Transferrin receptor antigen-binding domain and methods of use thereof

Abstract

The present disclosure relates generally to antigen-binding domains that specifically bind to the human transferrin receptor (TfR) and their use in transport across the blood-brain barrier (BBB).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application Nos. 63 / 369,898, filed July 29, 2022, 63 / 374,967, filed September 8, 2022, 63 / 489,693, filed March 10, 2023, 63 / 495,511, filed April 11, 2023, and 63 / 513,820, filed July 14, 2023, each of which is incorporated herein by reference in its entirety.

[0002] Reference to electronically submitted sequence listing The electronically submitted sequence listing (Name: 4503_022PC05_SeqListing_ST26.xml, Size: 660,697 bytes, Created: July 13, 2023) is incorporated herein by reference in its entirety.

[0003] Field of the Disclosure The present disclosure relates to antigen-binding domains that specifically bind to the human transferrin receptor (TfR), which are capable of crossing the blood-brain barrier and transporting other agents (e.g., therapeutically active agents) bound to the antigen-binding domain across the blood-brain barrier. [Background technology]

[0004] background The passive movement of substances from the blood to the brain is restricted by the blood-brain barrier (BBB). The BBB precisely regulates central nervous system (CNS) homeostasis, enabling proper neuronal function and protecting nervous tissue from toxins and pathogens. Alterations to the BBB are important factors in the pathogenesis and progression of various neurological disorders. However, the BBB poses challenges for the delivery of therapeutic agents to the CNS. While recombinant protein and antibody therapeutics have been highly successful outside the CNS, these biologics do not efficiently cross the BBB. As a result, delivery of some therapeutic agents to the CNS relies on direct injection of the therapeutic agent into the CNS. However, such injections are an invasive procedure whose effectiveness is limited by the rapid transport of therapeutic-containing cerebrospinal fluid (CSF) from the brain to the blood. Alternatively, CNS-targeted therapeutic agents can be administered systemically at high doses that allow the therapeutic agent to fully penetrate the BBB. However, this approach can have unintended effects due to high peripheral doses or the increased manufacturing and formulation burdens associated with achieving such high doses. Therefore, there is a need for improved products and methods for delivering therapeutic agents across the BBB. Summary of the Invention

[0005] Summary of the Disclosure Provided herein are antigen-binding domains, fusion proteins, antibodies, and multispecific proteins that specifically bind to the human transferrin receptor (TfR), and methods of making and using them.

[0006] In some aspects, provided herein is an antigen-binding domain that specifically binds to the apical domain of the human transferrin receptor (TfR), wherein the antigen-binding domain localizes to the brain parenchyma of a subject after peripheral injection, and is not within a modified CH3 domain.

[0007] In some aspects, provided herein are antigen-binding domains that specifically bind to human transferrin receptor (TfR), and are represented by SEQ ID NOs: 94, 415, 418, 423, 150, and 151, respectively; SEQ ID NOs: 74 to 76 and 135 to 137, respectively; SEQ ID NOs: 77 to 79 and 138 to 140, respectively; SEQ ID NOs: 80 to 82 and 135 to 137, respectively; SEQ ID NOs: 83 to 85 and 141 to 143, respectively; SEQ ID NOs: 74, 86, 76, and 135 to 137, respectively; SEQ ID NOs: 87 to 89 and 143 to 145, respectively; and SEQ ID NO: 9. 0 to 92 and 146 to 148, respectively, SEQ ID NOs: 83, 93, 85 and 141 to 143, respectively, SEQ ID NOs: 94 to 96 and 149 to 151, respectively, SEQ ID NOs: 97, 86, 98 and 135 to 137, respectively, SEQ ID NOs: 99, 100, 101 and 152 to 154, respectively, SEQ ID NOs: 99, 102, 103 and 155 to 157, respectively, SEQ ID NOs: 104 to 106, 138, 158 and 159, respectively, SEQ ID NOs: 107 to 109 and 160 to 162, respectively, SEQ ID NOs: 99, 110, 103 and 155 to 157, respectively, SEQ ID NOs: 9 SEQ ID NOs: 90, 113, 114, 165, 166, and 157, respectively; SEQ ID NOs: 83, 115, 85, 141, 142, and 167, respectively; SEQ ID NOs: 116, 110, 103, and 155-157, respectively; SEQ ID NOs: 117-119 and 168-170, respectively; SEQ ID NOs: 116, 110, 103, and 168-170, respectively; SEQ ID NOs: 90, 120, 114, 165, 166, and 157, respectively; SEQ ID NOs: 90, 113, 114, 171, 166, and 157, respectively; SEQ ID NOs: 97, 121, 122, 135, 136, and 172, respectively; SEQ ID NOs: 74, 123, 124, 173, 136, and 137, respectively; SEQ ID NOs: 74, 125, 126, 174, 136, and 137, respectively; SEQ ID NOs: 99, 127, 128, 155, 175, and 176, respectively; SEQ ID NOs: 90, 129, 130, 165, 166, and 157, respectively; SEQ ID NOs: 131, 132, 133, 177, 166, and 178, respectively; SEQ ID NOs: 131, 132, 133, and 179 to 181, respectively; SEQ ID NOs: 90, 134,114, 165, 166, and 157, respectively; SEQ ID NOs: 83, 302, 303, 141, 142, and 182, respectively; SEQ ID NOs: 99, 305, 103, 307, 308, and 157, respectively; SEQ ID NOs: 99, 306, 103, 307, 308, and 157, respectively; SEQ ID NOs: 99, 306, 103, 307, 309, and 157, respectively; SEQ ID NOs: 99, 306, 103, 310, 311, and 157, respectively; SEQ ID NOs: 99, 305, 103, 310, 311, and 157, respectively; SEQ ID NOs: 99, 312, 103, 307, 308, respectively. , and 157, respectively, SEQ ID NOs: 99, 312, 103, 307, 309, and 157, respectively, SEQ ID NOs: 99, 312, 103, 310, 311, and 157, respectively, SEQ ID NOs: 94, 95, 418, 421, 150, and 151, respectively, SEQ ID NOs: 94, 412, 419, 422, 150, and 151, respectively, SEQ ID NOs: 94, 412, 420, 422, 150, and 151, respectively, SEQ ID NOs: 94, 413, 418, 422, 150, and 151, respectively, SEQ ID NOs: 94, 414, 418, 422, 150, and 151, respectively, Sequence numbers 94, 416, 418, 422, 150, and 151, respectively; sequence numbers 94, 417, 418, 424, 150, and 151, respectively; sequence numbers 460, 312, 103, 307, 309, and 157, respectively; sequence numbers 460, 312, 461, 307, 309, and 157, respectively; sequence numbers 460, 312, 462, 307, 309, and 157, respectively; sequence numbers 460, 312, 463, 307, 309, and 157, respectively; sequence numbers 460, 312, 464, 307, 309, and 157, respectively; sequence number 460 , 312, 465, 307, 309, and 157, respectively; SEQ ID NOs: 460, 312, 466, 307, 309, and 157, respectively; SEQ ID NOs: 460, 312, 467, 307, 309, and 157, respectively; SEQ ID NOs: 460, 312, 468, 307, 309, and 157, respectively; SEQ ID NOs: 460, 312, 469, 307, 309, and 157, respectively; SEQ ID NOs: 460, 312, 470, 307, 309, and 157, respectively; SEQ ID NOs: 460, 312, 103, 471, 309, and 157, respectively; SEQ ID NOs: 460, 312,and 157, respectively; SEQ ID NOs: 460, 312, 103, 473, 309, and 157, respectively; SEQ ID NOs: 460, 312, 103, 474, 309, and 157, respectively; SEQ ID NOs: 460, 312, 103, 475, 309, and 157, respectively; SEQ ID NOs: 460, 312, 103, 476, 309, and 157, respectively; or SEQ ID NOs: 460, 312, 103, 471, 309, and 157, respectively.

[0008] In some embodiments, the antigen binding domain comprises a VH and a VL, wherein the VH and VL are selected from the group consisting of SEQ ID NOs: 399 and 400, respectively, SEQ ID NOs: 10 and 11, respectively, SEQ ID NOs: 12 and 13, respectively, SEQ ID NOs: 14 and 15, respectively, SEQ ID NOs: 16 and 17, respectively, SEQ ID NOs: 18 and 19, respectively, SEQ ID NOs: 20 and 21, respectively, SEQ ID NOs: 22 and 23, respectively, SEQ ID NOs: 24 and 25, respectively, SEQ ID NOs: 26 and 27, respectively, SEQ ID NOs: 28 and 29, respectively, SEQ ID NOs: 30 and 31, respectively, SEQ ID NOs: 32 and 33, respectively, SEQ ID NOs: 34 and 35, respectively, SEQ ID NOs: 35 and 36, respectively, SEQ ID NOs: 37 and 38, respectively, SEQ ID NOs: 39 and 400, respectively, SEQ ID NOs: 10 and 11, respectively, SEQ ID NOs: 12 and 13, respectively, SEQ ID NOs: 14 and 15, respectively, SEQ ID NOs: 16 and 17, respectively, SEQ ID NOs: 18 and 19, respectively, SEQ ID NOs: 20 and 21, respectively, SEQ ID NOs: 22 and 23, respectively, SEQ ID NOs: 24 and 25, respectively, SEQ ID NOs: 26 and 27, respectively, SEQ ID NOs: 28 and 29, respectively, SEQ ID NOs: 30 and 31, respectively, SEQ ID NOs: 32 and 33, respectively, SEQ ID NOs: 34 and 35 5, SEQ ID NOs: 36 and 37, respectively, SEQ ID NOs: 38 and 39, respectively, SEQ ID NOs: 40 and 41, respectively, SEQ ID NOs: 42 and 43, respectively, SEQ ID NOs: 44 and 45, respectively, SEQ ID NOs: 46 and 47, respectively, SEQ ID NOs: 48 and 49, respectively, SEQ ID NOs: 50 and 51, respectively, SEQ ID NOs: 52 and 53, respectively, SEQ ID NOs: 54 and 55, respectively, SEQ ID NOs: 56 and 57, respectively, SEQ ID NOs: 58 and 59, respectively, SEQ ID NOs: 60 and 61, respectively, SEQ ID NOs: 62 and 63, respectively, SEQ ID NOs: 64 and 65, respectively, SEQ ID NOs: 66 and 67, respectively. Nos. 68 and 69, SEQ ID NOs: 70 and 71, respectively, SEQ ID NOs: 72 and 73, respectively, SEQ ID NOs: 313 and 314, respectively, SEQ ID NOs: 315 and 316, respectively, SEQ ID NOs: 317 and 314, respectively, SEQ ID NOs: 318 and 316, respectively, SEQ ID NOs: 319 and 314, SEQ ID NOs: 320 and 316, respectively, SEQ ID NOs: 321 and 314, respectively, SEQ ID NOs: 322 and 316, respectively, SEQ ID NOs: 323 and 314, respectively, SEQ ID NOs: 324 and 316, respectively, SEQ ID NOs: 325 and 326, respectively, SEQ ID NOs: 327 and 328, respectively Sequence numbers 325 and 329, SEQ ID NOs: 330 and 331, respectively, SEQ ID NOs: 325 and 332, respectively, SEQ ID NOs: 330 and 333, respectively, SEQ ID NOs: 334 and 332, respectively, SEQ ID NOs: 335 and 336, respectively, SEQ ID NOs: 337 and 338, respectively, SEQ ID NOs: 38 and 33, respectively, SEQ ID NOs: 339 and 340, respectively, SEQ ID NOs: 339 and 341, respectively, SEQ ID NOs: 339 and 342, respectively, SEQ ID NOs: 343 and 340, respectively, SEQ ID NOs: 343 and 342, respectively, SEQ ID NOs: 344 and 340, respectively, SEQ ID NOs: 344 and 341, respectively,SEQ ID NOs: 344 and 342, respectively, SEQ ID NOs: 345 and 340, respectively, SEQ ID NOs: 345 and 341, respectively, SEQ ID NOs: 345 and 342, respectively, SEQ ID NOs: 346 and 347, respectively, SEQ ID NOs: 348 and 316, respectively, SEQ ID NOs: 349 and 347, respectively, SEQ ID NOs: 349 and 350, respectively, SEQ ID NOs: 351 and 352, respectively, SEQ ID NOs: 349 and 353, respectively, SEQ ID NOs: 346 and 354, respectively, SEQ ID NOs: 349 and 355, respectively, SEQ ID NOs: 349 and 356, respectively, SEQ ID NOs: 357 and 316, respectively 349 and 358, SEQ ID NOs: 349 and 316, respectively, SEQ ID NOs: 359 and 360, respectively, SEQ ID NOs: 361 and 362, respectively, SEQ ID NOs: 363 and 316, respectively, SEQ ID NOs: 364 and 365, respectively, SEQ ID NOs: 349 and 366, respectively, SEQ ID NOs: 346 and 316, respectively, SEQ ID NOs: 346 and 367, respectively, SEQ ID NOs: 349 and 368, respectively, SEQ ID NOs: 369 and 316, respectively, SEQ ID NOs: 346 and 370, respectively, SEQ ID NOs: 371 and 316, respectively, SEQ ID NOs: 372 and 356, respectively, SEQ ID NOs: 357 and 358, respectively, SEQ ID NOs: 349 and 373, respectively, SEQ ID NOs: 346 and 374, respectively, SEQ ID NOs: 375 and 316, respectively, SEQ ID NOs: 376 and 316, respectively, SEQ ID NOs: 346 and 377, respectively, SEQ ID NOs: 378 and 379, respectively, SEQ ID NOs: 380 and 381, respectively, SEQ ID NOs: 349 and 382, ​​respectively, SEQ ID NOs: 357 and 383, respectively, SEQ ID NOs: 349 and 358, respectively, SEQ ID NOs: 384 and 316, respectively, SEQ ID NOs: 385 and 316, respectively, SEQ ID NOs: 357 and 386, respectively, SEQ ID NOs: 387 and 388, respectively 359 and 316, SEQ ID NOs: 389 and 316, respectively, SEQ ID NOs: 390 and 316, respectively, SEQ ID NOs: 391 and 392, respectively, SEQ ID NOs: 393 and 356, respectively, SEQ ID NOs: 390 and 392, respectively, SEQ ID NOs: 357 and 386, SEQ ID NOs: 394 and 395, respectively, SEQ ID NOs: 396 and 395, respectively, SEQ ID NOs: 397 and 395, respectively, SEQ ID NOs: 398 and 395, respectively, SEQ ID NOs: 401 and 395, respectively, SEQ ID NOs: 402 and 403, respectively, SEQ ID NOs: 443 and 341, respectively, SEQ ID NOs: 444 and 341, respectively,SEQ ID NOs: 445 and 341, respectively, SEQ ID NOs: 446 and 341, respectively, SEQ ID NOs: 447 and 341, respectively, SEQ ID NOs: 448 and 341, respectively, SEQ ID NOs: 449 and 341, respectively, SEQ ID NOs: 450 and 341, respectively, SEQ ID NOs: 451 and 341, respectively, SEQ ID NOs: 452 and 341, respectively, SEQ ID NOs: 453 and 341, respectively, SEQ ID NOs: 443 and 454, respectively, SEQ ID NOs: 443 and 455, respectively, SEQ ID NOs: 443 and 456, respectively, SEQ ID NOs: 443 and 457, respectively, SEQ ID NOs: 443 and 458, respectively, or SEQ ID NOs: 443 and 459, respectively.

[0009]

[0023] In some aspects, provided herein is an antigen binding domain that specifically binds to a human TfR, comprising a VH and a VL, wherein the VH is selected from the group consisting of SEQ ID NOs: 399, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 313, 315, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383 Antigen-binding domains are provided comprising the amino acid sequence of 34, 335, 337, 38, 339, 343, 344, 345, 346, 348, 349, 351, 357, 359, 361, 363, 364, 369, 371, 372, 375, 376, 378, 380, 384, 385, 387, 389, 390, 391, 393, 394, 396, 397, 398, 401, 402, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, or 453.

[0010]

[0020] In some aspects, provided herein is an antigen binding domain that specifically binds to human TfR, comprising a VH and a VL, wherein the VL is selected from the group consisting of: 400, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 314, 316, 326, 328, 329, 331, 332, 333, 334, 335, 336, 337, 338, 339, 440, 441, 442, 443, 444, 445, 446, 447, 4 ...8, 449, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 Antigen-binding domains are provided comprising the amino acid sequence of 332, 333, 336, 338, 340, 341, 342, 347, 350, 352, 353, 354, 355, 356, 358, 360, 362, 365, 366, 367, 368, 370, 373, 374, 377, 379, 381, 382, ​​383, 386, 388, 392, 395, 403, 454, 455, 456, 457, 458, or 459.

