QKANSFPYT (SEQ ID NO: 249) 69323 (REGN16816) HCVR (V) H nucleotide sequence GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGACTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTTACATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCGAGAACTCCCTACATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAGAGGGGGATCTACTCTGGTTCGGGGAGTTAAGGGAGGCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO: 250) HCVR (V) H amino acid sequence EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAENSLHLQMNSLRAEDTALYYCARGGSTLVRGVKGGYYGMDVWGQGTTVTVSS(SEQ ID NO: 251) HCDR1:GFTFDDYA(SEQ ID NO: 252) HCDR2:ISWNSGYI(SEQ ID NO: 253) HCDR3:ARGGSTLVRGVKGGYYGMDV(SEQ ID NO: 254) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATAAGTAGCTATTTAAATTGGTATCAGCAGAAACCAGGTAAAGCCCCTAAGGTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTATTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA(SEQ ID NO: 255) LCVR (V) L amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKVLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPLTFGGGTKVEIK(SEQ ID NO: 256) LCDR1:QSISSY(SEQ ID NO: 257) LCDR2:AAS(SEQ ID NO: 258) LCDR3:QQSYSIPLT(SEQ ID NO: 259) 69305 HCVR (V) H nucleotide sequence CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACATTTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGGGTCAACTGGATCTCTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 260) HCVR (V) H amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDISKNTLYLQMNSLRAEDTAVYYCAGQLDLFFDYWGQGTLVTVSS (SEQ ID NO: 261) Or E VQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDISKNTLYLQMNSLRAEDTAVYYCAGQLDLFFDYWGQGTLVTVSS (SEQ ID NO: 691) HCDR1: GFTFSSYG (SEQ ID NO: 262) HCDR2: IWYDGSNK (SEQ ID NO: 263) HCDR3: AGQLDLFFDY (SEQ ID NO: 264) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGACAGGTATTTAAATTGGTATCGGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATACTACATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCCTCAGCAGTCTGCAGCCTGAAGATTTTGCAACTTACTACTGTCAGCAGAGTTACAGTCCCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA(SEQ ID NO: 265) LCVR (V) L amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSIDRYLNWYRQKPGKAPKLLIYTTSSLQSGVPSRFSGSGSGTDFTLTLSSLQPEDFATYYCQQSYSPPLTFGGGTKVEIK(SEQ ID NO: 266) LCDR1:QSIDRY(SEQ ID NO: 267) LCDR2:TTS(SEQ ID NO: 268) LCDR3:QQSYSPPLT(SEQ ID NO: 269) 69307 (REGN16817) HCVR (V) H nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGATTCACCTTTAGTAACTATTGGATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGGAAGATGGAAGTGAGAAAGAGTATGTGGACTCTGTGAAGGGCCGGTTCACCATCTCCAGAGACAACGCCAAGAATTCACTGTATCTGCAAATGAACAGCCTGAGAGGCGAGGACACGGCTGTATATTACTGTGCGAGAGATGGGGAGCAGCTCGTCGATTACTATTACTACTACGTTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO: 270) HCVR (V) H amino acid sequence EVQLVESGGGLVQPGGSLRLSCTASGFTFSNYWMTWVRQAPGKGLEWVANIKEDGSEKEYVDSVKGRFTISRDNAKNSLYLQMNSLRGEDTAVYYCARDGEQLVDYYYYYVMDVWGQGTTVTVSS(SEQ ID NO: 271) HCDR1:GFTFSNYW(SEQ ID NO: 272) HCDR2:IKEDGSEK(SEQ ID NO: 273) HCDR3:ARDGEQLVDYYYYYVMDV(SEQ ID NO: 274) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTTGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAAAAGGCTGACAGTCTCCCGTACGCTTTTGGCCAGGGGACCAAGCTGGAGATCAAA(SEQ ID NO: 275) LCVR (V) L amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQKADSLPYAFGQGTKLEIK(SEQ ID NO: 276) LCDR1:QGISSW(SEQ ID NO: 277) LCDR2:AAS(SEQ ID NO: 278) LCDR3:QKADSLPYA(SEQ ID NO: 279) 12795B HCVR (V) H nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTTCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAACCTCTGGATTCACCTTTACCAGCTATGACATGAAGTGGGTCCGCCAGGCTCCAGGGCTGGGCCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAACACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAGGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTACGAGGTCCCATGACTTCGGTGCCTTCGACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ IDNO: 280) HCVR (V) H amino acid sequence EVQLVESGGGLVQPGGSLRLSCATSGFTFTSYDMKWVRQAPGLGLEWVSAISGSGGNTYYADSVKGRFTISRDNSRNTLYLQMNSLRAEDTAVYYCTRSHDFGAFDYFDYWGQGTLVTVSS (SEQ ID NO: 281) Or EVQLV Q SGGGLVQPGGSLRLSCATSGFTFTSYDMKWVRQAPGLGLEWVSAISGSGGNTYYADSVKGRFTISRDNSRNTLYLQMNSLRAEDTAVYYCTRSHDFGAFDYFDYWGQGT M VTVSS (SEQ ID NO: 692) HCDR1: GFTFTSYD (SEQ ID NO: 282) HCDR2: ISGSGGNT (SEQ ID NO: 283) HCDR3: TRSHDFGAFDYFDY (SEQ ID NO: 284) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAGATCATTTTGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCAGTTTGCACAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCTTGCAGCCTGAAGATTTTGCAACCTATTACTGTCTACAGTATGATACTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO: 285) LCVR (V) L amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQGIRDHFGWYQQKPGKAPKRLIYAASSLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDTYPLTFGGGTKVEIK (SEQ ID NO: 286) Or DIQ L TQSPSSLSASVGDRVTITCRASQGIRDHFGWYQQKPGKAPKRLIYAASSLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDTYPLTFGGGTKVEIK (SEQ ID NO: 693) LCDR1: QGIRDH (SEQ ID NO: 287) LCDR2: AAS (SEQ ID NO: 288) LCDR3: LQYDTYPLT (SEQ ID NO: 289) 12798B (REGN17078 Fab; REGN17072 scFv; REGN16818) HCVR (V) H nucleotide sequence GAAGTGCAGCTGGTGGAGTCTGGGGGAGACTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGCTACCAGAGTCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAATTTCCTGTATCTGCAAATGAACAGTCTGAGATCTGAGGACACGGCCTTGTATCACTGTGCAAAAGATATGGATATCTCGCTAGGGTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ IDNO: 290) HCVR (V) H amino acid sequence EVQLVESGGDLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSATRVYADSVKGRFTISRDNAKNFLYLQMNSLRSEDTALYHCAKDMDISLGYYGLDVWGQGTTVTVSS(SEQ ID NO: 291) HCDR1:GFTFDDYA(SEQ ID NO: 292) HCDR2:ISWNSATR(SEQ ID NO: 293) HCDR3:AKDMDISLGYYGLDV(SEQ ID NO: 294) LCVR (V) L nucleotide sequence GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGACTGTTAGCAGCAACTTAGCCTGGTATCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTTCATCCTCCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCTCCCTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA(SEQ ID NO: 295) LCVR (V) L amino acid sequence EIVMTQSPATLSVSPGERATLSCRASQTVSSNLAWYQQKPGQAPRLLIYGSSSRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPPYTFGQGTKLEIK(SEQ ID NO: 296) LCDR1:QTVSSN(SEQ ID NO: 297) LCDR2:GSS(SEQ ID NO: 298) LCDR3:QQYNNWPPYT(SEQ ID NO: 299) 12799B (REGN17079 Fab; REGN17073 scFv; REGN16819) HCVR (V) H nucleotide sequence CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTGGAGTGGGTGTGGTCTGGATCCGTCAGCCCCCCGGAAAGGCCCTGGAGTGGCTTGCACTCATTTATTGGAATGATCATAAGCGGTACAGCCCATCTCTGGGGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACACTACAGTGGGAGCTATTCCTACTACTACTATGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO: 300) HCVR (V) H amino acid sequence QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVVWIRQPPGKALEWLALIYWNDHKRYSPSLGSRLTITKDTSKNQVVLTMTNMDPVDTATYYCAHYSGSYSYYYYGLDVWGQGTTVTVSS(SEQ ID NO: 301) HCDR1:GFSLSTSGVG(SEQ ID NO: 302) HCDR2:IYWNDHK(SEQ ID NO: 303) HCDR3:AHYSGSYSYYYYGLDV(SEQ ID NO: 304) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTGCCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTGAGCTCCTGATCTATGCTGCATCCAGTTTGCAAGGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAATTTACTATTGTCAACAGGCTAACTATTTCCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA(SEQ ID NO: 305) LCVR (V) L amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGIASWLAWYQQKPGKAPELLIYAASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFAIYYCQQANYFPWTFGQGTKVEIK(SEQ ID NO: 306) LCDR1:QGIASW(SEQ ID NO: 307) LCDR2:AAS(SEQ ID NO: 308) LCDR3:QQANYFPWT(SEQ ID NO: 309) 12801B HCVR (V) H nucleotide sequence GAGGTGCAGCTGTTGGAGTCTGGGGGAGCCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTACCTCCTATGCCATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCATCTATTAGAGGTAGTGGTGGTGGCACATACTCCGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAGGGACACTCTATATCTGCAAATGAACAGTGTGAGAGCCGAGGACACGGCCGTTTATTACTGTGCGAGGTCCCATGACTACGGTGCCTTCGACTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ IDNO: 310) HCVR (V) H amino acid sequence EVQLLESGGALVQPGGSLRLSCAASGFTFTSYAMHWVRQAPGKGLEWVSSIRGSGGGTYSADSVKGRFTISRDNSRDTLYLQMNSVRAEDTAVYYCARSHDYGAFDFFDYWGQGTLVTVSS (SEQ ID NO: 311) Or EVQLLESGGALVQPGGSLRLSCAASGFTFTSYAMHWVRQAPGKGLEWVSSIRGSGGGTYSADSVKGRFTISRDNSRDTLYLQMNSVRAEDTAVYYCARSHDYGAFDFFDYWGQGT T VTVSS (SEQ ID NO: 694) HCDR1: GFTFTSYA (SEQ ID NO: 312) HCDR2: IRGSGGGT (SEQ ID NO: 313) HCDR3: ARSHDYGAFDFFDY (SEQ ID NO: 314) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAACTGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCGGCCTGAAGATTTTGCAACTTTTTACTGTCTACAGTATAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO: 315) LCVR (V) L amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQGIRTDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLRPEDFATFYCLQYNSYPLTFGGGTKVEIK (SEQ ID NO: 316) Or DIQMTQSPSSLSASVGDRVTITCRASQGIRTDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLRPEDFATFYCLQYNSYPLTFGGGTKV D IK (SEQ ID NO: 695) LCDR1: QGIRTD (SEQ ID NO: 317) ​​​​​​​​​​CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTTCATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTACTGGTAGTACCATAAATTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAATGTCAAGAATTCACTGTATCTGCAAATGACCAGCCTGAGAGTCGAGGACACGGCCGTGTATTACTGTACGAGAGATAACTGGAACTATGAATACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA(SEQ ID NO: 320) HCVR (V) H amino acid sequence QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYFMSWIRQAPGKGLEWVSYISSTGSTINYADSVKGRFTISRDNVKNSLYLQMTSLRVEDTAVYYCTRDNWNYEYWGQGTLVTVSS(SEQ ID NO: 321) HCDR1:GFTFSDYF(SEQ ID NO: 322) HCDR2:ISSTGSTI(SEQ ID NO: 323) HCDR3:TRDNWNYEY(SEQ ID NO: 324) LCVR (V) L nucleotide sequence GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCATCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTTTGTTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAACTTATTACTGTCAGCAGTATGATATCTGGCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA(SEQ ID NO: 325) LCVR (V) L amino acid sequence EIVMTQSPATLSVSPGERATLSCRASQSVSINLAWYQQKPGQAPRLLIFVASTRATGIPARFSGSGSGTEFTLTISSLQSEDFATYYCQQYDIWPYTFGQGTKLEIK(SEQ ID NO: 326) LCDR1:QSVSIN(SEQ ID NO: 327) LCDR2:VAS(SEQ ID NO: 328) LCDR3:QQYDIWPYT(SEQ ID NO: 329) 12808B HCVR (V) H nucleotide sequence CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTGTCTGGTGAATCCATCAGCAGTAATACTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAATGGATTGGGAGTATCGATTATAGTGGGACCACCAATTATAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAGGAATCACTTCTCCCTGAGGCTGAGGTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGAGAGTGGGGAAACTACGGCTACTATTACGGTATGGACGTTTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 330) HCVR (V) H amino acid sequence QLQLQESGPGLVKPSETLSLTCTVSGESISSNTYYWGWIRQPPGKGLEWIGSIDYSGTTNYNPSLKSRVTISVDTSRNHFSLRLRSVTAADTAVYYCAREWGNYGYYYGMDVWGQGTTVTVSS (SEQ ID NO: 331) Or Q V QL V ESGPGLVKPSETLSLTCTVSGESISSNTYYWGWIRQPPGKGLEWIGSIDYSGTTNYNPSLKSRVTISVDTSRNHFSLRLRSVTAADTAVYYCAREWGNYGYYYGMDVWGQGTTVTVSS (SEQ ID NO: 696) HCDR1: GESISSNTYY (SEQ ID NO: 332) HCDR2: IDYSGTT (SEQ ID NO: 333) HCDR3: AREWGNYGYYYGMDV (SEQ ID NO: 334) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATTAAGGTTCAGTGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAACAACCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTATCGCATAATAGTTACCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA(SEQ ID NO: 335) LCVR (V) L amino acid sequence DIQMTQSPSSLSASVGDRVTINCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPLRFSGSGSGTEFTLTINNLQPEDFATYYCLSHNSYPWTFGQGTKVEIK(SEQ ID NO: 336) LCDR1:QGIRND(SEQ ID NO: 337) LCDR2:AAS(SEQ ID NO: 338) LCDR3:LSHNSYPWT(SEQ ID NO: 339) 12812B (REGN16821) HCVR (V) H nucleotide sequence CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAGGGTCTCCTGCAAGGCTTCTAGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATGGGAGGGATCATCCCCATCTTTGGTACAGCAAACTACGCACAGAAGTTCCTGGCCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAGAAGGGGTGGAACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA(SEQ ID NO: 340) HCVR (V) H amino acid sequence QVQLVQSGAEVKKPGSSVRVSCKASRGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFLARVTITADESTSTAYMELSSLRSEDTAVYYCAREKGWNYFDYWGQGTLVTVSS(SEQ ID NO: 341) HCDR1:RGTFSSYA(SEQ ID NO: 342) HCDR2:IIPIFGTA(SEQ ID NO: 343) HCDR3:AREKGWNYFDY(SEQ ID NO: 344) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCACCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA(SEQ ID NO: 345) LCVR (V) L amino acid sequence DIQMTQSPPSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPRTFGQGTKVEIK(SEQ ID NO: 346) LCDR1:QGISSW(SEQ ID NO: 347) LCDR2:AAS(SEQ ID NO: 348) LCDR3:QQANSFPRT(SEQ ID NO: 349) 12816B HCVR (V) H nucleotide sequence CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAACTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGGACTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAAATCACTGTATCTGGAGATGAACAGCCTCAGAGCCGAGGACACGGCCGTGTACTACTGTGCGAGAGAGGGGTACGGTAATGACTACTATTACTACGGTATAGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 350) HCVR (V) H amino acid sequence QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMNWIRQAPGKGLEWVSYISSSGTTIYYADSVKGRFTISRDNAKKSLYLEMNSLRAEDTAVYYCAREGYGNDYYYYGIDVWGQGTTVTVSS (SEQ ID NO: 351) Or E VQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMNWIRQAPGKGLEWVSYISSSGTTIYYADSVKGRFTISRDNAKKSLYLEMNSLRAEDTAVYYCAREGYGNDYYYYGIDVWGQGTTVTVSS (SEQ ID NO: 697) HCDR1: GFTFSDYY (SEQ ID NO: 352) HCDR2: ISSSGTTI (SEQ ID NO: 353) HCDR3: AREGYGNDYYYYGIDV (SEQ ID NO: 354) LCVR (V) L nucleotide sequence GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATGGTAATGGATACAACTATTTGACTTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 355) LCVR (V) L amino acid sequence DIVMTQSPLSLPVTPGEPASISCRSSQSLLHGNGYNYLTWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTKLEIK (SEQ ID NO: 356) Or DI QL TQSPLSLPVTPGEPASISCRSSQSLLHGNGYNYLTWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK V EIK (SEQ ID NO: 698) LCDR1: QSLLHGNGYNY (SEQ ID NO: 357) LCDR2: LGS (SEQ ID NO: 358) LCDR3: MQALQTPYT (SEQ ID NO: 359) 12833B HCVR (V) H nucleotide sequence CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTTTGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGATATTTATATCATATGATGGAAGTGATAAATACTATGCAGACTCCGTGAAGGGCCGATTCGCCATCTCCAGAGACAGTTCCAAGAACACGCTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGAAAACGGTATTTTGACTGATTCCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ IDNO: 360) HCVR (V) H amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVIFISYDGSDKYYADSVKGRFAISRDSSKNTLYLQMNSLRAEDTAVYYCAKENGILTDSYGMDVWGQGTTVTVSS (SEQ ID NO: 361) Or E VQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVIFISYDGSDKYYADSVKGRFAISRDSSKNTLYLQMNSLRAEDTAVYYCAKENGILTDSYGMDVWGQGTTVTVSS (SEQ ID NO: 699) HCDR1: GFTFSSFG (SEQ ID NO: 362) HCDR2: ISYDGSDK (SEQ ID NO: 363) HCDR3: AKENGILTDSYGMDV (SEQ ID NO: 364) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO: 365) LCVR (V) L amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK(SEQ ID NO: 366) LCDR1:QSISSY(SEQ ID NO: 367) LCDR2:AAS(SEQ ID NO: 368) LCDR3:QQSYSTPPIT(SEQ ID NO: 369) 12834B HCVR (V) H nucleotide sequence CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCTGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGTGTTTACCATGGTAACACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGAGGGGTATTACGATTTTTGGAGTGGTTATTACCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 370) HCVR (V) H amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISVYHGNTNYAQKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAREGYYDFWSGYYPFDYWGQGTLVTVSS (SEQ ID NO: 371) Or E VQLV E SGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISVYHGNTNYAQKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAREGYYDFWSGYYPFDYWGQGT T VTVSS (SEQ ID NO: 700) HCDR1: GYTFTSYG (SEQ ID NO: 372) HCDR2: ISVYHGNT (SEQ ID NO: 373) HCDR3: AREGYYDFWSGYYPFDY (SEQ ID NO: 374) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO: 375) LCVR (V) L amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK(SEQ ID NO: 376) LCDR1:QSISSY(SEQ ID NO: 377) LCDR2:AAS(SEQ ID NO: 378) LCDR3:QQSYSTPPIT(SEQ ID NO: 379) 12835B HCVR (V) H nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGATACAACCTGGAGGGTCCCTGAGACTCTCCTGTGAAGCCTCTGGATTCACCTTCAGAAATTATGAAATGAATTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATATATTAGTAGTAGTGGTAATATGAAAGACTACGCAGAGTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGTCAAGAATTCACTGCAGCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTTTATTACTGTGCGAGAGACGAGTTTCCTTACGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO: 380) HCVR (V) H amino acid sequence EVQLVESGGGLIQPGGSLRLSCEASGFTFRNYEMNWVRQAPGKGLEWVSYISSSGNMKDYAESVKGRFTISRDNVKNSLQLQMNSLRVEDTAVYYCARDEFPYGMDVWGQGTTVTVSS(SEQ ID NO: 381) HCDR1:GFTFRNYE(SEQ ID NO: 382) HCDR2:ISSSGNMK(SEQ ID NO: 383) HCDR3:ARDEFPYGMDV(SEQ ID NO: 384) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO: 385) LCVR (V) L amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK(SEQ ID NO: 386) LCDR1:QSISSY(SEQ ID NO: 387) LCDR2:AAS(SEQ ID NO: 388) LCDR3:QQSYSTPPIT(SEQ ID NO: 389) 12847B (REGN17083 anti-hTfR Fab; REGN17077 anti-hTfR scFv; REGN16826) HCVR (V) H nucleotide sequence GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTTCAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGAACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAGTAGTGGTAGCATGGACTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAAAACTCCCTGTATCTGCAAATGAACAGTCTGAGAACTGAGGACACGGCCTTATATTACTGTGCAAAAGCTAGGGAAGTTGGAGACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ IDNO: 390) HCVR (V) H amino acid sequence EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMNWVRQAPGKGLEWVSGISWSSGSMDYADSVKGRFTISRDNAKNSLYLQMNSLRTEDTALYYCAKAREVGDYYGMDVWGQGTTVTVSS(SEQ ID NO: 391) HCDR1:GFTFDDYA(SEQ ID NO: 392) HCDR2:ISWSSGSM(SEQ ID NO: 393) HCDR3:AKAREVGDYYGMDV(SEQ ID NO: 394) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO: 395) LCVR (V) L amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK(SEQ ID NO: 396) LCDR1:QSISSY(SEQ ID NO: 397) LCDR2:AAS(SEQ ID NO: 398) LCDR3:QQSYSTPPIT(SEQ ID NO: 399) 12848B (REGN16827) HCVR (V) H nucleotide sequence GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGACACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATAATTTTGGCATGCACTGGGTCCGGCAAGGTCCAGGGAAGGGCCTGGAATGGGTCTCAGGTCTTACTTGGAATAGTGGTGTCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGACCTGAGGACACGGCCTTATATTACTGTGCAAAAGATATACGGAATTACGGCCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA(SEQ ID NO: 400) HCVR (V) H amino acid sequence EVQLVESGGGLVQPGRSLTLSCAASGFTFDNFGMHWVRQGPGKGLEWVSGLTWNSGVIGYADSVKGRFTISRDNAKNSLYLQMNSLRPEDTALYYCAKDIRNYGPFDYWGQGTLVTVSS(SEQ ID NO: 401) HCDR1:GFTFDNFG(SEQ ID NO: 402) HCDR2:LTWNSGVI(SEQ ID NO: 403) HCDR3:AKDIRNYGPFDY(SEQ ID NO: 404) LCVR (V) L nucleotide sequence GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA(SEQ ID NO: 405) LCVR (V) L amino acid sequence EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK(SEQ ID NO: 406) LCDR1:QSVSSSY(SEQ ID NO: 407) LCDR2:GAS(SEQ ID NO: 408) LCDR3:QQYGSSPWT(SEQ ID NO: 409) 12843B (REGN17075 anti-hTfR scFv; REGN16824; REGN17081 anti-hTfR Fab) HCVR (V) H nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTACAGCCTGGAGGGTCCCTAAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAATATTTTTGAAATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATTTCCTACATTAGTAGTCGTGGAACTACCACATACTACGCAGACTCTGTGAGGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAGATTATGAAGCAACAATCCCTTTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA(SEQ ID NO: 410) HCVR (V) H amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFNIFEMNWVRQAPGKGLEWISYISSRGTTTYYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDYEATIPFDFWGQGTLVTVSS(SEQ ID NO: 411) HCDR1:GFTFNIFE(SEQ ID NO: 412) HCDR2:ISSRGTTT(SEQ ID NO: 413) HCDR3:ARDYEATIPFDF(SEQ ID NO: 414) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO: 415) LCVR (V) L amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK(SEQ ID NO: 416) LCDR1:QSISSY(SEQ ID NO: 417) LCDR2:AAS(SEQ ID NO: 418) LCDR3:QQSYSTPPIT(SEQ ID NO: 419) 12844B HCVR (V) H nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAAGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGAAGCCTCTGGATTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGATCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGATAGAACAAATTATGCAGACTCTGTGAAGGGCCGATTCATCATTTCCAGAGACAACGCCAAGAACTCTGTGTATCTACAAATGAACAGTCTGAGAGCGGAGGACTCGGCCTTGTATCACTGTGCGAGAGATCAGGGACTCGGAGTGGCAGCTACCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ IDNO: 420) HCVR (V) H amino acid sequence EVQLVESGGSVVRPGGSLRLSCEASGFTFDDYGMSWVRQDPGKGLEWVSGINWNGDRTNYADSVKGRFIISRDNAKNSVYLQMNSLRAEDSALYHCARDQGLGVAATLDYWGQGTLVTVSS (SEQ ID NO: 421) Or EVQLVESGGSVVRPGGSLRLSCEASGFTFDDYGMSWVRQDPGKGLEWVSGINWNGDRTNYADSVKGRFIISRDNAKNSVYLQMNSLRAEDSALYHCARDQGLGVAATLDYWGQGT M VTVSS (SEQ ID NO: 701) HCDR1: GFTFDDYG (SEQ ID NO: 422) HCDR2: INWNGDRT (SEQ ID NO: 423) HCDR3: ARDQGLGVAATLDY (SEQ ID NO: 424) LCVR (V) L nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATT...