[0011] In some aspects, the antigen binding domains provided herein are selected from the group consisting of SEQ ID NOs: 399 and 400, respectively, SEQ ID NOs: 10 and 11, respectively, SEQ ID NOs: 12 and 13, respectively, SEQ ID NOs: 14 and 15, respectively, SEQ ID NOs: 16 and 17, respectively, SEQ ID NOs: 18 and 19, respectively, SEQ ID NOs: 20 and 21, respectively, SEQ ID NOs: 22 and 23, respectively, SEQ ID NOs: 24 and 25, respectively, SEQ ID NOs: 26 and 27, respectively, SEQ ID NOs: 28 and 29, respectively, SEQ ID NOs: 30 and 31, respectively, SEQ ID NOs: 32 and 33, respectively, SEQ ID NOs: 34 and 35, respectively, SEQ ID NOs: 36 and 37, SEQ ID NOs:38 and 39, SEQ ID NOs:40 and 41, SEQ ID NOs:42 and 43, SEQ ID NOs:44 and 45, SEQ ID NOs:46 and 47, SEQ ID NOs:48 and 49, SEQ ID NOs:50 and 51, SEQ ID NOs:52 and 53, SEQ ID NOs:54 and 55, SEQ ID NOs:56 and 57, SEQ ID NOs:58 and 59, SEQ ID NOs:60 and 61, SEQ ID NOs:62 and 63, SEQ ID NOs:64 and 65, SEQ ID NOs:66 and 67, SEQ ID NOs:68 and 69, respectively SEQ ID NOs: 70 and 71, respectively, SEQ ID NOs: 72 and 73, SEQ ID NOs: 313 and 314, respectively, SEQ ID NOs: 315 and 316, respectively, SEQ ID NOs: 317 and 314, respectively, SEQ ID NOs: 318 and 316, respectively, SEQ ID NOs: 319 and 314, respectively, SEQ ID NOs: 320 and 316, respectively, SEQ ID NOs: 321 and 314, respectively, SEQ ID NOs: 322 and 316, respectively, SEQ ID NOs: 323 and 314, respectively, SEQ ID NOs: 324 and 316, respectively, SEQ ID NOs: 325 and 326, respectively, SEQ ID NOs: 327 and 328, respectively, SEQ ID NOs: 325 and 329, respectively, SEQ ID NOs: 330 and 331, respectively, SEQ ID NOs: 325 and 332, respectively, SEQ ID NOs: 330 and 333, respectively, SEQ ID NOs: 334 and 332, respectively, SEQ ID NOs: 335 and 336, respectively, SEQ ID NOs: 337 and 338, respectively, SEQ ID NOs: 38 and 33, respectively, SEQ ID NOs: 339 and 340, respectively, SEQ ID NOs: 339 and 341, respectively, SEQ ID NOs: 339 and 342, respectively, SEQ ID NOs: 343 and 340, respectively, SEQ ID NOs: 343 and 342, respectively, SEQ ID NOs: 344 and 340, respectively, SEQ ID NOs: 344 and 341, respectively, SEQ ID NOs: 344 and 342,SEQ ID NOs: 345 and 340, respectively, SEQ ID NOs: 345 and 341, respectively, SEQ ID NOs: 345 and 342, respectively, SEQ ID NOs: 346 and 347, respectively, SEQ ID NOs: 348 and 316, SEQ ID NOs: 349 and 347, respectively, SEQ ID NOs: 349 and 350, respectively, SEQ ID NOs: 351 and 352, respectively, SEQ ID NOs: 349 and 353, respectively, SEQ ID NOs: 346 and 354, respectively, SEQ ID NOs: 349 and 355, respectively, SEQ ID NOs: 349 and 356, respectively, SEQ ID NOs: 357 and 316, respectively, SEQ ID NOs: 349 and 358, respectively 349 and 316, SEQ ID NOs: 359 and 360, respectively, SEQ ID NOs: 361 and 362, respectively, SEQ ID NOs: 363 and 316, respectively, SEQ ID NOs: 364 and 365, respectively, SEQ ID NOs: 349 and 366, respectively, SEQ ID NOs: 346 and 316, respectively, SEQ ID NOs: 346 and 367, respectively, SEQ ID NOs: 349 and 368, respectively, SEQ ID NOs: 369 and 316, respectively, SEQ ID NOs: 346 and 370, respectively, SEQ ID NOs: 371 and 316, respectively, SEQ ID NOs: 372 and 356, respectively, SEQ ID NOs: 357 and 358, respectively, SEQ ID NOs: 349 and 373, respectively, SEQ ID NOs: 346 and 374, respectively, SEQ ID NOs: 375 and 316, respectively, SEQ ID NOs: 376 and 316, respectively, SEQ ID NOs: 346 and 377, respectively, SEQ ID NOs: 378 and 379, respectively, SEQ ID NOs: 380 and 381, respectively, SEQ ID NOs: 349 and 382, ​​respectively, SEQ ID NOs: 357 and 383, respectively, SEQ ID NOs: 349 and 358, respectively, SEQ ID NOs: 384 and 316, respectively, SEQ ID NOs: 385 and 316, respectively, SEQ ID NOs: 357 and 386, respectively, SEQ ID NOs: 387 and 388, respectively, SEQ ID NOs: 359 and 316, respectively 389 and 316, SEQ ID NOs: 390 and 316, respectively, SEQ ID NOs: 391 and 392, respectively, SEQ ID NOs: 393 and 356, respectively, SEQ ID NOs: 390 and 392, respectively, SEQ ID NOs: 357 and 386, SEQ ID NOs: 394 and 395, respectively, SEQ ID NOs: 396 and 395, respectively, SEQ ID NOs: 397 and 395, respectively, SEQ ID NOs: 398 and 395, respectively, SEQ ID NOs: 401 and 395, respectively, SEQ ID NOs: 402 and 403, respectively, SEQ ID NOs: 443 and 341, respectively, SEQ ID NOs: 444 and 341, respectively, SEQ ID NOs: 445 and 341, respectively,The VH and VL comprise the amino acid sequences of SEQ ID NOs: 446 and 341, respectively, SEQ ID NOs: 447 and 341, respectively, SEQ ID NOs: 448 and 341, respectively, SEQ ID NOs: 449 and 341, respectively, SEQ ID NOs: 450 and 341, respectively, SEQ ID NOs: 451 and 341, respectively, SEQ ID NOs: 452 and 341, respectively, SEQ ID NOs: 453 and 341, respectively, SEQ ID NOs: 443 and 454, respectively, SEQ ID NOs: 443 and 455, respectively, SEQ ID NOs: 443 and 456, respectively, SEQ ID NOs: 443 and 457, respectively, SEQ ID NOs: 443 and 458, respectively, or SEQ ID NOs: 443 and 459, respectively.

[0012] In some embodiments, the antigen-binding domain specifically binds to the apical domain of the human transferrin receptor (TfR), the antigen-binding domain localizes to the brain parenchyma of the subject after peripheral injection, and the antigen-binding domain is not within the modified CH3 domain.

[0013] In some embodiments, the antigen-binding domain can cross the blood-brain barrier (BBB). In some embodiments, the antigen-binding domain binds to cynomolgus monkey TfR. In some embodiments, the antigen-binding domain is internalized into blood-brain barrier epithelial cells. In some embodiments, the blood-brain barrier epithelial cells are HCMEC / D3 cells.

[0014] In some embodiments, the antigen-binding domain binds to human TfR with an affinity of 500 nM to 10 μM. In some embodiments, the antigen-binding domain binds to human TfR with an affinity of 2 μM to 8 μM. In some embodiments, the antigen-binding domain binds to human TfR with an affinity of 2 μM to 5 μM. In some embodiments, the antigen-binding domain binds to human TfR with an affinity of 750 nM to 2 μM. In some embodiments, the antigen-binding domain binds to human TfR with an affinity of 50 nM to 500 nM. In some embodiments, the antigen-binding domain binds to human TfR with an affinity of 100 nM to 250 nM. In some embodiments, the antigen-binding domain binds to human TfR with an affinity of 1 nM to 50 nM. In some embodiments, the antigen-binding domain binds to human TfR with an affinity of 6.7 nM to 3.5 μM. In some aspects, the antigen-binding domain binds to cynomolgus monkey TfR with an affinity of 38 nM to 2.3 μM. In some embodiments, the antigen-binding domain binds to human TfR with an affinity of about 3.5 μM. In some aspects, the antigen-binding domain binds to cynomolgus monkey TfR with an affinity of about 1.5 μM. In some aspects, affinity is measured by high-throughput surface plasmon resonance (SPR) detection.

[0015] In some embodiments, the antigen binding domain does not reduce cell surface expression of TfR on HCMEC / D3 cells by more than 60% compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. In some embodiments, the antigen binding domain does not reduce cell surface expression of TfR on HCMEC / D3 cells by more than 40% compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. In some embodiments, the antigen binding domain does not significantly increase cell surface expression of TfR on HCMEC / D3 cells compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. In some embodiments, the antigen binding domain accumulates in vascular-depleted mouse brains at least four-fold or at least five-fold more than the isotype control.

[0016] In some embodiments, the antigen-binding domain specifically binds to human TfR at least 5-fold more than it binds to an unrelated protein. In some embodiments, the antigen-binding domain specifically binds to cynomolgus monkey TfR at least 5-fold more than it binds to an unrelated protein. In some embodiments, the antigen-binding domain specifically binds to human TfR at least 5-fold more than it binds to an unrelated protein and / or specifically binds to cynomolgus monkey TfR at least 5-fold more than it binds to an unrelated protein. In some embodiments, the antigen-binding domain does not significantly reduce TfR expression levels in the brain of a primate following intravenous administration of the antigen-binding domain.

[0017] In some embodiments, the antigen-binding domain comprises a VH and a VL on a single polypeptide chain. In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv is oriented as VH-linker-VL. In some embodiments, the scFv is oriented as VL-linker-VH. In some embodiments, the linker is about 5 to about 25 amino acids, about 5 to about 20 amino acids, about 10 to about 25 amino acids, or about 10 to about 20 amino acids. In some embodiments, the linker comprises the amino acid sequence GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 183) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 288). In some embodiments, the antigen-binding domain comprises a VH on a first polypeptide and a VL on a second polypeptide.

[0018] In some embodiments, the antigen-binding domain comprises the amino acid sequence of any one of SEQ ID NOs: 404, 185, 186, 189, 190, 192, 193, 195-259, and 261-284.

[0019] In some aspects, the antigen-binding domain is a murine antigen-binding domain, a chimeric antigen-binding domain, a humanized antigen-binding domain, or a human antigen-binding domain; optionally, the antigen-binding domain is a humanized antigen-binding domain.

[0020] In some aspects, provided herein is an antigen-binding domain that specifically binds to human TfR, wherein the antigen-binding domain is a VHH and comprises (i) a VH CDR1, a VH CDR2, and a VH CDR3 of the antigen-binding domain provided herein, or (ii) a VH of the antigen-binding domain provided herein, wherein optionally the VHH is capable of crossing the blood-brain barrier (BBB).

[0021] In some embodiments, provided herein are fusion proteins comprising an antigen-binding domain provided herein and a heterologous protein or peptide, such as beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebromocriptine (β-glucocerebromocriptine), and the like. enzymes (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), glycoprotein non-transferase B (GPNMB), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 1 (SIG1), and sialic acid-binding Ig-like lectin 2 (SIG2). Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4-domain A4A (MS4A4A), transmembrane 4-domain A6A (MS4A6A), or transmembrane protein 106B (TMEM106b), clusterin (APOJ), reelin, ubiquitin protein ligase E3A (UBE3A), tripeptidyl peptidase 1 Heterologous proteins or peptides are provided that include the amino acid sequence of (CLN2 / TPP1), alpha-L-iduronidase (IDUA), iduronate 2-sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosamide N-acetyltransferase (HGSNAT), N-acetyl-alpha-glucosaminidase (NAGLU), or N-sulfoglucosamine sulfohydrolase (SGSH), or portions thereof.

[0022] In some embodiments, provided herein are fusion proteins further comprising an Fc domain, hi some embodiments, the Fc domain is capable of binding to FcRn.

[0023] In some embodiments, provided herein are fusion proteins comprising (i) a single scFv, VHH, or Fab antigen-binding domain that binds to human TfR and (ii) two copies of a heterologous protein or peptide. In some embodiments, a fusion protein comprising a single scFv, Fab, or VHH antigen-binding domain that binds to human TfR is linked to the C-terminus of one of the two copies of the heterologous protein or peptide. In some embodiments, the two copies of the heterologous protein or peptide are linked to the N-terminus of the Fc domain. In some embodiments, a single scFv, Fab, or VHH antigen-binding domain that binds to human TfR is linked to the N-terminus of the Fc domain of the fusion proteins provided herein. In some embodiments, two copies of the heterologous protein or peptide are linked to the C-terminus of the Fc domain of the fusion proteins provided herein.

[0024] In some aspects, provided herein is a fusion protein comprising: (i) an antibody that binds to human TfR, the antibody comprising two heavy chains and two light chains; and (ii) two copies of a heterologous protein or peptide, wherein each copy of the heterologous protein or peptide is linked to the C-terminus of one of the two antibody heavy chains. In some aspects, provided herein is a fusion protein comprising: (i) two scFv, Fab, or VHH antigen-binding domains that bind to human TfR, (ii) an Fc domain, and (iii) two copies of a heterologous protein or peptide, wherein the two scFv, Fab, or VHH antigen-binding domains that bind to human TfR are linked to the C-terminus of the Fc domain, and the two copies of the heterologous protein or peptide are linked to the N-terminus of the Fc domain. In some embodiments, provided herein is a fusion protein comprising (i) a single scFv, VHH, or Fab antigen-binding domain that binds to human TfR, (ii) an Fc domain, and (iii) a single copy of a heterologous protein or peptide, wherein the single scFv, VHH, or Fab antigen-binding domain that binds to human TfR is linked to the C-terminus of the Fc domain and the heterologous protein or polypeptide is linked to the N-terminus of the Fc domain. In some embodiments, provided herein is a fusion protein comprising (i) a single scFv, VHH, or Fab antigen-binding domain that binds to human TfR, (ii) an Fc domain, and (iii) a single copy of a heterologous protein or peptide, wherein the single scFv, VHH, or Fab antigen-binding domain that binds to human TfR is linked to the N-terminus of the Fc domain and the heterologous protein or polypeptide is linked to the C-terminus of the Fc domain.In some embodiments, provided herein are fusion proteins comprising (i) a single scFv, VHH, or Fab antigen-binding domain that binds to human TfR, (ii) an Fc domain, and (iii) a single copy of a heterologous protein or peptide, wherein the single scFv, VHH, or Fab antigen-binding domain that binds to human TfR and the heterologous protein or polypeptide are both linked to the N-terminus of the Fc domain.

[0025] In some embodiments, the Fc domain is a heterodimeric Fc, optionally comprising knob-and-hole mutations. In some embodiments, the Fc is a single-chain, monovalent Fc. In some embodiments, the Fc is a modified Fc having one or more modifications set forth in Table 1 or 2. In some embodiments, the Fc comprises mutations that reduce effector function, optionally wherein the mutations that reduce effector function include (i) L234A, L235A, and / or P331S, and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S.

[0026] In some embodiments, provided herein are antibodies comprising the antigen-binding domains provided herein. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that bind to the same human TfR epitope as the antigen-binding domains provided herein. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that competitively inhibit the binding of the antigen-binding domains provided herein to human TfR.

[0027] In some embodiments, provided herein are multispecific proteins comprising a first antigen-binding domain provided herein linked to a second antigen-binding domain. In some embodiments, the second antigen-binding domain specifically binds to a CNS antigen. In some embodiments, provided herein are multispecific proteins comprising an antigen-binding domain provided herein linked to an antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a CNS antigen. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region. In some embodiments, the antigen-binding domain provided herein is linked to the C-terminus of the heavy chain constant region, optionally via an amino acid linker. In some embodiments, the multispecific protein is bispecific. In some embodiments, the multispecific protein is bivalent, trivalent, or tetravalent. In some embodiments, the multispecific protein is bivalent. In some embodiments, the multispecific protein is trivalent, and optionally the trivalent protein comprises one antigen-binding domain that binds to human TfR and two antigen-binding domains that bind to CNS antigens. In some embodiments, the multispecific protein is tetravalent, and optionally the tetravalent protein comprises two antigen-binding domains that bind to human TfR and two antigen-binding domains that bind to CNS antigens.

[0028] In some aspects, provided herein are multispecific proteins that are trivalent and bispecific and comprise an antigen-binding domain provided herein linked to an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, and wherein the antigen-binding domain is an scFv, and wherein the scFv is linked to the C-terminus of one of the two antibody heavy chains, optionally via an amino acid linker.

[0029] In some aspects, provided herein are multispecific proteins that are trivalent and bispecific and comprise an antigen-binding domain provided herein linked to an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, and wherein the antigen-binding domain is an scFv, and wherein the scFv is linked to the N-terminus of one of the two antibody heavy chains, optionally via an amino acid linker.

[0030] In some aspects, provided herein are multispecific proteins that are tetravalent and bispecific, comprising two antigen-binding domains provided herein and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, each of the two antigen-binding domains being an scFv, Fab, or VHH, and wherein one of the two antigen-binding domains is linked, optionally via an amino acid linker, to the C-terminus of one antibody heavy chain, and the other of the antigen-binding domains is linked, optionally via an amino acid linker, to the C-terminus of the other antibody heavy chain.

[0031] In some aspects, provided herein are multispecific proteins that are tetravalent and bispecific, comprising two antigen-binding domains provided herein and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, each of the two antigen-binding domains being an scFv, Fab, or VHH, and wherein one of the two antigen-binding domains is linked, optionally via an amino acid linker, to the N-terminus of one antibody heavy chain, and the other of the antigen-binding domains is linked, optionally via an amino acid linker, to the N-terminus of the other antibody heavy chain.

[0032] In some aspects, provided herein are multispecific proteins that are tetravalent and bispecific, comprising two antigen-binding domains provided herein and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, each of the two antigen-binding domains being an scFv, Fab, or VHH, and wherein one of the two antigen-binding domains is linked, optionally via an amino acid linker, to the N-terminus of one antibody heavy chain, and the other of the antigen-binding domains is linked, optionally via an amino acid linker, to the C-terminus of the other antibody heavy chain.

[0033] In some embodiments, the two antigen-binding domains provided herein are two copies of the same antigen-binding domain.

[0034] In some aspects, provided herein are multispecific proteins that are bivalent and bispecific and comprise: (i) an Fc domain; (ii) a single antigen-binding domain provided herein that binds to human TfR and is a single scFv, VHH, or Fab antigen-binding domain linked to the N-terminus of the Fc domain; and (iii) a second antigen-binding domain that specifically binds to a CNS antigen, which is a single scFv, VHH, or Fab linked to the C-terminus of the Fc domain.

[0035] In some aspects, provided herein are multispecific proteins that are bivalent and bispecific and comprise: (i) an Fc domain; (ii) a single antigen-binding domain provided herein that binds to human TfR and is a single scFv, VHH, or Fab antigen-binding domain linked to the C-terminus of the Fc domain; and (iii) a second antigen-binding domain that specifically binds to a CNS antigen, wherein the second antigen-binding domain is a single scFv, VHH, or Fab linked to the C-terminus of the Fc domain.

[0036] In some embodiments, the Fc domain is a heterodimeric Fc that optionally comprises a knob-and-hole mutation. In some embodiments, the Fc is a modified Fc having one or more modifications set forth in Table 1 or 2. In some embodiments, the Fc comprises a mutation that reduces effector function, optionally comprising (i) L234A, L235A, and / or P331S, and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S. In some embodiments, the antibody or antigen-binding fragment thereof comprises a mutation that reduces effector function, optionally comprising (i) L234A, L235A, and / or P331S, and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S. In some embodiments, the antibody or antigen-binding fragment thereof comprises a constant region comprising knob mutations and mutations that reduce effector function, optionally wherein the mutations that reduce effector function comprise (i) L234A, L235A, and / or P331S, and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S. In some embodiments, the antibody or antigen-binding fragment thereof comprises a constant region comprising hole mutations and mutations that reduce effector function, optionally wherein the mutations that reduce effector function comprise (i) L234A, L235A, and / or P331S, and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S.