Claims

1. A multi-domain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide.

2. The multi-domain therapeutic protein of claim 1, wherein the C-terminus of the TfR binding delivery domain is fused to the N-terminus of the acid sphingomyelinase polypeptide.

3. The multi-domain therapeutic protein according to claim 1 or 2, wherein the TfR binding and delivery domain is fused to the acidic sphingomyelinase polypeptide via a peptide linker, optionally wherein the linker comprises, is substantially composed of, or is composed of the following sequences: The sequence listed in any one of SEQ ID NO: 617, 808 and 616; optionally the connector comprises, is substantially composed of or consists of the following sequences: the sequence listed in any one of SEQ ID NO: 617 and 808.

4. The multidomain therapeutic protein according to any one of claims 1 to 3, wherein the acid sphingomyelinase polypeptide lacks the acid sphingomyelinase signal peptide.

5. The multi-domain therapeutic protein according to any one of claims 1 to 4, wherein the acidic sphingomyelinase polypeptide comprises, is substantially composed of, or is composed of the following sequences: The sequence listed in SEQ ID NO: 733, 731 or 728; optionally, the acid sphingomyelinase polypeptide comprises, is substantially composed of or consists of the sequence listed in SEQ ID NO:

733.

6. The multi-domain therapeutic protein according to any one of claims 1 to 5, wherein the TfR binding and delivery domain comprises an anti-TfR antigen-binding protein, optionally wherein the antigen-binding protein is in the form of about 41 nM K. D Or with a stronger affinity, it binds to the human transferrin receptor, optionally wherein the antigen-binding protein binds at approximately 3 nM K. D Or, with a stronger affinity, bind to the human transferrin receptor, or optionally, wherein the antigen-binding protein binds at a K+ of about 0.45 nM to 3 nM. D It binds to the human transferrin receptor.

7. The multi-domain therapeutic protein of claim 6, wherein the anti-TfR antigen-binding protein comprises: (i) HCVR, wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, and comprises an amino acid sequence (or a variant thereof) listed in SEQ ID NO: 391, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, 401, 411, 421, 431, 441, 451, 461, 471, or 481; and / or (ii) LCVR, wherein the LCVR comprises LCDR1, LCDR2 and LCDR3, and comprises an amino acid sequence (or a variant thereof) listed in SEQ ID NO: 396, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 406, 416, 426, 436, 446, 456, 466, 476 or 486.

8. The multi-domain therapeutic protein according to claim 6 or 7, wherein the anti-TfR antigen-binding protein comprises: (1) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 391; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

396. (2) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 171; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

176. (3) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 181; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

186. (4) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 191; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

196. (5) HCVR, wherein the HCVR comprises HCDR1, HCDR2 and HCDR3 and comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 201; and LCVR, wherein the LCVR comprises LCDR1, LCDR2 and LCDR3 and comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

206. (6) HCVR, wherein the HCVR comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 211; and LCVR, wherein the LCVR comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

216. (7) HCVR, wherein the HCVR comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 221; and LCVR, wherein the LCVR comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

226. (8) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 231; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

236. (9) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 241; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

246. (10) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 251; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

256. (11) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 261; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

266. (12) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 271; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

276. (13) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 281; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

286. (14) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 291; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

296. (15) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 301; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

306. (16) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 311; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

316. (17) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 321; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

326. (18) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 331; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

336. (19) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 341; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

346. (20) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 351; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

356. (21) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 361; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

366. (22) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 371; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

376. (23) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 381; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

386. (24) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 401; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

406. (25) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 411; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

416. (26) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 421; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

426. (27) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 431; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

436. (28) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 441; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

446. (29) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 451; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

456. (30) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 461; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

466. (31) HCVR, wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, and comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 471; and LCVR, wherein the LCVR comprises LCDR1, LCDR2, and LCDR3, and comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 476; or (32) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 481; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

486.

9. The multidomain therapeutic protein according to any one of claims 6 to 8, wherein the anti-TfR antigen-binding protein comprises: (1) HCVR, comprising HCDR1, HCDR2, and HCDR3, and containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 391; and LCVR, comprising LCDR1, LCDR2, and LCDR3, and containing the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 396; or (2) HCVR, which comprises HCDR1, HCDR2 and HCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 411; and LCVR, which comprises LCDR1, LCDR2 and LCDR3 and contains the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

416.

10. The multidomain therapeutic protein according to any one of claims 6 to 9, wherein the anti-TfR antigen-binding protein comprises: (a) HCVR, comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 392, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 393, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 394; and LCVR, comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 397, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 398, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 399; (b) HCVR, comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 172, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 173, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 174; and LCVR, comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 177, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 178, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 179; (c) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 182, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 183, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 184; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 187, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 188, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 189; (d) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 192, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 193, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 194; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 197, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 198, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 199; (e) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 202, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 203, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 204; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 207, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 208, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 209; (f) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 212, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 213, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 214; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 217, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 218, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 219; (g) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 222, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 223, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 224; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 227, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 228, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 229; (h) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 232, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 233, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 234; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 237, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 238, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 239; (i) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 242, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 243, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 244; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 247, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 248, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 249; (j) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 252, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 253, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 254; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 257, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 258, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 259; (k) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 262, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 263, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 264; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 267, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 268, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 269; (l) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 272, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 273, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 274; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 277, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 278, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 279; (m)HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 282, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 283, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 284; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 287, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 288, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 289; (n) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 292, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 293, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 294; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 297, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 298, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 299; (o) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 302, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 303, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 304; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 307, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 308, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 309; (p)HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 312, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 313, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 314; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 317, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 318, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 319; (q) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 322, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 323, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 324; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 327, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 328, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 329; (r)HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 332, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 333, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 334; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 337, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 338, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 339; (s) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 342, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 343, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 344; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 347, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 348, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 349; (t)HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 352, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 353, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 354; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 357, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 358, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 359; (u)HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 362, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 363, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 364; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 367, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 368, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 369; (v) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 372, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 373, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 374; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 377, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 378, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 379; (w) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 382, ​​HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 383, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 384; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 387, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 388, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 389; (x) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 402, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 403, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 404; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 407, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 408, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 409; (y)HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 412, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 413, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 414; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 417, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 418, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 419; (z) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 422, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 423, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 424; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 427, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 428, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 429; (aa) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 432, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 433, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 434; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 437, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 438, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 439; (ab)HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 442, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 443, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 444; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 447, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 448, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 449; (ac)HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 452, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 453, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 454; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 457, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 458, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 459; (ad)HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 462, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 463, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 464; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 467, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 468, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 469; (ae)HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 472, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 473, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 474; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 477, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 478, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 479; and / or (af) HCVR, the HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 482, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 483, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 484; and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 487, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 488, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

489.

11. The multidomain therapeutic protein according to any one of claims 6 to 10, wherein the anti-TfR antigen-binding protein comprises: (a) an HCVR comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 392, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 393, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 394; and an LCVR comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 397, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 398, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 399; or (b) HCVR, comprising: HCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 412, HCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 413, and HCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 414; and LCVR, comprising: LCDR1 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 417, LCDR2 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 418, and LCDR3 comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

419.

12. The multidomain therapeutic protein according to any one of claims 6 to 11, wherein the anti-TfR antigen-binding protein comprises: (i) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 391; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

396. (ii) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 171; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

176. (iii) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 181; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

186. (iv) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 191; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

196. (v) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 201; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

206. (vi) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 211; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

216. (vii) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 221; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

226. (viii) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 231; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

236. (ix) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 241; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

246. (x) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 251; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

256. (xi)HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 261; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

266. (xii) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 271; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

276. (xiii) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 281; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

286. (xiv)HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 291; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

296. (xv)HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 301; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

306. (xvi) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 311; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

316. (xvii) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 321; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

326. (xviii) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 331; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

336. (xix)HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 341; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

346. (xx)HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 351; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

356. (xxi)HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 361; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

366. (xxii) HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 371; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

376. (xxiii) HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 381; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

386. (xxiv)HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 401; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

406. (xxv)HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 411; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

416. (xxvi)HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 421; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

426. (xxvii)HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 431; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

436. (xxviii) HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 441; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

446. (xxix)HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 451; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

456. (xxx)HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 461; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

466. (xxxi)HCVR, the HCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 471; and LCVR, the LCVR comprising the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 476; and / or (xxxii) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 481; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

486.