[0037] In some aspects, the antibody or antigen-binding fragment thereof comprises a constant region comprising a knob mutation and a constant region comprising a hole mutation. In some aspects, the antigen-binding domain is linked to the constant region comprising the hole mutation, optionally via an amino acid linker. In some aspects, the antigen-binding domain is linked to the constant region comprising the knob mutation, optionally via an amino acid linker.

[0038] In some embodiments, the amino acid linker is a glycine-serine linker. In some embodiments, the glycine-serine linker comprises the amino acid sequence (GGGGS)x3 (SEQ ID NO: 184) or the amino acid sequence (GGSGG)x3 (SEQ ID NO: 289).

[0039] In some embodiments, the CNS antigen is a brain antigen. In some embodiments, the CNS antigen is not TfR.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof. In some embodiments, the IgG antibody or antigen-binding fragment thereof is an IgG1 antibody or antigen-binding fragment thereof or an IgG4 antibody or antigen-binding fragment thereof.

[0041] In some embodiments, the multispecific protein binds to human TfR with an equilibrium dissociation constant (KD) of about 65 nM to about 4 μM and / or binds to cynomolgus monkey TfR with a KD of about 37 nM to about 1.3 μM.

[0042] In some embodiments, the multispecific protein is internalized into blood-brain barrier epithelial cells at a greater than 10-fold or greater than 40-fold increase compared to internalization by an isotype control, and optionally the blood-brain barrier epithelial cells are HCMEC / D3 cells. In some embodiments, the multispecific protein does not reduce cell surface expression of TfR on HCMEC / D3 cells by more than 60% compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. In some embodiments, the multispecific protein does not reduce cell surface expression of TfR on HCMEC / D3 cells by more than 40% compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. In some embodiments, the multispecific protein does not significantly increase cell surface expression of TfR on HCMEC / D3 cells compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control.

[0043] In some embodiments, the multispecific protein accumulates in vascular-depleted mouse brains at least 4-fold or at least 5-fold more than an isotype control. In some embodiments, the second antigen-binding domain specifically binds to human TfR at least 5-fold more than it binds to an unrelated protein. In some embodiments, the second antigen-binding domain specifically binds to cynomolgus monkey TfR at least 5-fold more than it binds to an unrelated protein. In some embodiments, the second antigen-binding domain specifically binds to human TfR at least 5-fold more than it binds to an unrelated protein and / or specifically binds to cynomolgus monkey TfR at least 5-fold more than it binds to an unrelated protein.

[0044] In some embodiments, the CNS antigen is beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, beta-glucocerebromide (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), trigger expressed in myeloid cells receptor 2 (TREM2), sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), sialic acid-binding Ig-like lectin 11 (Siglec11), glycoprotein non-metastatic melanoma protein B (GPNMB), paired immunoglobulin-like type 2 Receptor alpha (PILRA), transmembrane 4-domain A4A (MS4A4A), transmembrane 4-domain A6A (MS4A6A), MS4A4E, transmembrane protein 106B (TMEM106b), ubiquitin protein ligase E3A (UBE3A), CR1, ABCA1, ABCA7, HLA-DR1, HLA-DR5, IL1RAP, TREML2, IL-34, SORL1, or ADAM1. In some embodiments, the CNS antigen is MS4A4A, and optionally, (i) the antigen binding domain, antibody, or antigen binding domain that binds MS4A4A comprises a VH comprising the MS4A4A-binding VH sequence of SEQ ID NO: 406 and / or a VL comprising the VL sequence of SEQ ID NO: 407, and / or (ii) the antigen binding domain that binds human TfR comprises the scFv sequence of SEQ ID NO: 406.

[0045] In some embodiments, the multispecific protein comprises the amino acid sequence of SEQ ID NOs: 405-407.

[0046] In some embodiments, the multispecific protein is capable of binding to FcRn.

[0047] In some aspects, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are linked to an imaging agent.

[0048] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are capable of crossing the BBB.

[0049] In some aspects, provided herein are compositions comprising a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first polynucleotide, the second polynucleotide, and the third polynucleotide encode a multispecific protein provided herein, wherein the first polynucleotide encodes a first heavy chain, the second polynucleotide encodes a second heavy chain and an antigen-binding domain that specifically binds to human TfR, and the third polynucleotide encodes a light chain.

[0050] In some aspects, provided herein are compositions comprising a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first polynucleotide, the second polynucleotide, and the third polynucleotide encode a multispecific protein provided herein, wherein the first polynucleotide encodes a first heavy chain and a first antigen-binding domain that specifically binds to human TfR, the second polynucleotide encodes a second heavy chain and a second antigen-binding domain that specifically binds to human TfR, and the third polynucleotide encodes a light chain, and optionally, the first antigen-binding domain that binds to human TfR and the second antigen-binding domain comprise the same amino acid sequence.

[0051] In some embodiments, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation.

[0052] In some embodiments, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation, hi some embodiments, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0053] In some embodiments, the ratio of the first polynucleotide, the second polynucleotide, and the third polynucleotide is about 1:3:6.

[0054] In some embodiments, provided herein are compositions comprising a first polynucleotide and a second polynucleotide, wherein the first and second polynucleotides encode a multispecific protein provided herein, wherein the first polynucleotide encodes a heavy chain and an antigen-binding domain that binds to human TfR, and the second polynucleotide encodes a light chain.

[0055] In some aspects, provided herein are host cells comprising the compositions provided herein.

[0056] In some aspects, provided herein is an isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain of an antigen-binding domain provided herein. In some aspects, provided herein is an isolated polynucleotide comprising a nucleic acid molecule encoding the light chain variable region of an antigen-binding domain provided herein.

[0057] In some aspects, provided herein is an isolated vector comprising a polynucleotide provided herein.

[0058] In some aspects, provided herein is an isolated vector comprising a nucleic acid molecule encoding a heavy chain variable region of an antigen-binding domain provided herein and a nucleic acid molecule encoding a light chain variable region of the antigen-binding domain.

[0059] In some aspects, provided herein is a host cell comprising a polynucleotide provided herein or a vector provided herein. In some aspects, the host cell is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS1, COS7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture.

[0060] In some aspects, provided herein are methods of producing an antigen-binding domain or a multispecific protein, comprising culturing a host cell provided herein such that the antigen-binding domain or multispecific protein is produced, and optionally further comprising isolating the antigen-binding domain or multispecific protein from the culture.

[0061] In some aspects, provided herein is an isolated antigen-binding domain or multispecific protein thereof produced by the methods provided herein.

[0062] In some aspects, provided herein are pharmaceutical compositions comprising (i) an antigen-binding domain, fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein, and (ii) a pharmaceutically acceptable carrier. In some aspects, the concentration of the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein is increased in the brain after administration to a subject compared to an isotype control. In some aspects, administration increases delivery of the fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition into the brain by at least 50%, at least 100%, at least 200%, at least 500%, or at least 1000% compared to an isotype control.

[0063] In some aspects, provided herein are methods of treating a neurological disease or disorder in a subject, comprising administering to the subject a fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition provided herein. In some aspects, administration increases delivery of the fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition into the brain by at least 50%, at least 100%, at least 200%, at least 500%, or at least 1000% compared to an isotype control. In some aspects, administration increases delivery of the fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition to the frontal cortex, entorhinal cortex, and / or hippocampus.

[0064] In some aspects, the neurological disease or disorder is selected from neuropathy disorders, neurodegenerative diseases, cancer, ophthalmic disorders, seizure disorders, lysosomal storage disorders, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, and CNS inflammation. In some aspects, the neurological disease or disorder is selected from Alzheimer's disease (AD), Huntington's disease, dystonia, ataxia, Bell's palsy, stroke, dementia, dementia with Lewy bodies, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, encephalitis, traumatic brain injury, and limbic-predominant age-related TDP-43 encephalopathy (LATE). In some aspects, the dementia is frontotemporal dementia (FTD). In some embodiments, the neurological disease or disorder is Alzheimer's disease. In some aspects, the Alzheimer's disease is early-onset Alzheimer's disease, prodromal Alzheimer's disease, mild Alzheimer's disease, or late-onset Alzheimer's disease. In some embodiments, the neurological disease or disorder is Parkinson's disease. In some aspects, the neurological disease or disorder is frontotemporal epilepsy. In some aspects, the neurological disease or disorder is autism. In some aspects, the neurological disease or disorder is lissencephaly.

[0065] In some aspects, provided herein are methods of treating a lysosomal storage disease in a subject, comprising administering to the subject a fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition provided herein. In some aspects, the lysosomal storage disease is selected from Gaucher disease, ceroid lipofuscinosis (Batten disease), mucopolysaccharidosis (MPS) type I, MPS type II, and MPS type III.

[0066] In some aspects, provided herein are methods of transporting a fusion protein, an antibody or antigen-binding fragment thereof, or a multispecific protein across the BBB in a subject, the method comprising administering to the subject a fusion protein, an antibody or antigen-binding fragment thereof, a multispecific protein, or a pharmaceutical composition provided herein.

[0067] In some embodiments, the concentration of the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein is increased in the brain after administration compared to an isotype control, hi some embodiments, the concentration of the fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition in the brain is increased by at least 50%, at least 100%, at least 200%, at least 500%, or at least 1000% compared to an isotype control.

[0068] In some embodiments, administration of the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein does not result in more than a 10% decrease in reticulocyte count in the subject compared to administration of an isotype control. In some embodiments, administration of the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein does not result in a decrease in reticulocyte count in the subject compared to an isotype control.

[0069] In some aspects, provided herein are methods of increasing the concentration of a CNS-binding antigen in the CSF of a subject, comprising administering to the subject a multispecific protein provided herein, wherein the concentration of the CNS-binding antigen is increased compared to administering the CNS-binding antigen alone to the subject.

[0070] In some aspects, provided herein are methods of imaging a CNS antigen in a subject, the method comprising administering to the subject a fusion protein, an antibody or antigen-binding fragment thereof, or a multispecific protein provided herein, and locating an imaging agent within the subject.

[0071] In some aspects, provided herein are methods of detecting a CNS antigen in vitro, comprising contacting an in vitro sample with a fusion protein, an antibody or antigen-binding fragment thereof, or a multispecific protein provided herein, and locating an imaging agent in the sample.

[0072] In some aspects, provided herein is the use of a fusion protein, an antibody or antigen-binding fragment thereof, a multispecific protein, or a pharmaceutical composition provided herein in a method provided herein.

[0073] In some aspects, provided herein are fusion proteins, antibodies or antigen-binding fragments thereof, multispecific proteins, or pharmaceutical compositions for use in the methods provided herein.

[0074] It will be understood that one, some, or all of the features of the various aspects described herein may be combined to form other aspects of the present disclosure. These and other aspects of the present disclosure will be readily apparent to those skilled in the art. These and other embodiments of the present disclosure are further described in the detailed description that follows. [Brief explanation of the drawings]

[0075] [Figure 1A] An example of a 2+1 bispecific antibody is shown. [Figure 1B] An example of a 2+2 bispecific antibody is shown. [Figure 1C] An example of a 2+2 bispecific antibody with two different scFvs is shown. [Figure 1D] Exemplary formats are shown, including a TfR antigen-binding domain. (i) shows an exemplary 2+1 bispecific antibody format. (ii) shows an exemplary 1+1 antibody format. (iii) shows another exemplary 1+1 antibody format with two different VHH domains. [Figure 1E]Figures 1A-1C show exemplary formats of fusion proteins comprising a TfR antigen-binding domain and a heterologous protein or polypeptide. The circular "Pac-Man" shape represents the heterologous protein or polypeptide. (i), (ii), (iii), and (iv) show exemplary 1+1 fusion protein formats. (v) and (vi) show exemplary 2+1 fusion protein formats. (vii) shows an exemplary 2+2 fusion protein format. [Figure 2] 1 shows TfR surface levels on hCMEC / D3 cells after treatment with 2+1 anti-TfR bispecific antibodies as assessed by FACS (see Example 13). [Figure 3] 1 shows TfR surface levels on hCMEC / D3 cells after treatment with a humanized 2+1 anti-TfR bispecific antibody, as assessed by FACS (see Example 13). [Figure 4] 1 shows total TfR protein levels in hCMEC / D3 cells after treatment with 2+1 anti-TfR bispecific antibodies as assessed by Western blot (see Example 13). [Figure 5] Brain PK of 2+1 anti-TfR bispecific antibodies is shown as fold change relative to isotype control (see Example 17). [Figure 6] 1 shows serum PK of a 2+1 anti-TfR bispecific antibody in wild-type and hTfR ECD+ / - knock-in mice (see Example 19). [Figure 7] Whole vessel TfR levels after injection of hu-TfR+ / -KI mice with anti-TfR antibodies (i.e., TfR-9.1B.39 and TfR-15.WH8) are shown compared to values ​​obtained in isotype-treated animals (see Example 20). [Figure 8] 1 shows blood reticulocyte levels after 2+1 anti-TfR bispecific antibody treatment (see Example 21). [Figure 9] 1 shows the moderate antibody-dependent cellular cytotoxicity (ADCC) response of a 2+1 anti-TfR bispecific antibody against BBB cell lines (see Example 22). [Figure 10]We show that NSLF and LALAPS strongly improve the ADCC signal from a 2+1 anti-TfR bispecific antibody (see Example 22). [Figure 11] 2 shows 2+1 anti-TfR bispecific antibody activity as measured by sTREM2 levels in an in vitro assay (see Example 24). [Figure 12] 1 shows absolute reticulocyte counts in non-human primates (NHPs) after administration of a 2+1 anti-TfR bispecific antibody (see Example 26). [Figure 13] Serum and CSF levels of 2+1 anti-TfR bispecific antibody after the first and second doses are shown (see Example 27). [Figure 14] 1 shows antibody concentrations in NHP brain fractions after administration of a 2+1 anti-TfR bispecific antibody (see Example 28). [Figure 15] 1 shows levels of soluble TREM2 (sTREM2) in serum and CSF of NHPs following administration of a 2+1 anti-TfR bispecific antibody (see Example 29). [Figure 16] 1 shows the levels of CSF-1 in the CSF of NHPs after administration of a 2+1 anti-TfR bispecific antibody (see Example 29). [Figure 17] Figure 1 shows relative TfR protein levels (measured by sandwich Meso Scale Discovery method) in cynomolgus monkey brain sections 48 hours after the second dose of 2+1 anti-TfR antibody. TfR protein concentrations in each animal were normalized and presented as a percentage of the mean TfR level (hIgG1 isotype) in control-treated animals (see Example 30). DETAILED DESCRIPTION OF THE INVENTION

[0076] Detailed Description of the Disclosure The present disclosure relates to antigen-binding domains that specifically bind to the human transferrin receptor (TfR), antibodies and antigen-binding fragments thereof comprising such antigen-binding domains, methods for making and using such antigen-binding domains, antibodies, and antigen-binding fragments thereof, pharmaceutical compositions comprising such antigen-binding domains, antibodies, and antigen-binding fragments thereof, nucleic acids encoding such antigen-binding domains, antibodies, and antigen-binding fragments thereof, and host cells comprising nucleic acids encoding such antigen-binding domains, antibodies, and antigen-binding fragments thereof.

[0077] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are commonly employed by those skilled in the art using conventional methods, for example, widely used methods such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M.A.usubel, et al. eds., (2003); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000).

[0078] definition The terms "central nervous system" and "CNS" refer to the complex of nervous tissue that controls bodily functions and includes the brain and spinal cord.

[0079] The terms "blood-brain barrier" and "BBB" refer to the network of brain capillary endothelial cells that are tightly sealed by tight junctions.

[0080] A "central nervous system antigen" or "CNS antigen" is an antigen expressed in the CNS, including the brain, that can be targeted by an antibody or small molecule. Examples of such antigens include, but are not limited to, beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebroidase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interleukin 6 receptor (IL6), and the like. R), TNF receptor 1 (TNFR1), interleukin 1β (IL1(3)), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), glycoprotein nonmetastatic melanoma protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4-domain A4A (MS4A4A), transmembrane 4-domain A6A (MS4A6A), ubiquitin protein ligase E3A (UBE3A), or transmembrane protein 106B (TMEM106b).

[0081] A "brain antigen" is a CNS antigen that is expressed in the brain.

[0082] "Transferrin receptor," "TfR," "TfR polypeptide," and "TfR protein" are used interchangeably herein and, unless otherwise specified, refer to any native TfR from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (cyno)) and rodents (e.g., mice and rats). TfR is also referred to as transferrin receptor protein 1, TR, tfR1, Trfr, T9, and p90. In some embodiments, the term encompasses both wild-type sequences and naturally occurring variant sequences, e.g., splice variants or allelic variants. In some embodiments, the term encompasses "full-length," unprocessed TfR, as well as any form of TfR that results from processing within a cell. In some embodiments, the TfR is a human TfR. As used herein, the term "human TfR" refers to a polypeptide having the amino acid sequence of SEQ ID NO: 8. (SEQ ID NO: 8)

[0083] The term "antibody" refers to an immunoglobulin molecule that recognizes and binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, via at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term "antibody" encompasses monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific (e.g., bispecific) antibodies, and any other immunoglobulin molecule so long as it exhibits the desired biological activity. Antibodies can be of any of five major immunoglobulin classes—IgA, IgD, IgE, IgG, and IgM—or their subclasses (isotypes) (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2)—based on the type of heavy chain constant region, designated alpha, delta, epsilon, gamma, and mu, respectively. Different classes of antibodies have distinct and well-known subunit structures and three-dimensional structures. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0084] The terms "anti-TfR antibody," "antibody that binds to TfR," and "antibody that specifically binds to TfR" refer to an antibody that can bind to TfR with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting TfR. In one embodiment, the extent of binding of an anti-TfR antibody to an unrelated, non-TfR polypeptide is less than about 10% of the binding of the antibody to TfR, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to TfR has an affinity of <20 μM, <15 μM, <12 μM, <10 μM, <7.5 μM, <5 μM, <2.5 μM, <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10-13 M, e.g., 10 -9 M~10 -13 M) In certain embodiments, the anti-TfR antibody binds to an epitope of TfR that is conserved among TfRs from different species.

[0085] The term "antibody fragment" refers to a portion of an antibody. An "antigen-binding fragment" refers to a portion of an antibody that binds to an antigen. An antigen-binding fragment of an antibody may include an antigenic determining region of the antibody (e.g., a complementarity-determining region (CDR)). Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. An antigen-binding fragment of an antibody may be monovalent or multivalent (e.g., bivalent). An antigen-binding fragment of an antibody may be monospecific or multispecific (e.g., bispecific). An antigen-binding fragment of an antibody may be derived from any animal species, such as, for example, rodents (e.g., mice, rats, or hamsters) and humans, or may be artificially generated.