13. The multidomain therapeutic protein according to any one of claims 6 to 12, wherein the anti-TfR antigen-binding protein comprises: (i) HCVR, wherein the HCVR comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 391; and LCVR, wherein the LCVR comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 396; or (ii) HCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 411; and LCVR, which comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO:

416.

14. The multidomain therapeutic protein according to any one of claims 1 to 13, wherein the TfR binding delivery domain comprises an anti-TfR antibody, an antibody fragment, or a single-chain variable fragment (scFv).

15. The multi-domain therapeutic protein of claim 14, wherein the TfR binding delivery domain is the single-chain variable fragment (scFv), optionally wherein the multi-domain therapeutic protein comprises domains arranged in the following orientation: N'-heavy chain variable region-light chain variable region-acid sphingomyelinase polypeptide-C' or N'-light chain variable region-heavy chain variable region-acid sphingomyelinase polypeptide-C'. Optionally, the scFv and the acid sphingomyelinase polypeptide are linked by a peptide linker, and optionally, the peptide linker is -(GGGGS). m - (SEQ ID NO: 537); where m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, Optionally, the scFv variable region is linked via a peptide linker, and optionally, the peptide linker is -(GGGGS). m - (SEQ ID NO: 537); where m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

16. The multi-domain therapeutic protein of claim 15, wherein the multi-domain therapeutic protein comprises a heavy chain variable region (V... H ) and light chain variable region (V L ) and acid sphingomyelinase polypeptide, wherein the V H V L The polypeptide arrangement of acid sphingomyelinase is as follows: (i)V L -V H - Acidic sphingomyelinase polypeptide; (ii)V H -V L - Acidic sphingomyelinase polypeptide; (iii)V L -[(GGGGS)3(SEQ ID NO: 616)]-V H -[(GGGGS)2(SEQ ID NO: 617)]-acidic sphingomyelinase polypeptide; or (iv)V H -[(GGGGS)3(SEQ ID NO: 616)]-V L -[(GGGGS)2(SEQ ID NO: 617)]-acidic sphingomyelinase polypeptide.

17. The multi-domain therapeutic protein of claim 15 or 16, wherein the scFv comprises, is substantially composed of, or is composed of the following sequences: The sequences listed as any one of SEQ ID NO: 508, 494, 503, and 505, Optionally, the scFv comprises, is substantially composed of, or consists of the sequences listed in SEQ ID NO: 508 or 505. Optionally, the scFv may comprise, consist substantially of, or consist of the sequence listed in SEQ ID NO:

508.

18. The multi-domain therapeutic protein according to any one of claims 1 to 17, wherein the multi-domain therapeutic protein comprises, is substantially composed of, or is composed of the following sequences: The sequence listed in SEQ ID NO: 837, 839, 841, 737 or 739; optionally, the multidomain therapeutic protein comprises, is substantially composed of or consists of the sequence listed in SEQ ID NO: 837 or 839.

19. The multi-domain therapeutic protein according to any one of claims 1 to 14, wherein the TfR binding and delivery domain is a Fab protein, the Fab protein comprising a complete light chain, a heavy chain variable region, and a heavy chain constant region CH1 domain. Optionally, the Fab protein comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 584 and 635, or comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 588 and 636. Optionally, the Fab protein comprises the amino acid sequence (or a variant thereof) listed in SEQ ID NO: 584 and 635. Optionally, the Fab protein comprises, is substantially composed of, or is composed of the following sequences: The sequence listed in SEQ ID NO: 815 or 817, and Optionally, the C-terminus of the CH1 domain is linked to the N-terminus of the acid sphingomyelinase polypeptide, or optionally, the C-terminus of the light chain is linked to the N-terminus of the acid sphingomyelinase polypeptide. Optionally, the multidomain therapeutic protein comprises, is substantially composed of, or is composed of the sequence listed in SEQ ID NO: 833 or 835.

20. The multi-domain therapeutic protein according to any one of claims 1 to 19, wherein the TfR binding and delivery domain is an antigen-binding protein that binds to one or more hTfR epitopes selected from the group consisting of: (a) Epitopes containing the sequence LLNE (SEQ ID NO: 752) and / or epitopes containing the sequence TYKEL (SEQ ID NO: 706); (b) Epitopes containing the sequence DSTDFTGT (SEQ ID NO: 753) and / or epitopes containing the sequence VKHPVTGQF (SEQ ID NO: 754) and / or epitopes containing the sequence IERIPEL (SEQ ID NO: 755); (c) Epitopes containing the sequence LNENSYVPREAGSQKDEN (SEQ ID NO: 756); (d) Epitopes containing the sequence FEDL (SEQ ID NO: 718); (e) Epitopes containing the sequence IVDKNGRL (SEQ ID NO: 757); (f) Epitopes containing the sequence IVDKNGRLVY (SEQ ID NO: 758); (g) Epitopes containing the sequence DQTKF (SEQ ID NO: 759); (h) Epitopes containing the sequence LVENPGGY (SEQ ID NO: 760) and / or epitopes containing the sequence PIVNAELSF (SEQ ID NO: 761) and / or epitopes containing the sequence PYLGTTMDT (SEQ ID NO: 762); (i) Epitopes containing the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or epitopes containing the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or epitopes containing the sequence TYKEL (SEQ ID NO: 706); (j) Epitopes containing the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 707) and / or epitopes containing the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 708) and / or epitopes containing the sequence LIERIPELNKVARAAAE (SEQ ID NO: 709); (k) contains epitopes of the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 710); (l) Epitopes containing the sequence GTKKDFEDL (SEQ ID NO: 711); (m) contains an epitope of the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 712); (n) Epitopes containing the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or epitopes containing the sequence DQTKFPIVNAEL (SEQ ID NO: 714) and / or epitopes containing the sequence TYKELIERIPELNK (SEQ ID NO: 715); (o) Epitopes containing the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or epitopes containing the sequence TYKELIERIPELNK (SEQ ID NO: 715); (p) contains epitopes of the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 716); (q) Epitopes containing the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or epitopes containing the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 717); (r) Epitopes containing the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or epitopes containing the sequence LVENPGYVAYSKAATVTGKL (SEQ ID NO: 719) and / or epitopes containing the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 720) and / or epitopes containing the sequence ISRAAAEKL (SEQ ID NO: 721) and / or epitopes containing the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 722) and / or epitopes containing the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 723); (s) Epitopes contained in or overlapping with the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or epitopes contained in or overlapping with the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or epitopes contained in or overlapping with the sequence TYKEL (SEQ ID NO: 706); (t) Epitopes contained in or overlapping with the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 707) and / or epitopes contained in or overlapping with the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 708) and / or epitopes contained in or overlapping with the sequence LIERIPELNKVARAAAE (SEQ ID NO: 709); (u) Epitopes contained in or overlapping with the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 710); (v) Epitopes contained in or overlapping with the sequence GTKKDFEDL (SEQ ID NO: 711); (w) Epitopes contained in or overlapping with the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 712); (x) Epitopes contained in or overlapping with the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or epitopes contained in or overlapping with the sequence DQTKFPIVNAEL (SEQ ID NO: 714) and / or epitopes contained in or overlapping with the sequence TYKELIERIPELNK (SEQ ID NO: 715); (y) Epitopes contained in or overlapping with the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or epitopes contained in or overlapping with the sequence TYKELIERIPELNK (SEQ ID NO: 715); (z) Epitopes contained in or overlapping with the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 716); (aa) Epitopes contained in or overlapping with the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or epitopes contained in or overlapping with the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 717); and (bb) Epitopes contained in or overlapping with the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or epitopes contained in or overlapping with the sequence LVENPGYVAYSKAATVTGKL (SEQ ID NO: 719) and / or epitopes contained in or overlapping with the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 720) and / or epitopes contained in or overlapping with the sequence ISRAAAEKL (SEQ ID NO: 721) and / or epitopes contained in or overlapping with the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 722) and / or epitopes contained in or overlapping with the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 723) 21. The multi-domain therapeutic protein of claim 20, wherein the TfR binding and delivery domain comprises an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof binding to one or more hTfR epitopes selected from the group consisting of: (a) Epitopes consisting of the sequence LLNE (SEQ ID NO: 752) and / or epitopes consisting of the sequence TYKEL (SEQ ID NO: 706); (b) Epitopes consisting of the sequence DSTDFTGT (SEQ ID NO: 753) and / or the sequence VKHPVTGQF (SEQ ID NO: 754) and / or the sequence IERIPEL (SEQ ID NO: 755); (c) An epitope consisting of the sequence LNENSYVPREAGSQKDEN (SEQ ID NO: 756); (d) Epitopes consisting of the sequence FEDL (SEQ ID NO: 718); (e) Epitopes consisting of the sequence IVDKNGRL (SEQ ID NO: 757); (f) Epitopes consisting of the sequence IVDKNGRLVY (SEQ ID NO: 758); (g) Epitopes consisting of the sequence DQTKF (SEQ ID NO: 759); (h) Epitopes consisting of the sequence LVENPGGY (SEQ ID NO: 760) and / or epitopes consisting of the sequence PIVNAELSF (SEQ ID NO: 761) and / or epitopes consisting of the sequence PYLGTTMDT (SEQ ID NO: 762); (i) Epitopes consisting of the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or epitopes consisting of the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or epitopes consisting of the sequence TYKEL (SEQ ID NO: 706); (j) Epitopes consisting of the sequence KRKLSEKLDSTDFTGTIKL (SEQ ID NO: 707) and / or epitopes consisting of the sequence YTLIEKTMQNVKHPVTGQFL (SEQ ID NO: 708) and / or epitopes consisting of the sequence LIERIPELNKVARAAAE (SEQ ID NO: 709); (k) An epitope consisting of the sequence LNENSYVPREAGSQKDENL (SEQ ID NO: 710); (l) An epitope consisting of the sequence GTKKDFEDL (SEQ ID NO: 711); (m) An epitope consisting of the sequence SVIIVDKNGRLVYLVENPGGYVAYSK (SEQ ID NO: 712); (n) Epitopes consisting of the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or epitopes consisting of the sequence DQTKFPIVNAEL (SEQ ID NO: 714) and / or epitopes consisting of the sequence TYKELIERIPELNK (SEQ ID NO: 715); (o) Epitopes consisting of the sequence LLNENSYVPREAGSQKDEN (SEQ ID NO: 713) and / or epitopes consisting of the sequence TYKELIERIPELNK (SEQ ID NO: 715); (p) An epitope consisting of the sequence SVIIVDKNGRLVYLVENPGGYVAY (SEQ ID NO: 716); (q) an epitope consisting of the sequence IYMDQTKFPIVNAEL (SEQ ID NO: 705) and / or an epitope consisting of the sequence FGNMEGDCPSDWKTDSTCRM (SEQ ID NO: 717); and (r) Epitopes consisting of the sequence LLNENSYVPREAGSQKDENLAL (SEQ ID NO: 704) and / or epitopes consisting of the sequence LVENPGYVAYSKAATVTGKL (SEQ ID NO: 719) and / or epitopes consisting of the sequence IYMDQTKFPIVNAELSF (SEQ ID NO: 720) and / or epitopes consisting of the sequence ISRAAAEKL (SEQ ID NO: 721) and / or epitopes consisting of the sequence VTSESKNVKLTVSNVLKE (SEQ ID NO: 722) and / or epitopes consisting of the sequence FCEDTDYPYLGTTMDT (SEQ ID NO: 723).

22. A composition comprising a nucleic acid construct comprising a coding sequence for a multi-domain therapeutic protein according to any of the preceding claims.

23. The composition of claim 22, wherein the coding sequence of the TfR binding delivery domain is codon-optimized or CpG-depleted, the coding sequence of the acid sphingomyelinase polypeptide is codon-optimized or CpG-depleted, or the coding sequence of the multi-domain therapeutic protein is codon-optimized or CpG-depleted.

24. The composition of claim 22 or 23, wherein the coding sequence of the TfR binding delivery domain is codon-optimized and CpG-depleted, the coding sequence of the acid sphingomyelinase polypeptide is codon-optimized and CpG-depleted, or the coding sequence of the multi-domain therapeutic protein is codon-optimized and CpG-depleted.

25. The composition according to any one of claims 22 to 24, wherein the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 532, 530, 531, 524-529, and 533-536, and encodes an scFv comprising any one of SEQ ID NO: 508, 494, 503, or 505. Optionally, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 532, 530, and 531, and encodes an scFv containing SEQ ID NO: 508; or optionally, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 527-529, and encodes an scFv containing SEQ ID NO:

505. Optionally, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 532, and encodes an scFv containing SEQ ID NO: 508; or optionally, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 530, and encodes an scFv containing SEQ ID NO: 508; or optionally, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 527, and encodes an scFv containing SEQ ID NO:

505.

26. The composition according to any one of claims 22 to 25, wherein the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 532, 530, 531, 524-529, and 533-536, is codon-optimized and CpG-depleted, and encodes an scFv comprising any one of SEQ ID NO: 508, 494, 503, or 505. Optionally, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 532, 530, and 531, is codon-optimized and CpG-depleted, and encodes an scFv containing SEQ ID NO: 508; or optionally, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 527-529, is codon-optimized and CpG-depleted, and encodes an scFv containing SEQ ID NO:

505. Optionally, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 532, the scFv encoding sequence is codon-optimized and CpG-depleted, and encodes an scFv containing SEQ ID NO: 508, or optionally, the scFv encoding sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 530 ... 527 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, the scFv encoding sequence is codon-optimized and CpG-depleted, and the encoding contains SEQ ID NO: 505 of scFv.

27. The composition according to any one of claims 22 to 26, wherein the scFv encoding sequence comprises, is substantially composed of, or is composed of the following sequences: The sequences listed as any one of SEQ ID NO: 532, 530, 531, 524-529, and 533-536, Optionally, the scFv encoding sequence comprises, is substantially composed of, or is composed of any of the sequences listed in SEQ ID NO: 532, 530, and 531; or, optionally, the scFv encoding sequence comprises, is substantially composed of, or is composed of any of the sequences listed in SEQ ID NO: 527-529. Optionally, the scFv encoded sequence comprises, is substantially composed of, or is composed of the sequence listed in SEQ ID NO: 532; or optionally, the scFv encoded sequence comprises, is substantially composed of, or is composed of the sequence listed in SEQ ID NO: 530; or optionally, the scFv encoded sequence comprises, is substantially composed of, or is composed of the sequence listed in SEQ ID NO:

527.

28. The composition of any one of claims 22 to 27, wherein the nucleic acid construct comprises a splice acceptor upstream of the coding sequence of the multi-domain therapeutic protein, wherein the nucleic acid construct comprises a polyadenylation signal or sequence downstream of the coding sequence of the multi-domain therapeutic protein, or wherein the nucleic acid construct comprises a splice acceptor upstream of the coding sequence of the multi-domain therapeutic protein and comprises a polyadenylation signal or sequence downstream of the coding sequence of the multi-domain therapeutic protein.

29. The composition according to any one of claims 22 to 28, wherein the nucleic acid construct does not contain homologous arms.

30. The composition according to any one of claims 22 to 29, wherein the nucleic acid construct from 5' to 3' comprises: a splice acceptor, the coding sequence of the multi-domain therapeutic protein, and a polyadenylation signal or sequence. The nucleic acid construct wherein the nucleic acid construct does not contain the promoter that drives the expression of the multi-domain therapeutic protein, and The nucleic acid constructs described therein do not contain homologous arms.

31. The composition according to any one of claims 22 to 28, wherein the nucleic acid construct comprises a homologous arm.

32. The composition according to any one of claims 22 to 31, wherein the nucleic acid construct does not contain a promoter that drives the expression of the multi-domain therapeutic protein.

33. The composition according to any one of claims 22 to 31, wherein the coding sequence of the multi-domain therapeutic protein is operatively linked to a promoter, optionally wherein the promoter is a liver-specific promoter.

34. The composition according to any one of claims 22 to 33, wherein the nucleic acid construct is contained in a nucleic acid carrier or lipid nanoparticles.

35. The composition of claim 34, wherein the nucleic acid construct is contained in the nucleic acid vector, optionally wherein the nucleic acid vector is a viral vector.