[0086] An "antigen-binding fragment" or "antigen-binding region" refers to a monovalent portion of an antibody that binds to an antigen. An "antigen-binding domain" may comprise an antigenic determining region of an antibody (e.g., a complementarity-determining region (CDR)). An antibody or antigen-binding fragment thereof (including monospecific and multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof) can comprise an antigen-binding domain. In some embodiments, the antigen-binding domain is not present in the context of an antibody.

[0087] The terms "anti-TfR antigen-binding domain," "antigen-binding domain that binds to TfR," "anti-TfR antigen-binding region," "antigen-binding region that binds to TfR," and "TfR binding domain" refer to an antigen-binding domain that binds to TfR with sufficient affinity such that the antigen-binding domain is useful as a diagnostic and / or therapeutic agent in targeting TfR. In one embodiment, the extent of binding of an anti-TfR antigen-binding domain to an unrelated, non-TfR polypeptide is less than about 10% of the binding of the antigen-binding domain to TfR, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to TfR has an affinity of <20 μM, <15 μM, <12 μM, <10 μM, <7.5 μM, <5 μM, <2.5 μM, <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 M) In certain embodiments, the anti-TfR antigen binding domain binds to an epitope of TfR that is conserved among TfRs from different species.

[0088] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, whole antibodies include those having heavy and light chains, including the Fc region. The constant regions may be native-sequence constant regions (e.g., human native-sequence constant regions) or amino acid sequence variants thereof. In some cases, intact antibodies may have one or more effector functions.

[0089] "Native IgG antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains at one end a variable domain (V H ), followed by several constant domains. Each light chain has at one end a variable domain (V L ) and a constant domain at the other end, with the light-chain constant domain aligned with the first constant domain of the heavy chain, and the light-chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain and heavy-chain variable domains.

[0090] Papain digestion of antibodies yields two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, a designation reflecting its tendency to crystallize easily. The Fab fragment contains the entire light chain plus the variable region domain (V) of one heavy chain. H ) and the first constant domain of the heavy chain (C H 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking antigen. The Fab' fragment contains one or more cysteines from the antibody hinge region, C H F(ab')2 antibody fragments differ from Fab fragments by having several additional residues at the carboxy terminus of one domain. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) in the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical bonds for antibody fragments are also known.

[0091] The Fc fragment contains the carboxy-terminal portions of both heavy chains linked by disulfides. The effector functions of the antibody are determined by sequences within the Fc region, which is also recognized by Fc receptors (FcRs) found on certain types of cells.

[0092] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain and one light-chain variable domain in tight, non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the H and L chains) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for a given antigen) has the ability to recognize and bind to antigen, although with lower affinity than that of the entire binding site.

[0093] A "single-chain Fv," sometimes abbreviated as "sFv" or "scFv," is an antibody fragment comprising a VH and a VL antibody domain linked as a single polypeptide chain. In some embodiments, the scFv polypeptide comprises a VH and a VL antibody domain linked as a single polypeptide chain. H Domains and V L It further comprises a polypeptide linker between the domains which enables the scFv to form the desired structure for antigen binding.

[0094] The term "diabody" refers to a V-type antibody that binds to a V-type IgG antibody, such that inter-chain but not intra-chain variable domain pairing results in bivalent fragments, i.e., fragments with two antigen-binding sites. H Domains and V L These refer to small antibody fragments created by constructing scFv fragments (see previous paragraph) with short linkers (approximately 5-10 residues) between the domains. Bispecific diabodies are small antibody fragments created by constructing scFv fragments (see previous paragraph) with short linkers (approximately 5-10 residues) between the domains. H Domain and V L It is a heterodimer of two "crossover" scFv fragments in which the domains are present on different polypeptide chains.

[0095] As used herein, a "2+1 antibody format" of a TfR antibody refers to a trivalent, bispecific antibody format comprising (i) a single antigen-binding domain that binds to human TfR, and (ii) an antibody comprising two heavy chains and two light chains, wherein the single antigen-binding domain that binds to human TfR is linked to the C-terminus of one of the two antibody heavy chains. This format is exemplified in Figure 1A and Figure ID (i).

[0096] As used herein, a "2+2 antibody format" of a TfR antibody refers to a tetravalent, bispecific antibody format comprising (i) two antigen-binding domains that bind to human TfR and (ii) an antibody, wherein the antibody comprises two heavy chains and two light chains, wherein one of the antigen-binding domains that bind to human TfR is linked to the C-terminus of one of the two antibody heavy chains, and the other antigen-binding domain that binds to human TfR is linked to the C-terminus of the other of the two antibody heavy chains. The two antigen-binding domains that bind to human TfR may comprise the same amino acid sequence. The first and second antigen-binding domains that bind to human TfR may comprise different amino acid sequences. This format is illustrated in Figures 1B and 1C.

[0097] As used herein, a "1+1 antibody format" of a TfR antibody refers to a bivalent, bispecific antibody format that includes (i) one antigen-binding domain that binds to human TfR and (ii) an antigen-binding domain (optionally including an Fc domain) that binds to an antigen other than human TfR. This format is illustrated in Figure ID. An exemplary 1+1 fusion protein format is illustrated in Figure IE.

[0098] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a light or heavy chain, typically consisting of approximately 110-120 or 110-125 amino acids at the amino terminus of a mature heavy chain and approximately 90-115 amino acids in a mature light chain, which differs significantly in sequence among antibodies and is responsible for the binding and specificity of a particular antibody to a particular antigen. While sequence variability is concentrated in regions called complementarity-determining regions (CDRs), the more highly conserved regions of the variable domain are called framework regions (FRs). While not wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of an antibody with an antigen. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In some embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs). "Kabat numbering" and like terms are art-recognized and refer to a system for numbering amino acid residues within the heavy and light chain variable regions of an antibody or antigen-binding fragment thereof. In certain embodiments, CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).Using the Kabat numbering system, the CDRs in an antibody heavy chain molecule are typically located at amino acids 31-35 (optionally including one or two additional amino acids after 35, designated 35A and 35B in the Kabat numbering scheme) (CDRH1), 50-65 (CDRH2), and 95-102 (CDRH3). Using the Kabat numbering system, the CDRs in an antibody light chain molecule are typically located at amino acids 24-34 (CDRL1), 50-56 (CDRL2), and 89-97 (CDRL3). In some embodiments, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme. Chothia instead refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDRH1 loop, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the loop (this is due to insertions at H35A and H35B in the Kabat numbering scheme: if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. In some embodiments, the CDRs may also be "Contact" CDRs. The "Contact" CDRs are based on analysis of available complex crystal structures. Residues from each of these CDRs are listed below. TIFF2025528752000001.tif89165

[0099] Each CDR may comprise the following "extended CDRs": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) for VL, and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) for VH. The variable domain residues are numbered according to Kabat et al. (supra) for each of these extended CDR definitions.

[0100] The terms "VH" and "VH domain" are used interchangeably and refer to the heavy chain variable region of an antibody.

[0101] As used herein, the term "heavy chain," when used in reference to an antibody, can refer to any of the distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant region that gives rise to the IgA, IgD, IgE, IgG, and IgM antibody classes (including IgG subclasses, e.g., IgG1, IgG2, IgG3, and IgG4), respectively. Heavy chain amino acid sequences are well known in the art. In some embodiments, the heavy chain is a human heavy chain.

[0102] The terms "VL" and "VL domain" are used interchangeably and refer to the light chain variable region of an antibody.

[0103] As used herein, the term "light chain," when used in reference to an antibody, can refer to either distinct type based on the amino acid sequence of the constant region, e.g., kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In some embodiments, the heavy chain is a human light chain.

[0104] As used herein, the term "constant region" refers to a region of an antibody that is not the variable region of the antibody, e.g., the carboxyl-terminal portions of the light and / or heavy chains that are not directly involved in binding the antibody to an antigen but can exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a highly conserved amino acid sequence compared to the immunoglobulin variable domain. In certain embodiments, the antibody or antigen-binding fragment comprises a constant region or portion thereof sufficient for antibody-dependent cellular cytotoxicity (ADCC).

[0105] By "constant domain" is meant a domain within the constant region that is capable of forming an immunoglobulin fold. Constant domains include CH1, CH2, CH3, and CL domains.

[0106] The term "monoclonal," when referring to antibodies or antigen-binding fragments thereof, refers to a population of homogeneous antibodies or antigen-binding fragments involved in highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies directed against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof encompasses not only intact, full-length monoclonal antibodies, but also antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) variants, fusion proteins containing antibodies or antibody portions, and any other immunoglobulin molecule containing an antigen-recognition site. Furthermore, "monoclonal" antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof produced by any method, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0107] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequences are derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of an antibody or antigen-binding fragment thereof from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and function, while the constant regions are homologous to the sequences of an antibody or antigen-binding fragment thereof from another species (usually human) to avoid eliciting an immune response in that species.

[0108] The term "humanized" antibody or antigen-binding fragment thereof refers to a non-human (e.g., murine) antibody or antigen-binding fragment that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof that contains minimal non-human (e.g., murine) sequence. Typically, humanized antibodies or antigen-binding fragments thereof are human immunoglobulins in which residues from their complementarity-determining regions (CDRs) have been replaced ("CDR-grafted") by residues from a CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and function (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). Humanized antibodies or antigen-binding fragments thereof can be further modified by substitution of additional residues within the Fv framework regions and / or the replaced non-human residues to improve and optimize the specificity, affinity, and / or function of the antibody or antigen-binding fragment. Generally, a humanized antibody or antigen-binding fragment thereof comprises a VH and VL region that contains substantially all of at least one, and usually two or three, of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are those of a human immunoglobulin consensus region. A humanized antibody or antigen-binding fragment thereof can also comprise at least a portion of an immunoglobulin constant region or Fc region, typically that of a human immunoglobulin. Examples of methods used to make humanized antibodies are described in U.S. Patent No. 5,225,539, Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904(1996). In some embodiments, the antibody is a resurfaced antibody.

[0109] The term "human" antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin locus, and such antibodies or antigen-binding fragments are made using any technique known in the art. This definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0110] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as herein defined.

[0111] As used herein, an "acceptor human framework" refers to a V-type nucleotide sequence derived from a human immunoglobulin framework or a human consensus framework. L Framework or V H A framework comprising the amino acid sequence of a framework. An acceptor human framework "derived" from a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. If pre-existing amino acid changes are present in the VH, in some embodiments, those changes occur at only three, two, or one of positions 71H, 73H, and 78H, e.g., the amino acid residues at those positions may be 71A, 73T, and / or 78A. In some embodiments, a VL acceptor human framework is derived from a VL. L The sequence is identical to a human immunoglobulin framework sequence or a human consensus framework sequence.

[0112] The "human consensus framework" is based on human immunoglobulin V L or V H A framework that represents the most commonly occurring amino acid residues in a selection of framework sequences. Generally, human immunoglobulin V L or V HThe selection of sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). For example, V L For example, the subgroup may be subgroup kappa I, kappa II, kappa III, or kappa IV, such as those of Kabat et al., supra. H For example, the subgroup may be subgroup I, subgroup II, or subgroup III, such as in Kabat et al., supra.

[0113] An "amino acid modification" at a specified position (e.g., of an antibody of the present disclosure) refers to a substitution or deletion of the specified residue, or an insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent to" a particular residue means an insertion within 1-2 residues thereof. The insertion can be N-terminal or C-terminal to the particular residue. In some embodiments, the amino acid modification is a substitution.

[0114] Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary depending on the antibody isotype.

[0115] The terms "Fc region" or "fragment crystallizable region" are used herein to define the C-terminal region of an immunoglobulin heavy chain and include native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from Pro230, to the carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, an intact antibody composition can include an antibody population in which all K447 residues have been removed, an antibody population in which the K447 residue has not been removed, and an antibody population having a mixture of antibodies with and without the K447 residue. Native-sequence Fc regions suitable for use in the antibodies of the present disclosure include human IgG1, IgG2, IgG3, and IgG4.

[0116] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes), native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0117] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, and in some embodiments, one or more amino acid substitution(s). In some embodiments, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about 1 to about 10 amino acid substitutions in the native-sequence Fc region or in the Fc region of the parent polypeptide, and in some embodiments, about 1 to about 5 amino acid substitutions. In some embodiments, the variant Fc region has at least 80% homology, at least 90% homology, or at least 95% homology to the native-sequence Fc region and / or the Fc region of the parent polypeptide.

[0118] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native-sequence human FcR. In some embodiments, the FcR binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses (including allelic variants and alternatively spliced ​​forms of these receptors). FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences but differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif ("ITAM") in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. FcRs can also extend the serum half-life of antibodies.

[0119] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or its antigen-binding fragment) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity," as used herein, refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or its antigen-binding fragment and an antigen). The affinity of a molecule X for its partner Y is generally determined by the equilibrium dissociation constant (K D Affinity can be expressed by, but not limited to, the equilibrium dissociation constant (K D ) and the equilibrium binding constant (K A ) can be measured and / or expressed in many ways well known in the art. D is k off / k on It is calculated from the quotient of K A is k on / k off It is calculated from the quotient of k on refers to, for example, the binding rate constant of an antibody or its antigen-binding fragment with an antigen, and k off k refers to, for example, the dissociation rate constant of an antibody or antigen-binding fragment thereof from an antigen. on and k off can be determined by techniques well known to those skilled in the art, such as BIAcore® or KinExA.

[0120] With respect to the binding of an antibody to a target molecule, the terms "specific binding" or "specifically binds to" or "is specific for" a particular polypeptide or epitope on a particular polypeptide target refer to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target, e.g., an excess of unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by the excess of unlabeled target. The terms "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular polypeptide target, as used herein, refer to, for example, binding that has a KD for the target of about 10 -4 M or less, 10 -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less or KD is 10 -4 M~10 -6 M or 10 -6 M~10 -10 M or 10 -7 M~10 -9 M. As will be appreciated by those skilled in the art, affinity and KD value are inversely proportional. High affinity for an antigen is measured by a low KD value. In some embodiments, the term "specific binding" refers to the binding of a molecule to a specific polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0121] The term "linker" or "linked" refers to a covalent bond between two polypeptides or two heterologous molecules. In some embodiments, the linker is a chemical linker. In some embodiments, the linker comprises a peptide bond, and the two polypeptides or two heterologous molecules are linked to each other directly or via one or more additional amino acids. A glycine linker is one that contains one or more glycines but no other amino acids, e.g., GGGG (SEQ ID NO: 285). A glycine-rich linker is one that contains one or more glycines and can contain other amino acids as long as glycine is the predominant species of the linker, e.g., GGGNGG (where N is any amino acid) (SEQ ID NO: 286). A glycine-serine linker is a linker that contains both glycine and serine in any proportion, e.g., GGGS (SEQ ID NO: 287). Similarly, a proline linker is one that contains one or more prolines but no other amino acids. A proline-rich linker is one that contains one or more prolines and can contain other amino acids as long as proline is the predominant species of the linker.

[0122] As used herein, "percent amino acid sequence identity" and "homology," with respect to peptide, polypeptide, or antibody sequences, are defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a particular peptide or polypeptide sequence after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of those in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art that are needed to achieve maximal alignment over the full length of the sequences being compared.

[0123] The term "epitope" includes any determinant capable of binding by an antibody. An epitope is the region of an antigen bound by an antibody targeting that antigen, and, if the antigen is a polypeptide, includes specific amino acids that make direct contact with the antibody. In most cases, epitopes reside on polypeptides, but in some cases, they may reside on other types of molecules, such as nucleic acids. Epitopes may have chemically active molecular surface groups, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen selectively recognize epitopes on the target antigen in a complex mixture of polypeptides and / or macromolecules.

[0124] An antibody that "binds to the same epitope" as a reference antibody refers to an antibody that contacts the same amino acid residues on the antigen as the reference antibody. The ability of an antibody to bind to the same epitope as a reference antibody can be determined using peptide scanning mutagenesis or high-throughput alanine scanning mutagenesis. In the latter method, a comprehensive mutation library of an antigen or a portion thereof (e.g., the extracellular domain) can be generated by mutating each individual amino acid residue to alanine (or, if the amino acid residue is alanine, to another residue such as serine) and testing each mutant for binding to the target antibody or its antigen-binding fragment.

[0125] An antibody is said to "competitively inhibit" the binding of a reference antibody to a particular epitope if the antibody selectively binds to that epitope or an overlapping epitope, thereby blocking the binding of the reference antibody to that epitope to some extent. Competitive inhibition can be measured by any method known in the art, such as, for example, a competitive ELISA assay. An antibody can be said to competitively inhibit the binding of the reference antibody to a particular epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0126] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are not in a form in which they are found in nature. In some aspects, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0127] As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0128] The term "expression system" refers to one or more nucleic acid molecules comprising operably linked coding and control sequence(s), and a host cell and / or other in vitro transcription and translation machinery, such that one or more proteins encoded by the nucleic acid molecule(s) are produced.

[0129] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, into which additional DNA segments can be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" or simply, "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Since the plasmid is the most commonly used form of vector, the terms "plasmid" and "vector" may be used interchangeably herein.

[0130] "Polynucleotide" or "nucleic acid," used interchangeably herein, refer to a polymer of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or a synthetic reaction.

[0131] A "host cell" includes an individual cell or cell culture that can be or has been the recipient of a vector(s) for incorporating a polynucleotide insert. A host cell includes the progeny of a single host cell, although the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of the invention.

[0132] As used herein, a "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed at the dosages and concentrations employed.

[0133] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of a treated individual's clinical pathology. Desirable effects of treatment include a reduction in the rate of progression, an improvement or alleviation of the pathology, and an improvement in the remission or prognosis of a particular disease, disorder, or condition. For example, an individual is effectively "treated" if one or more symptoms associated with a particular disease, disorder, or condition are reduced or eliminated.

[0134] As used herein, the terms "administer," "administering," "administration," and the like refer to methods that can be used to deliver a drug, e.g., an anti-human antibody or antigen-binding fragment thereof, to a desired site of biological action.