36. The composition according to claim 34 or 35, wherein the nucleic acid vector is an adeno-associated virus (AAV) vector. Optionally, the nucleic acid construct is side-attached with an inverted terminal repeat (ITR) sequence at each end, and optionally, at least one of the ITRs comprises SEQ ID NO: 160, is substantially composed of SEQ ID NO: 160, or is composed of SEQ ID NO: 160, and optionally, the ITR at each end comprises SEQ ID NO: 160, is substantially composed of SEQ ID NO: 160, or is composed of SEQ ID NO:

160.

37. The composition of claim 36, wherein the AAV vector is a single-chain AAV (ssAAV) vector.

38. The composition according to claim 36 or 37, wherein the AAV vector is a recombinant AAV8 (rAAV8) vector, optionally wherein the AAV vector is a single-stranded rAAV8 vector.

39. The composition according to any one of claims 22 to 38, wherein the composition is combined with a nuclease agent targeting a nuclease target site in a target genomic locus.

40. The composition of claim 39, wherein the target genomic locus is an albumin gene, optionally wherein the albumin gene is a human albumin gene.

41. The composition of claim 40, wherein the nuclease target site is located in intron 1 of the albumin gene.

42. The composition according to any one of claims 39 to 41, wherein the nuclease agent comprises: (a) Zinc finger nucleases (ZFN); (b) Transcription activator-like effector nucleases (TALENs); or (c)(i) the Cas protein or the nucleic acid encoding the Cas protein; and (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA targeting segment that targets the guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

43. The composition according to any one of claims 39 to 41, wherein the nuclease agent comprises: (a) the Cas protein or the nucleic acid encoding the Cas protein; and (b) A guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA targeting segment that targets the guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

44. The composition of claim 43, wherein the guide RNA target sequence is located in intron 1 of the albumin gene.

45. The composition according to claim 43 or 44, wherein the DNA targeting segment comprises any one of SEQ ID NO: 36, 30-35, and 37-61, optionally wherein the DNA targeting segment comprises any one of SEQ ID NO: 36, 30, 33, and 41, or The DNA targeting segment is composed of any one of SEQ ID NO: 36, 30-35 and 37-61, and optionally the DNA targeting segment is composed of any one of SEQ ID NO: 36, 30, 33 and 41.

46. ​​The composition according to any one of claims 43 to 45, wherein the guide RNA comprises any one of SEQ ID NO: 68, 100, 62-67, 69-99 and 101-125, and optionally wherein the guide RNA comprises any one of SEQ ID NO: 68, 100, 62, 94, 65, 97, 73 and 105.

47. The composition according to any one of claims 43 to 46, wherein the DNA targeting segment comprises or is composed of SEQ ID NO:

36.

48. The composition according to any one of claims 43 to 47, wherein the guide RNA comprises SEQ ID NO: 68 or 100.

49. The composition according to any one of claims 43 to 48, wherein the composition comprises the guide RNA in the form of RNA.

50. The composition according to any one of claims 43 to 49, wherein the guide RNA comprises at least one modification.

51. The composition according to claim 50, wherein the at least one modification comprises: (i) the phosphate thioester bond between the first four nucleotides at the 5' end of the guide RNA; (ii) the phosphate thioester bond between the last four nucleotides at the 3' end of the guide RNA; (iii) the 2'-O-methyl modified nucleotides at the first three nucleotides at the 5' end of the guide RNA; and (iv) the 2'-O-methyl modified nucleotides at the last three nucleotides at the 3' end of the guide RNA.

52. The composition according to any one of claims 43 to 51, wherein the composition comprises the guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 100, and the guide RNA comprising: (i) a phosphate thioester bond between the first four nucleotides at the 5' end of the guide RNA; (ii) a phosphate thioester bond between the last four nucleotides at the 3' end of the guide RNA; (iii) a 2'-O-methyl modified nucleotide at the first three nucleotides at the 5' end of the guide RNA; and (iv) a 2'-O-methyl modified nucleotide at the last three nucleotides at the 3' end of the guide RNA.

53. The composition according to any one of claims 43 to 52, wherein the Cas protein is a Cas9 protein, optionally wherein the Cas protein is derived from Streptococcus pyogenes Cas9 protein.

54. The composition according to any one of claims 43 to 53, wherein the Cas protein comprises the sequence listed in SEQ ID NO:

11.

55. The composition according to any one of claims 43 to 54, wherein the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises mRNA encoding the Cas protein.

56. The composition of claim 55, wherein the mRNA encoding the Cas protein comprises at least one modification.

57. The composition of claim 56, wherein the mRNA encoding the Cas protein is completely replaced by N1-methyl-pseuuridine.

58. The composition according to any one of claims 55 to 57, wherein the mRNA encoding the Cas protein comprises the sequence listed in SEQ ID NO: 1 or 2.

59. The composition according to any one of claims 43 to 58, wherein the composition comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence listed in SEQ ID NO: 1 or 2, and the mRNA encoding the Cas protein is completely substituted with N1-methyl-pseuuridine, comprises a 5' cap, and comprises a poly(A) tail.

60. The composition according to any one of claims 43 to 59, wherein the composition comprises the guide RNA in RNA form, and the guide RNA comprises SEQ ID NO: 68 or 100, and The composition comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence listed in SEQ ID NO: 1 or 2.

61. The composition according to any one of claims 43 to 60, wherein the composition comprises the guide RNA in RNA form, the guide RNA comprising SEQ ID NO: 100, and the guide RNA comprising: (i) a phosphate thioester bond between the first four nucleotides at the 5' end of the guide RNA; (ii) a phosphate thioester bond between the last four nucleotides at the 3' end of the guide RNA; (iii) a 2'-O-methyl-modified nucleotide at the first three nucleotides at the 5' end of the guide RNA; and (iv) a 2'-O-methyl-modified nucleotide at the last three nucleotides at the 3' end of the guide RNA, and The composition therein comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence listed in SEQ ID NO: 1 or 2, and the mRNA encoding the Cas protein is completely replaced by N1-methyl-pseuuridine, comprises a 5' cap, and comprises a poly(A) tail.

62. The composition according to any one of claims 43 to 61, wherein the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNA are associated with lipid nanoparticles.

63. The composition of claim 62, wherein the lipid nanoparticles comprise cationic lipids, neutral lipids, accessory lipids, and occult lipids.

64. The composition according to claim 63, wherein the cationic lipid is lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadec-9,12-dienoate), and / or The neutral lipids mentioned above are distearylphosphatidylcholine or 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), and / or The accessory lipid mentioned above is cholesterol, and / or The elusive lipid is 1,2-dimyristic-rac-glycerol-3-methoxypolyethylene glycol-2000.

65. The composition of claim 64, wherein the cationic lipid is lipid A, the neutral lipid is DSPC, the accessory lipid is cholesterol, and the occult lipid is PEG2k-DMG.

66. The composition according to any one of claims 63 to 65, wherein the lipid nanoparticles comprise four lipids in the following molar ratio: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

67. A cell comprising a multidomain therapeutic protein according to any one of claims 1 to 21 or a composition according to any one of claims 22 to 66.

68. The cell of claim 67, wherein the coding sequence of the nucleic acid construct or the multi-domain therapeutic protein is integrated into a target genomic locus, and wherein the multi-domain therapeutic protein is expressed from the target genomic locus, or wherein the coding sequence of the nucleic acid construct or the multi-domain therapeutic protein is integrated into intron 1 of an endogenous albumin locus, and wherein the multi-domain therapeutic protein is expressed from the endogenous albumin locus.

69. The cell according to claim 67 or 68, wherein the cell is a liver cell or hepatocyte.

70. The cell according to any one of claims 67 to 69, wherein the cell is a human cell.

71. A method comprising administering to cells or a population of cells a multidomain therapeutic protein according to any one of claims 1 to 21.

72. A method of inserting a nucleic acid encoding a multi-domain therapeutic protein into a target genomic locus in a cell or cell population, said multi-domain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide, said method comprising administering to said cell or cell population the composition according to any one of claims 39 to 66. The nuclease agent cleaves the nuclease target site in the target genomic locus, and the nucleic acid construct or the nucleic acid encoding the multi-domain therapeutic protein is inserted into the target genomic locus.

73. A method for expressing a multi-domain therapeutic protein in cells or a population of cells, said multi-domain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide, said method comprising administering to said cells or said population a composition according to any one of claims 22 to 31 and 33 to 38. The coding sequence of the multi-domain therapeutic protein is operatively linked to a promoter in the nucleic acid construct and expressed in the cell or cell population.