[0135] The term "effective amount" refers to an amount effective, at least at the dosage and for the duration necessary, to achieve a desired therapeutic result. An effective amount may be provided in one or more administrations. An effective amount is also an amount in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. In therapeutic applications, beneficial or desired results include clinical results, such as a reduction in one or more symptoms attributable to the disease, an improvement in the quality of life of the person suffering from the disease, a reduction in the dosage of other drugs required to treat the disease, augmentation of other drug therapies, e.g., by targeting, a delay in disease progression, and / or an extension of survival. An effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve a therapeutic treatment directly or indirectly. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of the administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result can be or is achieved.

[0136] As used herein, the terms "subject" and "patient" are used interchangeably. A subject can be a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey, or other primate, etc.). In some embodiments, the subject is a cynomolgus monkey. In some embodiments, the subject is a human.

[0137] As used herein, administration "in conjunction with" or "in combination with" another compound or composition includes simultaneous administration and / or administration at different times. Co-administration also encompasses administration as a combined drug or as separate compositions, including at different dosing frequencies or intervals, and using the same or different routes of administration. In some embodiments, co-administration is administration as part of the same treatment regimen.

[0138] As used herein, "neurological disease" refers to a disease or disorder that affects and / or has an etiology in the CNS. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, eye disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, stroke, behavioral disorder, and lysosomal storage disease.

[0139] As used herein, "lysosomal storage disease" or (LSD) refers to an inherited metabolic disorder characterized by the excessive accumulation of undigested or partially digested substrates, such as macromolecules, in various cells of organs, ultimately resulting in cellular dysfunction and clinical abnormalities. LSDs are defined as defects in lysosomal function, generally classified by the accumulated substrate, and include sphingolipidoses, oligosaccharidoses, mucolipidoses, mucopolysaccharidoses, lipoprotein storage disorders, and neuronal ceroid lipofuscinoses. LSDs can also include other deficiencies or defects in proteins that result in the accumulation of macromolecules, such as proteins required for normal post-translational modification of lysosomal enzymes or proteins important for proper lysosomal trafficking. LSDs are diseases caused by defects in a single gene. Enzyme defects cause approximately 70 percent of LSDs, with the remainder being defects in enzyme activators or related proteins.

[0140] "Protein replacement therapy" or "PRT" refers to a medical procedure that supplements or replaces a specific protein in a patient in whom that protein is deficient or absent.

[0141] "Enzyme replacement therapy enzyme" or "ERT enzyme" refers to an enzyme deficient in a lysosomal storage disease. "ERT enzyme variant" refers to a functional variant, including allelic and splice variants, of a wild-type ERT enzyme or a fragment thereof, which, for example, has 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%, or at least 95% of the activity of the corresponding wild-type ERT enzyme or fragment thereof when assayed under identical conditions. A "catalytically active fragment" of an ERT enzyme refers to a portion of a full-length ERT enzyme or a variant thereof, which, for example, has 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%, or at least 95% of the activity of the corresponding full-length ERT enzyme or variant thereof when assayed under identical conditions.

[0142] As used herein, the terms "about" and "approximately," when used to modify a numerical value or numerical range, indicate that deviations of up to 10% above and below that value or range remain within the intended meaning of the stated value or range. When embodiments are described herein in terms of "about" or "approximately" (a numerical value or range), it is understood that otherwise similar embodiments that refer to that particular numerical value or range are also provided.

[0143] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "an antibody" refers to one to many antibodies, such as molar amounts, and equivalents thereof known to those skilled in the art.

[0144] Whenever an embodiment is described herein using the term "comprising," it should be understood that other similar embodiments are also provided that are described using the terms "consisting of" and / or "consisting essentially of." In this disclosure, the terms "comprises," "comprising," "containing," and "having" and similar terms can mean "including" and similar terms. "Consisting of" or "consisting essentially of" is open-ended, allowing for the presence of more than what is recited, but excluding prior art embodiments, so long as the basic or novel characteristics of what is recited are not altered by the presence of more than what is recited.

[0145] Anti-TfR antigen-binding domain Provided herein are antigen-binding domains that specifically bind to human TfR.

[0146] Such antigen-binding domains can cross the blood-brain barrier (BBB) ​​and transport other agents (e.g., therapeutically active agents) bound to the antigen-binding domain across the BBB. Thus, in some embodiments, provided herein are antigen-binding domains that specifically bind to human TfR and that are capable of being internalized by epithelial cells of the BBB, such as HCMEC / D3 cells.

[0147] In some embodiments, an antigen binding domain that specifically binds to human TfR comprises six CDRs of an antibody listed in Table 12 and Table 13 (i.e., three VH CDRs of an antibody listed in Table 12 and three VL CDRs of the same antibody listed in Table 13).

[0148] In some embodiments, an antigen-binding domain that specifically binds to human TfR comprises the six CDRs of an antibody listed in Tables 12 and 13 and Tables 19, 21, 24, and 25. In some embodiments, the CDRs of such an antigen-binding domain can be determined according to the Chothia numbering scheme, which refers to the location of the structural loops of an immunoglobulin (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226). Typically, using the Kabat numbering convention, the Chothia CDR-H1 loop is located at amino acids 26-32, 33, or 34 in the heavy chain; the Chothia CDR-H2 loop is located at amino acids 52-56 in the heavy chain; the Chothia CDR-H3 loop is located at amino acids 95-102 in the heavy chain; the Chothia CDR-L1 loop is located at amino acids 24-34 in the light chain; the Chothia CDR-L2 loop is located at amino acids 50-56 in the light chain; and the Chothia CDR-L3 loop is located at amino acids 89-97 in the light chain. The ends of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, vary between H32 and H34 depending on the length of the loop (this is due to the insertions at H35A and H35B in the Kabat numbering scheme; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34).

[0149] In some embodiments, an antigen binding domain that specifically binds to human TfR comprises six CDRs of an antibody listed in Table 12, Table 13, Table 19, Table 21, Table 24, and Table 25. In some embodiments, such an antigen binding domain that specifically binds to human TfR comprises one or more CDRs, wherein the Chothia and Kabat CDRs have the same amino acid sequence. In some embodiments, provided herein is an antigen binding domain that specifically binds to human TfR, comprising a combination of Kabat and Chothia CDRs.

[0150] In some embodiments, the CDRs of an antigen-binding domain that specifically binds to human TfR can be determined according to MacCallum RM et al., (1996) J Mol Biol 262: 732-745. See also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In some embodiments, provided herein is an antigen-binding domain that specifically binds to human TfR, comprising the VH and VL CDRs of an antibody listed in Table 12, Table 13, Table 19, Table 21, Table 24, and Table 25, as determined by the method of MacCallum RM et al.

[0151] In some embodiments, the CDRs of an antigen-binding domain that specifically binds to human TfR are a compromise between Kabat CDRs and Chothia structural loops, and can be determined according to the AbM numbering scheme, which refers to AbM hypervariable regions, as used in Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In some embodiments, provided herein is an antigen-binding domain that specifically binds to human TfR, comprising the VH and VL CDRs of an antibody listed in Table 12, Table 13, Table 19, Table 21, Table 24, and Table 25, as determined by the AbM numbering scheme.

[0152] In some embodiments, an antigen-binding domain that specifically binds to human TfR comprises the six IMGT CDRs of an antibody listed in Table 12, Table 13, Table 19, Table 21, Table 24, and Table 25 according to the IMGT numbering scheme described in Lefranc MP, (1999) The Immunologist 7: 132-136 and Lefranc MP et al., (1999) Nucleic Acids Res 27: 209-212. According to the IMGT numbering scheme, VH-CDR1 is located at positions 26-35, VH-CDR2 is located at positions 51-57, VH-CDR3 is located at positions 93-102, VL-CDR1 is located at positions 27-32, VL-CDR2 is located at positions 50-52, and VL-CDR3 is located at positions 89-97.

[0153] In some embodiments, the antigen-binding domains provided herein that specifically bind to human TfR are described by their VL domain alone, their VH domain alone, their three VL CDRs alone, or their three VH CDRs alone. See, for example, Rader C et al., (1998) PNAS 95:8910-8915, the entire contents of which are incorporated herein by reference. This document describes the humanization of a murine anti-αvβ3 antibody, obtaining humanized antibody variants with affinity comparable to or higher than that of the original antibody by identifying complementary light or heavy chains from a human light or heavy chain library, respectively. See also Clackson T et al., (1991) Nature 352:624-628, the entire contents of which are incorporated herein by reference. This document describes a method for generating antibodies that bind to a specific antigen by using a particular VL domain (or VH domain) to screen a library for complementary variable domains. This screening yielded 14 new partners for a particular VH domain and 13 new partners for a particular VL domain that were shown to be strong binders by ELISA. See also Kim SJ & Hong HJ, (2007) J Microbiol 45: 572-577, which is incorporated herein by reference in its entirety. This document describes a method for generating antibodies that bind to a specific antigen by using a particular VH domain to screen a library (e.g., a human VL library) for complementary VL domains, and the selected VL domain can then be used to select additional complementary (e.g., human) VH domains.

[0154] In some embodiments, the antigen binding domain that specifically binds to human TfR comprises the VH of an antibody listed in Table 19, Table 21, Table 24, and Table 25.

[0155] In some embodiments, the antigen binding domain that specifically binds to human TfR comprises the VL of an antibody listed in Table 19, Table 21, Table 24, and Table 25.

[0156] In some embodiments, the antigen-binding domain that specifically binds to human TfR comprises the VH and VL of an antibody listed in Table 11 (i.e., the VH of an antibody listed in Table 11 and the VL of the same antibody listed in Table 11) or the VH and VL of an antibody listed in Tables 19, 21, 24, and 25 (i.e., the VH of an antibody listed in Tables 19, 21, 24, and 25 and the VL of the same antibody listed in Tables 19, 21, 24, and 25).

[0157] In some embodiments, an antigen binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 80% identical to the VH amino acid sequence of an antibody of Table 11, Table 19, Table 21, Table 24, and Table 25, and (ii) a VL comprising an amino acid sequence that is at least 80% identical to the VL amino acid sequence of the same antibody of Table 11, Table 19, Table 21, Table 24, and Table 25. In some embodiments, an antigen binding domain that specifically binds to human TfR also comprises the CDRs of an antibody of Table 12, Table 13, Table 19, Table 21, Table 24, and Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0158] In some embodiments, an antigen binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 85% identical to the VH amino acid sequence of an antibody of Table 11, Table 19, Table 21, Table 24, and Table 25, and (ii) a VL comprising an amino acid sequence that is at least 85% identical to the VL amino acid sequence of the same antibody of Table 11, Table 19, Table 21, Table 24, and Table 25. In some embodiments, an antigen binding domain that specifically binds to human TfR also comprises the CDRs of an antibody of Table 12, Table 13, Table 19, Table 21, Table 24, and Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0159] In some embodiments, an antigen binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 90% identical to the VH amino acid sequence of an antibody of Table 11, Table 19, Table 21, Table 24, and Table 25, and (ii) a VL comprising an amino acid sequence that is at least 90% identical to the VL amino acid sequence of the same antibody of Table 11, Table 19, Table 21, Table 24, and Table 25. In some embodiments, an antigen binding domain that specifically binds to human TfR also comprises the CDRs of an antibody of Table 12, Table 13, Table 19, Table 21, Table 24, and Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0160] In some embodiments, an antigen binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 95% identical to the VH amino acid sequence of an antibody of Table 11, Table 19, Table 21, Table 24, and Table 25, and (ii) a VL comprising an amino acid sequence that is at least 95% identical to the VL amino acid sequence of the same antibody of Table 11, Table 19, Table 21, Table 24, and Table 25. In some embodiments, an antigen binding domain that specifically binds to human TfR also comprises the CDRs of an antibody of Table 12, Table 13, Table 19, Table 21, Table 24, and Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0161] In some embodiments, an antigen binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 96% identical to the VH amino acid sequence of an antibody of Table 11, Table 19, Table 21, Table 24, and Table 25, and (ii) a VL comprising an amino acid sequence that is at least 96% identical to the VL amino acid sequence of the same antibody of Table 11, Table 19, Table 21, Table 24, and Table 25. In some embodiments, an antigen binding domain that specifically binds to human TfR also comprises the CDRs of an antibody of Table 12, Table 13, Table 19, Table 21, Table 24, and Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0162] In some embodiments, an antigen binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 97% identical to the VH amino acid sequence of an antibody of Table 11, Table 19, Table 21, Table 24, and Table 25, and (ii) a VL comprising an amino acid sequence that is at least 97% identical to the VL amino acid sequence of the same antibody of Table 11, Table 19, Table 21, Table 24, and Table 25. In some embodiments, an antigen binding domain that specifically binds to human TfR also comprises the CDRs of an antibody of Table 12, Table 13, Table 19, Table 21, Table 24, and Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0163] In some embodiments, an antigen binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 98% identical to the VH amino acid sequence of an antibody of Table 11, Table 19, Table 21, Table 24, and Table 25, and (ii) a VL comprising an amino acid sequence that is at least 98% identical to the VL amino acid sequence of the same antibody of Table 11, Table 19, Table 21, Table 24, and Table 25. In some embodiments, an antigen binding domain that specifically binds to human TfR also comprises the CDRs of an antibody of Table 12, Table 13, Table 19, Table 21, Table 24, and Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0164] In some embodiments, an antigen binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 99% identical to the VH amino acid sequence of an antibody of Table 11, Table 19, Table 21, Table 24, and Table 25, and (ii) a VL comprising an amino acid sequence that is at least 99% identical to the VL amino acid sequence of the same antibody of Table 11, Table 19, Table 21, Table 24, and Table 25. In some embodiments, an antigen binding domain that specifically binds to human TfR also comprises the CDRs of an antibody of Table 12, Table 13, Table 19, Table 21, Table 24, and Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0165] In some aspects, provided herein are antigen-binding domains that bind to the same TfR epitope as an antibody comprising the VH amino acid sequence of an antibody of Table 11, Table 19, Table 21, Table 24, and Table 25 and the VL amino acid sequence of the same antibody of Table 11, Table 19, Table 21, Table 24, and Table 25.

[0166] In some aspects, provided herein are antigen-binding domains that competitively inhibit binding of an antibody comprising the VH amino acid sequence of an antibody of Table 11, Table 19, Table 21, Table 24, and Table 25 and the VL amino acid sequence of the same antibody of Table 11, Table 19, Table 21, Table 24, and Table 25 to TfR.

[0167] In some embodiments, an antigen-binding domain that specifically binds to human TfR comprises a VH and a VL on a single polypeptide chain (e.g., a VH and a VL in Table 11, Table 19, Table 21, Table 24, and Table 25). In some embodiments, the antigen-binding domain comprises an scFv. An scFv may comprise a VH on the N-terminal side of a VL, or a VL on the N-terminal side of a VH. An scFv may comprise, for example, a linker between the VH and VL. Thus, an scFv may be oriented as VH-linker-VL or VL-linker-VH. Such a linker may be about 5 to 25 amino acids in length. Such a linker may be about 5 to 20 amino acids in length. Such a linker may be about 10 to 25 amino acids in length. Such a linker may be, for example, a glycine linker, a glycine-rich linker, or a glycine-serine linker. Such a linker may comprise the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 183). Such a linker may comprise the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 288).

[0168] In some embodiments, an antigen binding domain that specifically binds to human TfR comprises a VH on a first polypeptide and a VL on a second polypeptide (e.g., a Fab).

[0169] In some embodiments, the antigen-binding domain that specifically binds to human TfR comprises an antigen-binding fragment of a heavy chain-only antibody (e.g., a VHH or nanobody).

[0170] In some embodiments, the antigen-binding domain that specifically binds to human TfR is a mouse antigen-binding domain. In some embodiments, the antigen-binding domain that specifically binds to human TfR is a chimeric antigen-binding domain. In some embodiments, the antigen-binding domain that specifically binds to human TfR is a humanized antigen-binding domain. In some embodiments, the antigen-binding domain that specifically binds to human TfR is a human antigen-binding domain.

[0171] In some embodiments, the antigen binding domains provided herein that specifically bind to human TfR also bind to cynomolgus monkey TfR.

[0172] In some embodiments, the antigen-binding domains provided herein bind to human TfR with "low" affinity. In some embodiments, the antigen-binding domain binds to human TfR with an affinity of 500 nM to 10 μM. In some aspects, the antigen-binding domain binds to human TfR with an affinity of 2 μM to 5 μM. In some embodiments, the antigen-binding domain binds to human TfR with an affinity of 1 μM to 5 μM. In some aspects, the antigen-binding domain binds to human TfR with an affinity of 2 μM to 8 μM. In some aspects, the antigen-binding domain binds to human TfR with an affinity of 750 nM to 2 μM.

[0173] In some embodiments, the antigen binding domains provided herein bind to human TfR with "intermediate" affinity. In some embodiments, the antigen binding domains provided herein bind to human TfR with "intermediate" affinity. In some embodiments, the antigen binding domain binds to human TfR with an affinity of 10 nM to 500 nM. In some aspects, the antigen binding domain binds to human TfR with an affinity of 50 nM to 500 nM. In some aspects, the antigen binding domain binds to human TfR with an affinity of 100 nM to 250 nM. In some embodiments, the antigen binding domain binds to human TfR with an affinity of 10 nM to 100 nM. In some embodiments, the antigen binding domain binds to human TfR with an affinity of 250 nM to 500 nM.

[0174] In some embodiments, the antigen binding domains provided herein bind to human TfR with "high" affinity. In some embodiments, the antigen binding domain binds to human TfR with an affinity of less than 10 nM. In some embodiments, the antigen binding domain binds to human TfR with an affinity of 0.01 nM to 10 nM. In some embodiments, the antigen binding domain binds to human TfR with an affinity of 0.1 nM to 10 nM. In some embodiments, the antigen binding domain binds to human TfR with an affinity of 0.01 nM to 1 nM.

[0175] In some embodiments, antigen-binding domains provided herein that specifically bind to human TfR bind to human TfR with an affinity of 250 nM or less (e.g., 10 μM to 250 nM, 5 μM to 250 nM, 1 nM to 250 nM, or 3 nM to 250 nM), an affinity of 200 nM or less (e.g., 10 μM to 200 nM, 5 μM to 200 nM, 1 nM to 200 nM, or 3 nM to 200 nM), or an affinity of 150 nM or less (e.g., 10 μM to 150 nM, 5 μM to 150 nM, 1 nM to 150 nM, or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, for example, using a Carterra LSA platform. In some embodiments, the antigen-binding domains provided herein that specifically bind to human TfR are 450 The antibody binds to human TfR as measured by ELISA.

[0176] In some embodiments, an antigen binding domain provided herein that specifically binds to human TfR binds to cynomolgus monkey TfR with an affinity of 250 nM or less (e.g., 10 μM to 250 nM, 5 μM to 250 nM, 1 μM to 250 nM, 1 nM to 250 nM, or 3 nM to 250 nM), an affinity of 200 nM or less (e.g., 10 μM to 200 nM, 5 μM to 200 nM, 1 μM to 200 nM, 1 nM to 200 nM, or 3 nM to 200 nM), or an affinity of 150 nM or less (e.g., 10 μM to 150 nM, 5 μM to 150 nM, 1 μM to 150 nM, 1 nM to 150 nM, or 3 nM to 150 nM), optionally where the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, for example, using a Carterra LSA platform. In some embodiments, the antibody specifically binds to human TfR with an affinity of 0.1 μM to 10 μM, 0.1 μM to 100 μM, 0.1 μM to 100 μM, 0.1 μM to 1 nM, 1 μM to 10 μM, 1 μM to 100 μM, 1 μM to 1 nM, 1 μM to 10 nM, 1 μM to 100 nM, 1 μM to 150 nM, or 1 μM to 250 nM.

[0177] In some embodiments, the antigen-binding domains provided herein that specifically bind to human TfR bind to human TfR and cynomolgus monkey TfR, respectively, with an affinity of 250 nM or less (e.g., 1 μM to 250 nM, 10 μM to 250 nM, 1 nM to 250 nM, or 3 nM to 250 nM), an affinity of 200 nM or less (e.g., 1 μM to 200 nM, 10 μM to 250 nM, 1 nM to 200 nM, or 3 nM to 200 nM) or less, or an affinity of 150 nM or less (e.g., 1 μM to 150 nM, 10 μM to 250 nM, 1 nM to 150 nM, or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, for example, using a Carterra LSA platform. In some embodiments, antigen-binding domains provided herein that specifically bind to human TfR bind to human TfR and cynomolgus monkey TfR with an affinity of 1 μM to 100 μM, 1 μM to 1 nM, 1 μM to 10 nM, 1 μM to 100 nM, 1 μM to 150 nM, or 1 μM to 250 nM, respectively.

[0178] In some embodiments, antigen-binding domains provided herein that specifically bind to human TfR bind to human TfR with an affinity of 6.7 nM to 3.5 μM and to cynomolgus monkey TfR with an affinity of 38 nM to 2.3 μM. In some embodiments, antigen-binding domains provided herein that specifically bind to human TfR bind to human TfR with an affinity of 6.7 nM to 340 nM and to cynomolgus monkey TfR with an affinity of 18 nM to 870 nM, optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, for example, using a Carterra LSA platform.

[0179] In some embodiments, the antigen-binding domains provided herein that specifically bind to human TfR reduce cell surface expression of TfR on HCMED / D3 cells by 40-80% compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, for example, using Western blot or FACS.

[0180] In some embodiments, the antigen binding domains provided herein that specifically bind to human TfR do not significantly increase cell surface expression of TfR on HCMED / D3 cells compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, for example, using Western blot or FACS.

[0181] In some embodiments, antigen binding domains provided herein that specifically bind to human TfR do not reduce cell surface expression of TfR on HCMED / D3 cells by more than 40%, 60%, or 80% compared to cell surface expression of TfR on HCMED / D3 cells treated with an isotype control, and do not significantly increase cell surface expression of TfR on HCMED / D3 cells compared to cell surface expression of TfR on HCMED / D3 cells treated with an isotype control. Cell surface expression can be measured, for example, using Western blot or FACS.

[0182] In some embodiments, the antigen binding domains provided herein that specifically bind to human TfR accumulate in the brains of vascular-depleted human TfR knock-in mice after peripheral injection at least 4-fold or 5-fold more than isotype controls.

[0183] In some embodiments, an antigen binding domain provided herein that specifically binds to human TfR accumulates in the vascular-depleted non-human primate brain at least two-fold more than an isotype control after peripheral injection. In some embodiments, an antigen binding domain provided herein that specifically binds to human TfR accumulates in the vascular-depleted non-human primate brain at least four-fold or five-fold more than an isotype control after peripheral injection. In some embodiments, an antigen binding domain provided herein that specifically binds to human TfR accumulates in the vascular-depleted non-human primate brain at least eight-fold more than an isotype control after peripheral injection. In some embodiments, an antigen binding domain provided herein that specifically binds to human TfR accumulates in the vascular-depleted non-human primate brain at least 8-64-fold more than an isotype control after peripheral injection. In some embodiments, an antigen binding domain provided herein that specifically binds to human TfR accumulates in the vascular-depleted non-human primate brain at least 8-64-fold more than an isotype control after peripheral injection. In some embodiments, an antigen-binding domain provided herein that specifically binds to human TfR accumulates in the vascular-depleted non-human primate brain after peripheral injection at least 2-11 fold more than an isotype control.

[0184] In some embodiments, the antigen-binding domains provided herein specifically bind to human TfR at least 5-fold more than they bind to an unrelated protein. In some embodiments, the antigen-binding domains provided herein specifically bind to cynomolgus monkey TfR at least 5-fold more than they bind to an unrelated protein. In some embodiments, the antigen-binding domains provided herein specifically bind to human TfR at least 5-fold more than they bind to an unrelated protein and / or specifically bind to cynomolgus monkey TfR at least 5-fold more than they bind to an unrelated protein.

[0185] Also provided herein are antigen-binding domains that bind to the same epitope of TfR as the TfR antigen-binding domains provided herein. Also provided herein are antigen-binding domains that competitively inhibit the binding of the TfR antigen-binding domains provided herein to TfR.

[0186] Agents containing anti-TfR antigen-binding domains Provided herein are agents (e.g., fusion proteins, multispecific (e.g., bispecific) proteins, antibodies, antigen-binding fragments thereof, etc.) that comprise an antigen-binding domain that specifically binds to human TfR.

[0187] fusion proteins In some embodiments, the fusion proteins provided herein comprise an antigen-binding domain that specifically binds to human TfR and a heterologous protein. In some embodiments, the fusion proteins provided herein comprise (i) an antigen-binding domain that specifically binds to human TfR and (ii) a heterologous protein or polypeptide, or a fragment thereof. In some embodiments, the heterologous protein is a protein or polypeptide, or a fragment thereof, useful in protein replacement therapy (PRT). In some embodiments, the heterologous polypeptide is an enzyme (e.g., an enzyme for use in enzyme replacement therapy (ERT)) or a catalytically active fragment thereof. In some embodiments, the heterologous polypeptide or protein is an ERT enzyme or an ERT enzyme variant, or a catalytically active fragment thereof.

[0188] In some embodiments, the heterologous polypeptide in the fusion proteins provided herein is a growth factor. In some embodiments, the heterologous polypeptide in the fusion proteins provided herein is a decoy receptor. In some embodiments, the heterologous polypeptide in the fusion proteins provided herein is progranulin (PGRN), prosaposin (PSAP), or motor neuron survival protein (SMN). In some embodiments, the heterologous protein is ubiquitin protein ligase E3A (UBE3A), α-L iduronidase (IDUA), iduronate-2-sulfatase (IDS), N-acetylgalactosamine-6-sulfatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramide The enzyme is selected from beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta-hexosaminidase B, arylsulfatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme substitute thereof, such as larosinase, idursulfase, elosulfase alpha or galsulfase, or a variant or catalytically active fragment thereof.In some embodiments, the heterologous protein is selected from the group consisting of clusterin (APOJ), reelin, tripeptidyl peptidase 1 (CLN2 / TPP1), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), α-L iduronidase (IDUA), iduronate-2-sulfatase (IDS), N-acetylgalactosamine-6-sulfatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramide The protein or enzyme is selected from beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta-hexosaminidase B, arylsulfatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme substitute thereof, such as larosinase, idursulfase, elosulfase alpha or galsulfase, or a variant thereof, or a catalytically active fragment thereof.

[0189] An exemplary format of the fusion proteins disclosed herein is illustrated in FIG. 1E.

[0190] For example, in some embodiments, the heterologous protein or polypeptide in the fusion protein is located N-terminal to the antigen-binding domain that specifically binds to human TfR. In some embodiments, the heterologous protein or polypeptide in the fusion protein is located C-terminal to the antigen-binding domain that specifically binds to human TfR. In some embodiments, the heterologous fusion protein or polypeptide and the antigen-binding domain that specifically binds to human TfR are directly linked via a peptide bond. In some embodiments, the heterologous fusion protein or polypeptide and the antigen-binding domain that specifically binds to human TfR are linked via a linker (e.g., a peptide linker). In some embodiments, the fusion protein comprises an antigen-binding domain, a heterologous protein or polypeptide, and an Fc portion. In some embodiments, the antigen-binding domain and the heterologous protein or polypeptide are linked to the N-terminus of the Fc portion of the fusion protein. In some embodiments, the antigen-binding domain and the heterologous protein are linked to the N-terminus of the Fc portion of the fusion protein. In other embodiments, the antigen-binding domain is linked to the N-terminus of the Fc portion and the heterologous protein or polypeptide is linked to the C-terminus of the Fc portion of the fusion protein. In other embodiments, the antigen binding domain is linked to the C-terminus of the Fc portion and the heterologous protein or polypeptide is linked to the N-terminus of the Fc portion of the fusion protein.

[0191] In some embodiments, disclosed herein are fusion proteins comprising (i) a single scFv, VHH, or Fab antigen-binding domain that binds to human TfR and (ii) a heterologous protein or polypeptide, the fusion protein comprising two copies of the heterologous protein or polypeptide. In some embodiments, the fusion protein comprises an Fc domain. The Fc can be a heterodimeric Fc comprising a first Fc polypeptide with a knob mutation and a second Fc polypeptide with a hole mutation (a "knob-hole Fc" as described herein). In some embodiments, a single scFv, Fab, or VHH antigen-binding domain that binds to human TfR is linked to the C-terminus of the Fc domain (i.e., one of the two heavy chains of the Fc domain), and two copies of the heterologous protein or polypeptide are linked to the N-terminus of the Fc domain. In some embodiments, a single scFv, Fab, or VHH antigen-binding domain that binds to human TfR is linked to the N-terminus of an Fc domain (i.e., one of the two heavy chains of the Fc domain), and two copies of a heterologous protein or polypeptide are linked to the C-terminus of the Fc domain. Examples of this 2+1 format are shown in Figure IE as (v) and (vi).

[0192] In some embodiments, disclosed herein are fusion proteins comprising: (i) an antibody that binds to human TfR, the antibody comprising two heavy chains and two light chains; and (ii) two copies of a heterologous protein or polypeptide linked to the C-terminus of the two antibody heavy chains.

[0193] In some embodiments, the fusion protein comprises (i) two scFv, Fab, or VHH antigen-binding domains that bind to human TfR linked to the C-terminus of the heavy chain, and (ii) two copies of the fusion protein or fusion protein variant linked to the N-terminus of the Fc domain.

[0194] In some embodiments, disclosed herein is a fusion protein comprising (i) a single scFv, VHH, or Fab antigen-binding domain that binds to human TfR, (ii) an Fc domain, and (iii) a single copy of a heterologous protein or peptide, wherein the single scFv, VHH, or Fab antigen-binding domain that binds to human TfR is linked to the C-terminus of the Fc domain, and the heterologous protein or polypeptide is linked to the N-terminus of the Fc domain. In some embodiments, the Fc is a single-chain, engineered, monovalent Fc domain. An example of this single-chain 1+1 format of the fusion protein is shown as (i) in Figure IE.

[0195] In some embodiments, disclosed herein is a fusion protein comprising (i) a single scFv, VHH, or Fab antigen-binding domain that binds to human TfR, (ii) an Fc domain, and (iii) a single copy of a heterologous protein or polypeptide, wherein the single scFv, VHH, or Fab antigen-binding domain that binds to human TfR is linked to the N-terminus of the Fc domain, and the heterologous protein or polypeptide is linked to the C-terminus of the Fc domain. In some embodiments, the Fc is a single-chain engineered monovalent Fc. An example of this 1+1 format of the fusion protein is shown as (ii) in Figure IE.

[0196] In some embodiments, disclosed herein are fusion proteins comprising (i) a single scFv, VHH, or Fab antigen-binding domain that binds to human TfR, (ii) an Fc domain, and (iii) a single copy of a heterologous protein or polypeptide, wherein the single scFv, VHH, or Fab antigen-binding domain that binds to human TfR and the heterologous protein or polypeptide are both linked to the N-terminus or C-terminus of the Fc domain. An example of this 1+1 format of fusion protein is shown in Figure IE as (iv).

[0197] Bispecific and multispecific proteins In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise an antigen-binding domain that specifically binds to human TfR. In some embodiments, the antibodies or antigen-binding fragments thereof comprise an antigen-binding domain that specifically binds to human TfR and an antigen-binding domain that specifically binds to a CNS antigen or brain antigen. In some embodiments, the CNS antigen or brain antigen is not TfR. Also provided herein are antibodies or antigen-binding fragments thereof that bind to the same epitope on TfR as the TfR antigen-binding domains provided herein. Also provided herein are antibodies or antigen-binding fragments thereof that competitively inhibit the binding of the TfR antigen-binding domains provided herein to TfR.

[0198] In some embodiments, the multispecific proteins provided herein comprise a first antigen-binding domain that binds to human TfR and a second antigen-binding domain. The first antigen-binding domain that binds to human TfR can be any antigen-binding domain that binds to human TfR provided herein. The second antigen-binding domain can be an antigen-binding domain that specifically binds to a CNS antigen or a brain antigen. In some embodiments, the CNS antigen or brain antigen is not TfR.

[0199] In some embodiments, the multispecific proteins provided herein comprise an antigen-binding domain that binds to human TfR linked to an antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof can bind to a CNS antigen or a brain antigen. In some embodiments, the CNS antigen or brain antigen is not TfR. In some embodiments, such multispecific proteins can be in a 2+1 antibody format (shown in FIG. 1A) or a 2+2 antibody format (shown in FIGS. 1B and 1C).

[0200] In some embodiments, the multispecific proteins provided herein comprise a TfR antigen-binding domain that is an scFv linked to an antibody that binds to a CNS antigen, the antibody comprising two heavy chains and two light chains. In some embodiments, the scFv is linked to the C-terminus of one of the two antibody heavy chains, e.g., via a protein linker.

[0201] In some embodiments, a multispecific protein comprises (1) an antigen-binding domain that binds to TfR, (2) a second antigen-binding domain that binds a different CNS or brain antigen, and (3) an Fc region, wherein the TfR antigen-binding domain and the second antigen-binding domain are connected or linked to the Fc region of the multispecific protein. In other embodiments, a multispecific protein comprises (1) an antigen-binding domain that comprises a heavy chain variable region and binds to TfR, (2) a second antigen-binding domain that comprises a heavy chain variable region and binds a different CNS or brain antigen, and (3) an Fc region, wherein the TfR antigen-binding domain and the second antigen-binding domain are connected or linked to the Fc region of the multispecific protein. In some embodiments, a multispecific protein comprises an antigen-binding domain that binds to TfR, a second antigen-binding domain that binds a different CNS or brain antigen, and an Fc region. In some embodiments, the TfR antigen-binding domain and the second antigen-binding domain are connected or linked to the N-terminus of the Fc portion of the multispecific protein. In other embodiments, the TfR antigen binding domain is connected or linked to the N-terminus of the Fc portion of the multispecific protein and the second antigen binding domain is linked to the C-terminus of the Fc portion of the multispecific protein. In other embodiments, the TfR antigen binding domain is connected or linked to the C-terminus of the Fc portion of the multispecific protein and the second antigen binding domain is linked to the N-terminus of the Fc portion of the multispecific protein.

[0202] In some embodiments, the multispecific proteins provided herein comprise two copies of a TfR antigen-binding domain that is an scFv, and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, and one of the two copies of the antigen-binding domain is linked to the C-terminus of one of the antibody heavy chains, and the other copy of the antigen-binding domain is linked to the C-terminus of the other antibody heavy chain. In some embodiments, each scFv is linked to each heavy chain via a protein linker.

[0203] In some embodiments, disclosed herein are trivalent bispecific proteins comprising (i) a single scFv, Fab, or VHH antigen-binding domain that binds to human TfR, and (ii) an antibody, wherein the antibody comprises two heavy chains and two light chains, and the single scFv, Fab, or VHH antigen-binding domain that binds to human TfR is linked to the C-terminus or N-terminus of one of the two antibody heavy chains. The Fc can be a heterodimeric Fc, such as a knob-and-hole Fc. An example of this 2+1 format, including a knob-hole Fc, is shown as (i) in Figure 1D.

[0204] In some embodiments, disclosed herein are tetravalent bispecific proteins comprising (i) two scFv, Fab, or VHH antigen-binding domains that bind to human TfR, and (ii) an antibody, wherein the antibody comprises two heavy chains and two light chains, wherein one of the scFv, Fab, or VHH antigen-binding domains that bind to human TfR is linked to the C-terminus of one of the two antibody heavy chains, and the other of the scFv, Fab, or VHH antigen-binding domains that bind to human TfR is linked to the C-terminus or N-terminus of the other of the two antibody heavy chains. In some embodiments, the two scFv, Fab, or VHH antigen-binding domains that bind to human TfR may comprise the same amino acid sequence. In some embodiments, the Fc is a heterodimeric Fc, such as an Fc domain with a knob-and-hole mutation.

[0205] In some embodiments, disclosed herein are bivalent, bispecific proteins comprising (i) a single scFv, VHH, or Fab antigen-binding domain that binds to human TfR linked to the N-terminus of the Fc domain of the Fc dimer, and (ii) a second antigen-binding domain that does not specifically bind to human TfR and is linked to the N-terminus of the second Fc domain of the Fc dimer. In some embodiments, the second antigen-binding domain binds to a CNS or brain antigen other than TfR. An example of this 1+1 format is shown in Figure ID as (iii).

[0206] In some embodiments, bivalent bispecific proteins are disclosed herein, comprising: (i) a single scFv, VHH, or Fab antigen-binding domain that binds to human TfR linked to the N-terminus or C-terminus of the Fc domain of the Fc dimer; and (ii) a second antigen-binding domain, where the second antigen-binding domain does not specifically bind to human TfR, and the single scFv, VHH, or Fab linked to the N-terminus of the Fc or the C-terminus of the second Fc domain of the Fc dimer. In some embodiments, the Fc is a heterodimeric Fc, such as an Fc domain with a knob-and-hole mutation. An example of this 1+1 format is shown in Figure ID (ii), in which a single scFv is linked to the N-terminus of the Fc domain of the Fc region, and a second antigen-binding domain is linked to the N-terminus of the second Fc domain of the Fc region.