74. A method for expressing a multi-domain therapeutic protein from a target genomic locus in cells or a cell population, said multi-domain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide, said method comprising administering to said cells or said cell population a composition according to any one of claims 39 to 66, optionally wherein said nucleic acid construct is administered simultaneously with said nuclease agent or said one or more nucleic acids encoding said nuclease agent, or said nucleic acid construct is administered before or after said nuclease agent or said one or more nucleic acids encoding said nuclease agent. The nuclease agent cleaves the nuclease target site in the target genomic locus, the coding sequence of the nucleic acid construct or the multi-domain therapeutic protein is inserted into the target genomic locus to generate a modified target genomic locus, and the multi-domain therapeutic protein containing the TfR binding delivery domain fused with an acid sphingomyelinase polypeptide is expressed from the modified target genomic locus.

75. The method according to any one of claims 71 to 74, wherein the cell is a liver cell, or the cell population is a liver cell population, optionally wherein the cell is a hepatocyte, or the cell population is a hepatocyte population.

76. The method according to any one of claims 71 to 75, wherein the cell is a human cell, or the cell population is a human cell population.

77. The method according to any one of claims 71 to 76, wherein the cell is a neonatal cell, or the cell population is a neonatal cell population.

78. The method according to any one of claims 71 to 77, wherein the cells are in vitro or ex vivo, or the cell population is in vitro or ex vivo.

79. The method according to any one of claims 71 to 78, wherein the cells are in the body of the subject, or the cell population is in the body of the subject.

80. A method comprising administering to a subject a multidomain therapeutic protein according to any one of claims 1 to 21.

81. A method for inserting a nucleic acid encoding a multi-domain therapeutic protein into a target genomic locus in cells of a subject, said multi-domain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide, said method comprising administering to the subject the composition according to any one of claims 39 to 66. The nuclease agent cleaves the nuclease target site in the target genomic locus, and the coding sequence of the nucleic acid construct or the multi-domain therapeutic protein is inserted into the target genomic locus.

82. A method for expressing a multi-domain therapeutic protein in cells of a subject, said multi-domain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide protein, said method comprising administering to the subject the composition according to any one of claims 22 to 31 and 33 to 38. The coding sequence of the multi-domain therapeutic protein is operatively linked to a promoter in the nucleic acid construct and expressed in the cell.

83. A method for expressing a multi-domain therapeutic protein from a target genomic locus in in vivo cells of a subject, said multi-domain therapeutic protein comprising a TfR-binding delivery domain fused to an acid sphingomyelinase polypeptide protein, said method comprising administering to the subject a composition according to any one of claims 39 to 66, optionally wherein said nucleic acid construct is administered simultaneously with said nuclease agent or one or more nucleic acids encoding said nuclease agent, or said nucleic acid construct is administered before or after said nuclease agent or one or more nucleic acids encoding said nuclease agent. The nuclease agent cleaves the nuclease target site in the target genomic locus, the coding sequence of the nucleic acid construct or the multi-domain therapeutic protein is inserted into the target genomic locus to generate a modified target genomic locus, and the multi-domain therapeutic protein containing the TfR binding delivery domain fused with an acid sphingomyelinase polypeptide is expressed from the modified target genomic locus.

84. The method of claim 82 or 83, wherein the expressed multi-domain therapeutic protein is delivered to and internalized by the central nervous system tissue of the subject.

85. The method according to any one of claims 80 to 84, wherein the cell is a liver cell, optionally wherein the cell is a hepatocyte.

86. The method according to any one of claims 80 to 85, wherein the cell is a human cell.

87. The method according to any one of claims 80 to 86, wherein the cell is a neonatal cell.

88. A method for treating acid sphingomyelinase deficiency in a subject in need, the method comprising administering to the subject a multidomain therapeutic protein according to any one of claims 1 to 21.

89. A method of treating acid sphingomyelinase deficiency in a subject of need, the method comprising administering to the subject the composition according to any one of claims 22 to 31 and 33 to 38, The coding sequence of the multi-domain therapeutic protein is operatively linked to a promoter in the nucleic acid construct and expressed in the subject.

90. A method for treating acid sphingomyelinase deficiency in a subject in need, the method comprising administering to the subject a composition according to any one of claims 39 to 66, optionally wherein the nucleic acid construct is administered simultaneously with the nuclease agent or one or more nucleic acids encoding the nuclease agent, or the nucleic acid construct is administered before or after the nuclease agent or one or more nucleic acids encoding the nuclease agent. The nuclease agent cleaves the nuclease target site in the target genomic locus, the coding sequence of the nucleic acid construct or the multi-domain therapeutic protein is inserted into the target genomic locus to generate a modified target genomic locus, and the multi-domain therapeutic protein containing the TfR binding delivery domain fused with an acid sphingomyelinase polypeptide is expressed from the modified target genomic locus.

91. A method for preventing or alleviating the onset of signs or symptoms of acid sphingomyelinase deficiency in a subject in need, the method comprising administering to the subject a multi-domain therapeutic protein according to any one of claims 1 to 21, thereby preventing or alleviating the onset of signs or symptoms of said acid sphingomyelinase deficiency in the subject.

92. A method for preventing or alleviating the onset of signs or symptoms of acid sphingomyelinase deficiency in a subject of need, the method comprising administering to the subject the composition according to any one of claims 22 to 31 and 33 to 38. The coding sequence of the multi-domain therapeutic protein is operatively linked to a promoter in the nucleic acid construct and expressed in the subject, thereby preventing or alleviating the onset of signs or symptoms of acid sphingomyelinase deficiency in the subject.

93. A method for preventing or alleviating the onset of signs or symptoms of acid sphingomyelinase deficiency in a subject in need, the method comprising administering to the subject a composition according to any one of claims 39 to 66, optionally wherein the nucleic acid construct is administered simultaneously with the nuclease agent or one or more nucleic acids encoding the nuclease agent, or the nucleic acid construct is administered before or after the nuclease agent or one or more nucleic acids encoding the nuclease agent. The nuclease agent cleaves the nuclease target site, the coding sequence of the nucleic acid construct or the multi-domain therapeutic protein is inserted into the target genomic locus to generate a modified target genomic locus, and the multi-domain therapeutic protein containing the TfR binding delivery domain fused with an acid sphingomyelinase polypeptide is expressed from the modified target genomic locus, thereby preventing or alleviating the onset of signs or symptoms of the acid sphingomyelinase deficiency in the subject.

94. The method according to any one of claims 88 to 93, wherein the acid sphingomyelinase deficiency is Niemann-Pick disease type A.

95. The method according to any one of claims 80 to 94, wherein the subject is a human subject.

96. The method according to any one of claims 80 to 95, wherein the subject is a neonatal subject.

97. The method according to any one of claims 80 to 96, wherein the method results in a serum level of the multi-domain therapeutic protein in the subject of at least about 1 μg / mL, at least about 2 μg / mL, at least about 3 μg / mL, at least about 4 μg / mL, at least about 5 μg / mL, at least about 6 μg / mL, at least about 7 μg / mL, at least about 8 μg / mL, at least about 9 μg / mL, or at least about 10 μg / mL.

98. The method according to any one of claims 80 to 97, wherein the method results in a serum level of the multi-domain therapeutic protein in the subject of at least about 2 μg / mL or at least about 5 μg / mL.

99. The method according to any one of claims 80 to 98, wherein the method results in a serum level of the multidomain therapeutic protein in the subject between about 2 μg / mL and about 30 μg / mL or between about 2 μg / mL and about 20 μg / mL.

100. The method according to any one of claims 80 to 99, wherein the method results in a serum level of the multidomain therapeutic protein in the subject between about 5 μg / mL and about 30 μg / mL or between about 5 μg / mL and about 20 μg / mL.

101. The method according to any one of claims 80 to 100, wherein the acid sphingomyelinase activity level achieved by the method is at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% of the normal value.

102. The method according to any one of claims 80 to 101, wherein the method further comprises assessing the subject's pre-existing AAV immunity prior to administering the nucleic acid construct to the subject.

103. The method of claim 102, wherein the pre-existing AAV immunity is pre-existing AAV8 immunity.

104. The method of claim 102 or 103, wherein assessing the pre-existing AAV immunity comprises assessing immunogenicity using a total antibody immunoassay or a neutralizing antibody assay.

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