[0207] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein can be multispecific, e.g., bispecific. Many different formats and uses of bispecific binding molecules are known in the art (e.g., as reviewed in Kontermann; Drug Discovery Today, 2015 July;20(7):838-47; MAbs, 2012 March-April;4(2):182-97). Bispecific proteins according to the present invention are not limited to any particular bispecific format or method of production. Thus, bispecific proteins of the present disclosure can include a variety of configurations having a first antigen-binding domain that binds to human TfR and a second antigen-binding domain that binds, for example, to a CNS antigen or brain antigen.

[0208] Bispecific molecules include, for example, kappa / lambda bodies, dual affinity retargeting molecules (DARTs), knob-in-hole antibodies, strand-exchange engineered domain bodies (SEED bodies), and duobodies. In some aspects, the bispecific fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein comprise a knob mutation and a hole mutation. In some aspects, the knob mutation comprises the amino acid substitution T366W according to EU numbering. In some aspects, the hole mutation comprises the amino acid substitutions T366S, L368A, and Y407V according to EU numbering. Examples of bispecific molecules that can be used in the present disclosure include: (i) a single antibody with two arms containing different antigen-binding domains; (ii) a single-chain antibody with specificity for two different epitopes, for example, via two scFvs linked in tandem by an additional peptide linker; and (iii) a dual-variable domain antibody (DVD-Ig) in which each light and heavy chain contains two variable domains in tandem via a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig.™) Molecule, In: Antibody Engineering, Springer Berlin). Heidelberg (2010)), (iv) chemically linked bispecific (Fab') fragments, (v) Tandabs, which are fusions of two single-chain diabodies that give tetravalent bispecific antibodies with two binding sites for each target antigen, (vi) Flexibodies, which are combinations of scFvs and diabodies that give multivalent molecules, (vii) so-called "dock-and-lock" molecules based on the "dimerization and docking domain" of protein kinase A, which, when applied to Fabs, can give trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments, (viii) so-called Scorpion molecules, which, for example, contain two scFvs fused to either end of a human Fab arm, and (ix) diabodies. Other examples of antibody structures are described in WO 2019 / 246288, which is incorporated by reference.

[0209] In some embodiments, the bispecific proteins provided herein are selected from one of the following formats: CrossMab, DAF (2 in 1), DAF (4 in 1), DutaMab, DT-IgG, charge pair, Fab arm exchange, triomab, LUZ-Y, Fcab, kappalambda body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG (L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG (4-in-1), nanobody, nanobody-HAS, BiTE, TandAb, scdiabody-CH3, diabody-CH3, triplebody, miniantibody, minibody, TriBiminibody, scFv-CH3 KIH, Fab -scFv, scFV-CH-CL-scFV, F(ab')2, F(ab')2-scFv2, scFV-KIH, Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, tandem scFv -Fc, intrabody, ImmTAC, HSAbody, scdiabody-HAS, tandem scFv-toxin, IgG-IgG, Cov-X-body, and scFv1-PEG-scFv2 (described in Spiess, C., et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol. Immunol. (2015), incorporated herein by reference).

[0210] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are bivalent. In some embodiments, the multispecific proteins provided herein are multivalent (e.g., bivalent). In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are trivalent (e.g., a 2+1 antibody format). In some embodiments, the trivalent format comprises a single TfR antigen-binding domain provided herein and two antigen-binding domains that bind to CNS or brain antigens. The two antigen-binding domains that bind to CNS or brain antigens may comprise the same amino acid sequence or different amino acid sequences. In some embodiments, the TfR antigen-binding domain is an scFv. In some embodiments, the TfR antigen-binding domain is a VHH.

[0211] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are tetravalent (e.g., 2+2 antibody format). In some embodiments, the tetravalent format comprises two TfR antigen-binding domains provided herein and two antigen-binding domains that bind to CNS or brain antigens. The two TfR antigen-binding domains may comprise the same amino acid sequence or different amino acid sequences. In some embodiments, the two TfR antigen-binding domains comprise the same amino acid sequence. In some embodiments, one or both of the TfR antigen-binding domains is an scFv. The two antigen-binding domains that bind to CNS or brain antigens may comprise the same amino acid sequence or different amino acid sequences.

[0212] The fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein may include, for example, a linker that connects the TfR antigen-binding domain to a heterologous protein, antibody or antigen-binding fragment thereof, or other antigen-binding domain. The linker may be, for example, a glycine linker, a glycine-rich linker, or a glycine-serine linker. The linker may include the amino acid sequence of (GGGGS)x3 (SEQ ID NO: 184). The linker may include the amino acid sequence of (GGSGG)x3 (SEQ ID NO: 289).

[0213] The fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein may comprise a constant region. In some embodiments, the TfR antigen-binding domain provided herein is linked to a constant region, e.g., the C-terminus of the constant region. In some embodiments, the constant domain is a human constant domain. In some embodiments, the constant domain is a mouse, rat, rabbit, or monkey (e.g., cynomolgus monkey) constant domain. The constant region may be a heavy chain constant region. The constant region may be a human constant region. The constant region may be a human heavy chain constant region. The constant region may be an IgG constant region. The constant region may be an IgG1 constant region. The constant region may be an IgG2 constant region. The constant region may be an IgG4 constant region. The constant region may be a human IgG constant region. The constant region may be a human IgG1 constant region. The constant region may be a human IgG2 constant region. The constant region may be a human IgG4 constant region. The linker may comprise the amino acid sequence GGSGG (no repeats) (SEQ ID NO: 304). The linker may be from 1 to about 20 amino acids in length.

[0214] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein comprise a heavy chain and a light chain. With respect to the heavy chain, in some embodiments, the heavy chain of the antigen-binding proteins described herein can be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain can comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain comprises a human gamma (γ) heavy chain constant region. In some embodiments, the heavy chain comprises the amino acid sequence of an IgG1 heavy chain constant region. In some embodiments, the heavy chain comprises the amino acid sequence of an IgG2 (IgG2a or IgG2b) heavy chain constant region. In some embodiments, the heavy chain comprises the amino acid sequence of an IgG4 heavy chain constant region. With respect to the light chain, in some embodiments, the light chain is a kappa light chain. In some embodiments, the light chain is a lambda light chain. In some aspects, the light chain is a human kappa light chain or a human lambda light chain.

[0215] In some aspects, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein comprise a constant region comprising the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In some aspects, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein comprise a constant region comprising the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (IgG2a and IgG2b) of immunoglobulin molecule. In some embodiments, the constant region comprises the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (IgG2a and IgG2b).

[0216] Non-limiting examples of human constant region sequences are described, for example, in U.S. Pat. No. 5,693,780 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242.

[0217] In some aspects, the constant regions provided herein comprise a knob mutation. In some aspects, the constant regions provided herein comprise a hole mutation. Thus, in some aspects, a fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein can comprise a constant region that comprises a knob mutation and a constant region that comprises a hole mutation.

[0218] FC Domain The fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein can comprise an Fc domain or a fragment thereof. In some embodiments, the Fc domain is of the IgG class, the IgM class, or the IgA class. In some embodiments, the Fc domain or a fragment thereof is an IgG Fc domain or a fragment thereof. In some embodiments, the Fc domain or a fragment thereof is a human IgG Fc domain or a fragment thereof. In some embodiments, the Fc domain or a fragment thereof is a human IgG1 Fc domain or a fragment thereof. In some embodiments, the Fc domain or a fragment thereof is a human IgG2 Fc domain or a fragment thereof. In some embodiments, the Fc domain or a fragment thereof is a human IgG4 Fc domain or a fragment thereof. In some embodiments, the Fc domain or a fragment thereof is a monovalent Fc.

[0219] In some embodiments provided herein, the provided fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein comprises a modified Fc domain or fragment thereof. In some embodiments, the modified Fc domain or fragment thereof is a modified IgG1 Fc comprising one or more modifications. For example, in some embodiments, the IgG1 modified Fc comprises one or more amino acid substitutions (e.g., compared to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from the group consisting of N297A (Bolt S et al. (1993) Eur J Immunol 23:403-411), D265A (Shields et al. (2001) RJ Biol. Chem. 276, 6591-6604), L234A, L235A (Hutchins et al. (1995) Proc Natl Acad Sci USA, 92:11980-11984; Alegre et al., (1994) Transplantation 57:1537-1543.31; Xu et al., (2000) Cell Immunol, 200:16-26), G237A (Alegre et al. (1994) Transplantation 57:1537-1543.31; Xu et al. al.(2000)Cell Immunol,200:16-26), C226S, C229S, E233P, L234V, L234F, L235E(McEarchern et al.,(2007)Blood,109:1185-1192), P331S(Sazinsky et al.,(2008)Proc Natl Acad Sci USA 2008,105:20167-20172), K322A(Hezareh et al. (2001)J Virol. 75(24) 12161-12168), S267E, L328F, A330L, M252Y, S254T, E430G, and / or T256E, where amino acid positions are according to EU numbering. In some embodiments, the Fc comprises amino acid substitutions L234A, L235A, and P331S (LALAPS) according to EU numbering. In some embodiments, the Fc comprises N325S and L328F mutations according to EU numbering.In some embodiments, Fc comprises P329S or P329S according to EU numbering. In some embodiments, Fc comprises K322A according to EU numbering.

[0220] Table 1. Exemplary Fc domains TIFF2025528752000002.tif186165TIFF2025528752000003.tif141165

[0221] In some aspects provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are bispecific fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins. Bispecific molecules include, for example, kappa-lambda bodies, dual affinity retargeting molecules (DARTs), knob-in-hole antibodies, strand-exchange engineered domain bodies (SEED bodies), and duobodies. In some aspects, the bispecific fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein comprise a knob mutation and a hole mutation. In some aspects, the knob mutation comprises the amino acid substitution T366W according to EU numbering. In some aspects, the hole mutation comprises the amino acid substitutions T366S, L368A, and Y407V according to EU numbering.

[0222] In some embodiments provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein comprise one or more mutations to promote heterodimerization of the Fc domains. In some embodiments, the dimerization Fc domains of the bispecifics provided herein are formed by Fc domains containing amino acid mutations, substitutions, additions, or deletions that promote heterodimerization, such that different polypeptides comprising the different Fc domains dimerize to form a heterodimeric form. In some embodiments, the bispecifics of the present disclosure comprise a first Fc sequence comprising a first CH3 region and a second Fc sequence comprising a second CH3 region, wherein the sequences of the first CH3 region and the second CH3 region are different such that the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the homodimeric interaction between the first CH3 region and the second CH3 region, respectively.

[0223] Methods for promoting heterodimerization between Fc domains include amino acid deletions, additions, or substitutions in the amino acid sequence of the Fc domain, for example, by including a group of "knob-into-hole" deletions, additions, or substitutions, or by including amino acid deletions, additions, or substitutions that result in electrostatic guidance of the Fc, which favors attractive interactions between different polypeptide chains. Methods for promoting heterodimerization between complementary Fc polypeptides have been previously described, for example, in Ridgway et al., 1996, Protein Eng, 9:617-621; Merchant et al., 1998, Nature Biotechnol, 16:677-681; Moore et al., 2011, MAbs, 3:546-557; Von Kreudenstein et al., 2013, J Biol Chem, 285:19637-19464; Leaver-Fay et al., 2016, Structure, 24:641-651; Ha et al., 2016, Frontiers in Immunology, 7:1; Davis et al., 2010, Protein Eng Des Sel,23:195-202;WO1996 / 027011;WO1998 / 050431;WO2006 / 028936;WO2009 / 089004;WO2011 / 143545;WO2014 / 067011;WO2012 / 058768;WO2018 / 027025;US2014 / 0363426;US2015 / 03076 28; US2018 / 0016354; US2015 / 0239991; US2017 / 0058054; USPN5731168; USPN7183076; USPN9701759; USPN9605084; USPN9650446; USPN8216805; USPN8765412; and USPN8258268.

[0224] In some embodiments, the complementary Fc polypeptides of an Fc heterodimer contain mutations that alter the charge polarity across the Fc dimer interface, such that coexpression of electrostatically matched Fc domains favors favorable attractive interactions, thereby promoting the formation of desired Fc heterodimers, while unfavorable repulsive charge interactions suppress the formation of undesired Fc homodimers (Guneskaran et al., 2010, J Biol Chem, 285:19637-19646). When coexpressed in cells, association between polypeptide chains is possible, but the chains rarely self-associate due to charge repulsion.

[0225] Additionally, the complementary Fc polypeptides of an Fc heterodimer may contain a "knob-into-hole" structure that promotes heterodimerization between two Fc polypeptides. "Knob-into-hole" technology has been described, for example, in U.S. Patent Nos. 5,731,168, 7,695,936, 8,216,805, and 8,765,412; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protrusion ("knob") into the interface of a first polypeptide and a corresponding hole ("hole") into the interface of a second polypeptide, such that the protrusion lies within the hole, promoting heterodimer formation and preventing homodimer formation. The protrusion is formed by substituting a small amino acid side chain at the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary hole of the same or similar size as the protrusion is formed at the interface of the second polypeptide by substituting a large amino acid side chain with a smaller one (e.g., alanine or threonine). The protrusion and hole can be formed by modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or peptide synthesis. In some embodiments, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In some embodiments, the Fc domain subunit containing the knob modification further comprises the amino acid substitution S354C, and the Fc domain subunit containing the hole modification further comprises the amino acid substitution Y349C. Introduction of these two cysteine ​​residues forms disulfide bridges between the two subunits of the Fc region, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).Thus, in such a configuration, a first Fc polypeptide contains amino acid modifications to form the "knob" and a second Fc polypeptide contains amino acid modifications to form the "hole", thereby forming an Fc heterodimer consisting of the complementary Fc polypeptides.

[0226] Exemplary paired amino acid modifications of complementary Fc polypeptides in the Fc heterodimer form are shown in the table below (EU numbering).

[0227] Table 2. Exemplary paired Fc modifications of heterodimeric Fc domains TIFF2025528752000004.tif65165

[0228] Some agents provided herein comprise antigen-binding fragments of antibodies. Antigen-binding fragments of antibodies include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments, and scFv fragments, as well as other fragments described below. A review of specific antibody fragments is provided in Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., WO 93 / 16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and have increased in vivo half-lives, see U.S. Pat. No. 5,869,046.

[0229] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, e.g., EP 404,097, WO 1993 / 01161, Hudson et al. Nat. Med. 9:129-134 (2003). Triabodies and tetrabodies are also described in Hudson et al. Nat. Med. 9:129-134 (2003). Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In some embodiments, single-domain antibodies are human single-domain antibodies (see, e.g., U.S. Pat. No. 6,248,516).

[0230] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0231] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein may be chimeric. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, e.g., a monkey) and a human constant region. In a further example, the chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody.

[0232] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein may be humanized. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the non-human parent antibody. In some aspects, humanized antibodies are substantially non-immunogenic in humans. In some aspects, humanized antibodies have substantially the same affinity for a target as an antibody from another species from which the humanized antibody is derived. See, e.g., U.S. Patent Nos. 5,530,101, 5,693,761, 5,693,762, and 5,585,089. In some aspects, amino acids in antibody variable domains are identified that can be modified to reduce their immunogenicity without losing the native affinity of the antigen-binding domain. See, e.g., U.S. Patent Nos. 5,766,886 and 5,869,619. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the framework regions (FRs) are derived from human antibody sequences. A humanized antibody may also comprise at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues were derived), e.g., to restore or improve antibody specificity or affinity.

[0233] Humanized antibodies and methods for making them are reviewed, for example, in Almagro et al. Front. Biosci. 13:161 9-1633 (2008) and further described, for example, in U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409. Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)), framework regions derived from consensus sequences of human antibodies of specific subpopulations of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions obtained from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0234] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein may be human. Human antibodies can be produced using various techniques well known in the art. Human antibodies are generally described in van Dijk et al. Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg Curr. Opin. Immunol. 20:450-459 (2008).

[0235] Human antibodies can be prepared by administering immunogens to transgenic animals engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Large fragments of human Ig loci can be incorporated into mouse strains deficient in mouse antibody production, with the expectation that such mice will produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can preserve the diversity of large variable genes and the proper regulation of antibody production and expression. By exploiting mouse mechanisms for antibody diversification and selection and the lack of immune tolerance to human proteins, the recapitulated human antibody repertoire in these mouse strains can yield high-affinity, fully human antibodies against any antigen of interest, including human antigens. Hybridoma technology can be used to produce and select antigen-specific human MAbs with desired specificity. Certain exemplary methods are described in U.S. Pat. No. 5,545,807, EP 546073, and EP 546073. See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE™ technology), U.S. Patent No. 5,770,429 (describing HUMAB® technology), U.S. Patent No. 7,041,870 (describing KM MOUSE® technology), and U.S. Patent Application Publication No. 2007 / 0061900 (describing VELOCIMOUSE® technology). The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0236] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol. 133:3001 (1984) and Boerner et al. J. Immunol. 147:86 (1991)). Human antibodies generated via human B cell hybridoma technology are also described in Li et al. Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Pat. No. 7,189,826, which describes the production of monoclonal human IgM antibodies from hybridoma cell lines. Human hybridoma technology is also described in Vollmers et al., Histology and Histopathology, 20(3):927-937 (2005) and Vollmers et al., Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005). Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domains can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0237] In some embodiments provided herein, the antibodies are human antibodies isolated by in vitro methods and / or screening of combinatorial libraries for antibodies with the desired activity. Suitable examples include, but are not limited to, phage display (CAT, Morphosys, Dyax, Biosite / Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosome display (CAT), yeast display (Adimab), and the like. In certain phage display methods, repertoires of VH and VL genes are individually cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phage, as described in Winter et al. Ann. Rev. Immunol. 12:433-455 (1994). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. See also: Sidhu et al. J. Mol. Biol. 338(2):299-310, 2004; Lee et al. J. Mol. Biol. 340(5):1073-1093, 2004; Fellouse Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al. Methods 284(-2):119-132 (2004). Phages typically display antibody fragments, either as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies against the immunogen without the need to construct hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12:725-734 (1993), naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self antigens without immunization.Finally, naive libraries can also be synthetically generated by cloning unrearranged V gene segments from stem cells, using PCR primers containing random sequences to encode hypervariable CDR3 regions, and achieving in vitro rearrangement, as described in Hoogenboom et al., J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Application Publication Nos. 2007 / 0292936 and 2009 / 0002360. Antibodies isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0238] As provided herein, a fusion protein, an antibody or antigen-binding fragment thereof, or a multispecific protein can comprise an antigen-binding domain that binds to a CNS antigen or a brain antigen. As provided herein, an antibody or antigen-binding fragment thereof, or a multispecific protein provided herein can comprise an antigen-binding domain that binds to a CNS antigen or a brain antigen. In some embodiments, the CNS antigen or brain antigen is beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretagogue receptor 2 (GSP2), or leucine-rich repeat kinase 2 (LRRK2). The antigen-binding domain may be an antigen-binding domain that binds to ribosomal kinase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid-binding Ig-like lectin 3 (Siglec3), transmembrane 4-domain A4A (MS4A4A), transmembrane 4-domain A6A (MS4A6A), or transmembrane protein 106B (TMEM106b).In some embodiments, the CNS or brain antigen is beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, beta-glucocerebromide (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), ubiquitin protein ligase E3A (UBE3A), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, serotonin receptor 1 (SPR1), serotonin receptor 2 (SPR2), serotonin receptor 1 (SPR3), serotonin receptor 2 (SPR4), serotonin receptor 1 (SPR5), serotonin receptor 2 (SPR6), serotonin receptor 1 (SPR7), serotonin receptor 1 (SPR8), serotonin receptor 1 (SPR1), serotonin receptor 2 (SPR1), serotonin receptor 1 ... Rutilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33, sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), sialic acid-binding Ig-like lectin 11 (Siglec11), glycoprotein nonmetastatic melanoma The protein may be protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4-domain A4A (MS4A4A), transmembrane 4-domain A6A (MS4A6A), MSA4A4E, transmembrane protein 106B (TMEM106b), CR1, ABCA1, ABCA7, HLA-DR1, HLA-DR5, IL1RAP, TREML2, IL-34, SORL1, and ADAM1.

[0239] In some embodiments, the CNS or brain antigen is on a cancer cell within the central nervous system. In some embodiments, the CNS or brain antigen is a cell surface target on a hematological cancer cell selected from B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b. In some embodiments, the CNS or brain antigen is selected from siglec-3 or CD33, siglec-5, siglec-7, siglec-9, siglec14, PILRA, IL18-BP, MerTK, ACKR1, ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM (CD66E), EGFR, EGFR viii, a tumor cell target selected from ETBR, FGFR(1-4), folate receptor alpha, GAL-3BP (galectin-binding protein), GD2, GD3, GloboH (globohexacylceramide), gp100, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine-rich repeat-containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4, colon cancer kinase), RON, ROR1, TF (tissue factor), and TROP2.

[0240] As provided herein, a fusion protein, an antibody or antigen-binding fragment thereof, or a multispecific protein can comprise an antigen-binding domain that binds to a CNS antigen or a brain antigen. As provided herein, a multispecific protein can comprise an antigen-binding domain that binds to a CNS antigen or a brain antigen. The antigen-binding domain that binds to a CNS antigen or a brain antigen can comprise a VH and a VL. Exemplary CNS antigen-binding VH and VL sequences are shown below. Additional VH and VL and antigen-binding domain sequences can be found in US2017 / 0224702, US2018 / 0002433, US2021 / 0236634, and US2021 / 0238265, each of which is incorporated by reference in its entirety.

[0241] Table 3: Exemplary CNS antigens TIFF2025528752000005.tif211165

[0242] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are capable of crossing the BBB as a result of the anti-TfR antigen-binding domain in the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein being able to cross the BBB.

[0243] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are internalized into blood-brain barrier epithelial cells at a rate greater than 10-fold compared to internalization by an isotype control. The blood-brain barrier endothelial cells may be, for example, HCMEC / D3 cells.

[0244] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein do not reduce cell surface expression of TfR on HCMEC / D3 cells by more than 60% compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein do not reduce cell surface expression of TfR on HCMEC / D3 cells by more than 40% compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, for example, using Western blot or FACS.

[0245] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein do not significantly increase cell surface expression of TfR on HCMEC / D3 cells compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, for example, using Western blot or FACS.

[0246] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein do not reduce cell surface expression of TfR on HCMEC / D3 cells by more than 60% compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control, and do not significantly increase cell surface expression of TfR on HCMEC / D3 cells compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, for example, using Western blot or FACS. In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein do not reduce cell surface expression of TfR on HCMEC / D3 cells by more than 40% compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control, and do not significantly increase cell surface expression of TfR on HCMEC / D3 cells compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, for example, using Western blot or FACS.

[0247] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein accumulate in the brains of vascular-depleted mice at least 4-fold more than an isotype control. In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein accumulate in the brains of vascular-depleted mice at least 5-fold more than an isotype control.

[0248] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein bind to human TfR with an equilibrium dissociation constant (K D In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein bind to human TfR with an equilibrium dissociation constant (K) of about 6.7 nM to about 340 nM. D In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein bind to the cynomolgus monkey TfR with a K of about 38 nM to about 2.3 μM. D In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein bind to the cynomolgus monkey TfR with a K of about 18 nM to about 870 nM. D In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein bind to human TfR with an equilibrium dissociation constant (K D ) and binds to cynomolgus monkey TfR with a K of about 38 nM to about 2.3 μM. D In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein bind to human TfR with an equilibrium dissociation constant (K D ) and binds to cynomolgus monkey TfR with a K of approximately 18 nM to approximately 870 nM. D Combine with.

[0249] Polynucleotide, method for producing anti-TfR antigen-binding domain, and agent containing same In some aspects, provided herein are polynucleotides comprising nucleotide sequences encoding antigen-binding domains that specifically bind to human TfR, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins described herein or domains thereof described herein, and vectors (e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells)).

[0250] In some embodiments, the polynucleotides provided herein comprise a nucleic acid molecule encoding a heavy chain of an antigen-binding domain that specifically binds to the human TfR provided herein. In some embodiments, the polynucleotides provided herein comprise a nucleic acid molecule encoding a light chain of an antigen-binding domain that specifically binds to the human TfR provided herein. In some embodiments, the polynucleotides provided herein comprise a nucleic acid molecule encoding a heavy chain of an antigen-binding domain that specifically binds to the human TfR provided herein and a nucleic acid molecule encoding a light chain of an antigen-binding domain that specifically binds to the human TfR provided herein.

[0251] In some embodiments, provided herein are polynucleotide combinations or compositions. In some embodiments, the combinations or compositions comprise a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first polynucleotide, the second polynucleotide, and the third polynucleotide encode a multispecific protein provided herein, e.g., the first polynucleotide encodes a first heavy chain, the second polynucleotide encodes a second heavy chain and an antigen-binding domain that specifically binds to the human TfR provided herein, and the third polynucleotide encodes a light chain. In some embodiments, the antigen-binding domain that specifically binds to the human TfR is an scFv. In some embodiments, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation. In some embodiments, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0252] In some embodiments, the combination or composition comprises a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first polynucleotide, the second polynucleotide, and the third polynucleotide encode a multispecific protein provided herein, wherein the first polynucleotide encodes a first heavy chain and a first antigen-binding domain that specifically binds to human TfR, the second polynucleotide encodes a second heavy chain and a third antigen-binding domain that specifically binds to human TfR, and the third polynucleotide encodes a light chain. In some aspects, the first and second antigen-binding domains that bind to human TfR comprise the same amino acid sequence. In some aspects, the first and second antigen-binding domains that bind to human TfR comprise different amino acid sequences. In some aspects, the first and second antigen-binding domains that bind to human TfR are scFvs. In some aspects, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation. In some embodiments, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0253] In some embodiments, the combination or composition comprises a first polynucleotide and a second polynucleotide, wherein the first and second polynucleotides encode a multispecific protein provided herein, and the first polynucleotide encodes a heavy chain and an antigen-binding domain that binds to a human TfR provided herein, and the second polynucleotide encodes a light chain.

[0254] Also provided herein are antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human TfR as described herein, or polynucleotides comprising nucleotide sequences encoding the domains described herein, that have been optimized, for example, by codon / RNA optimization, substitution with a heterologous signal sequence, and / or removal of mRNA instability elements. Methods for generating nucleic acids optimized for recombinant expression by introducing codon changes (e.g., codon changes that encode the same amino acid due to the degeneracy of the genetic code) and / or removing inhibitory regions within the mRNA can be performed by applying the optimization methods described, for example, in U.S. Patent Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498.

[0255] Polynucleotides comprising nucleotide sequences encoding antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human TfR, or domains thereof, as described herein, can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the nucleic acid art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain, for example, nucleic acids comprising sequences encoding the light and / or heavy chains of the antigen-binding domain, antibody, or antigen-binding fragment thereof. The amplified nucleic acid can be cloned into a vector for expression in a host cell and for further cloning, for example, to generate antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human TfR, or domains thereof, as described herein.

[0256] The polynucleotides provided herein may be, for example, in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and the DNA may be double-stranded or single-stranded. If single-stranded, the DNA may be the coding strand or the non-coding (antisense) strand. In some embodiments, the polynucleotide is a cDNA or DNA lacking one or more endogenous introns. In some embodiments, the polynucleotide is a non-naturally occurring polynucleotide. In some embodiments, the polynucleotide is recombinantly produced. In some embodiments, the polynucleotide is isolated. In some embodiments, the polynucleotide is substantially pure.

[0257] In some embodiments, the polynucleotides provided herein are in the form of RNA. In some embodiments, the polynucleotides provided herein are in the form of RNA encoding the fusion proteins provided herein. In some embodiments, the polynucleotides provided herein are synthetic messenger RNA (mRNA). In some embodiments, the synthetic mRNA has at least one nucleoside modification. In some embodiments, the at least one nucleoside modification is pyridin-4-one ribonucleoside, 5-azauridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- Taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydrosh Pseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine cytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-Methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza 8-Aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine Bamoyl adenosine, N6,N6-dimethyl adenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guano In some embodiments, the guanosine is selected from 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0258] In some aspects, provided herein are polynucleotides encoding fusion proteins comprising an antigen binding protein provided herein and a heterologous polypeptide. In some aspects, the heterologous polypeptide is selected from the group consisting of beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebroidase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotransmitter (NPN), and the like. The heterologous polypeptide comprises an antigen-binding domain that binds to lophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), glycoprotein nonmetastatic melanoma protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4-domain A4A (MS4A4A), transmembrane 4-domain A6A (MS4A6A), or transmembrane protein 106B (TMEM106b), or a portion thereof. In some embodiments, the heterologous polypeptide comprises an antigen-binding domain that binds to ubiquitin protein ligase E3A (UBE3A). In some aspects, provided herein are polynucleotides encoding the multispecific proteins provided herein.In some embodiments, the multispecific antibody targets a target protein selected from the group consisting of beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, beta-glucocerebromide (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotransmitter (NPN), and / or NP-1 receptor 2 (NP-1 receptor 2). In some embodiments, the multispecific protein comprises an antigen-binding domain that binds to trophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), glycoprotein nonmetastatic melanoma protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4-domain A4A (MS4A4A), transmembrane 4-domain A6A (MS4A6A), or transmembrane protein 106B (TMEM106b), or a portion thereof. In some embodiments, the multispecific protein comprises an antigen-binding domain that binds to ubiquitin protein ligase E3A (UBE3A). In some embodiments, the polynucleotide is mRNA (eg, synthetic mRNA).

[0259] In some embodiments, the present disclosure provides a polynucleotide encoding a fusion protein disclosed herein that comprises a heterologous polypeptide. In some aspects, the heterologous polypeptide is an ERT enzyme or an ERT enzyme variant, or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide comprises β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide in the fusion protein provided herein is a growth factor. In some aspects, the heterologous polypeptide in the fusion protein provided herein is a decoy receptor. In some aspects, the heterologous polypeptide in the fusion protein provided herein is progranulin (PGRN), prosaposin (PSAP), or motor neuron survival protein (SMN). In some embodiments, the heterologous protein is selected from the group consisting of α-L iduronidase (IDUA), iduronate-2-sulfatase (IDS), N-acetylgalactosamine-6-sulfatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramideThe enzyme is selected from beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta-hexosaminidase B, arylsulfatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme substitute thereof, such as larosinase, idursulfase, elosulfase alpha or galsulfase, or a variant thereof, or a catalytically active fragment thereof. In some embodiments, the heterologous protein is selected from the group consisting of clusterin (APOJ), reelin, ubiquitin protein ligase E3A (UBE3A), tripeptidyl peptidase 1 (CLN2 / TPP1), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), α- L-iduronidase (IDUA), iduronate-2-sulfatase (IDS), N-acetylgalactosamine-6-sulfatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramidebeta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta-hexosaminidase B, arylsulfatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, their synthetic enzyme substitutes, e.g., larodinase, idursulfase, elosulfase alpha or galsulfase, or variants thereof, having catalytic activity These fragments include α-L iduronidase (IDUA), iduronate-2-sulfatase (IDS), N-acetylgalactosamine-6-sulfatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), and galactosylceramide. The polynucleotide is an enzyme selected from beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta-hexosaminidase B, arylsulfatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme substitute thereof, such as larodinase, idursulfase, elosulfase alpha, or galsulfase, or a variant or catalytically active fragment thereof. In some embodiments, the polynucleotide is an mRNA (e.g., a synthetic mRNA).

[0260] In certain aspects, provided herein are vectors (e.g., expression vectors) for recombinant expression in a host cell (e.g., a mammalian host cell) comprising a polynucleotide comprising a nucleotide sequence encoding an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human TfR described herein, or a domain thereof described herein. Vectors for producing an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human TfR described herein, or a domain thereof described herein, can be made, for example, by recombinant DNA technology using techniques well known in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors operably linked to a promoter comprising a nucleotide sequence encoding an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human TfR described herein, or a domain thereof described herein. Such vectors can include, for example, nucleotide sequences encoding an antigen-binding domain, a constant region of an antibody or antigen-binding fragment thereof (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036, and U.S. Pat. No. 5,122,464), and a variable domain of an antigen-binding domain, an antibody or antigen-binding fragment thereof; such nucleotide sequences can be cloned into such vectors to express an entire heavy chain, an entire light chain, or an entire heavy and light chain. In some embodiments, the vector is a gene therapy vector (e.g., an AAV or lentiviral vector).

[0261] In certain aspects, provided herein are expression systems comprising a polynucleotide comprising a nucleotide sequence encoding an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human TfR as described herein, or a domain thereof as described herein. The expression system can be contained in a vector. The expression system can also be integrated into the chromosome of a host cell. In some aspects, the expression system is a cell-free expression system. In some aspects, the expression system comprises a host cell comprising a polynucleotide and / or vector provided herein.

[0262] Accordingly, also provided herein are cells, e.g., host cells, comprising polynucleotides and / or vectors for recombinantly expressing an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human TfR as described herein, or a domain thereof as described herein. In some embodiments, to express a two-chain antigen-binding protein, vectors encoding the heavy and light chains separately can be co-expressed in the host cell to express the entire immunoglobulin. In some embodiments, the host cell comprises two different vectors: a first vector comprising a polynucleotide encoding the heavy chain of the antigen-binding protein described herein, and a second vector comprising a polynucleotide encoding the light chain of the antigen-binding protein. In some embodiments, a first host cell comprises a first vector comprising a polynucleotide encoding the heavy chain, and a second host cell comprises a second vector comprising a polynucleotide encoding the light chain. In some embodiments, a population of host cells comprising such a first host cell and such a second host cell is provided herein.

[0263] In some embodiments, provided herein are methods for producing an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human TfR as described herein, or a domain thereof as described herein, in a host cell. In some aspects, provided herein are methods for producing a single-chain antigen-binding domain that specifically binds to human TfR, an Fc domain, and a heterologous protein or polypeptide. In some aspects, provided herein are methods for producing a single-chain antigen-binding domain, an Fc domain, and a second antigen-binding domain that specifically binds to human TfR as described herein. The expression vector can be introduced into cells (e.g., host cells) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human TfR as described herein, or a domain thereof as described herein.

[0264] A variety of host-expression vector systems can be utilized to express the antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human TfR described herein, or domains thereof described herein (see, e.g., U.S. Patent No. 5,807,715). Such host-expression systems represent not only carriers in which coding sequences of interest can be produced and subsequently purified, but also cells that, when transformed or transfected with the appropriate nucleotide coding sequences, are capable of expressing the antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human TfR described herein, or domains thereof described herein.These host expression systems include, but are not limited to, bacteria (e.g., Escherichia coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; and plant cell systems (e.g., Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the coding sequences. reinhardtii); or mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, and NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells) harboring recombinant expression constructs containing promoters derived from the genomes of mammalian cells (e.g., metallothionein promoters) or mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters). In some embodiments, the cells for expressing an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human TfR described herein, or a domain thereof described herein, are CHO cells, e.g., CHO cells derived from CHO GS System™ (Lonza). In some embodiments, the cells for expressing an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human TfR described herein, or a domain thereof described herein, are human cells, e.g., human cell lines.In some embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In some embodiments, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells) are used to express the antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to the human TfR described herein, or domains thereof described herein. For example, mammalian cells such as Chinese hamster ovary cells (CHO) in combination with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus are effective expression systems for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-105; and Cockett MI et al., (1990) Biotechnology 8: 662-667). In some embodiments, an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human TfR as described herein, or a domain thereof as described herein, is produced by a CHO cell or an NSO cell.

[0265] In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product may contribute to the function of the protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells.

[0266] Once an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to the human TfR described herein, or domains thereof as described herein, is produced by recombinant expression, it can be purified by any purification method known in the art, such as chromatography (e.g., ion exchange, affinity, particularly affinity for a particular antigen following Protein A, and size exclusion column chromatography), centrifugation, differential solubility, or other standard methods for purifying proteins. Additionally, purification can be facilitated by fusing an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to the human TfR described herein, or domains thereof as described herein, to a heterologous polypeptide sequence.

[0267] In some embodiments, the antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human TfR described herein are isolated or purified. Generally, isolated or purified antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human TfR described herein are substantially free of other proteins. For example, in some embodiments, preparations of antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human TfR described herein are substantially free of cellular material and / or chemical precursors.

[0268] Pharmaceutical Composition Provided herein are compositions comprising an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human TfR described herein. In some embodiments, the antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human TfR with the desired degree of purification is present in a formulation containing, for example, a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed. Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives.

[0269] In some embodiments, the pharmaceutical composition comprises an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human TfR as described herein and a pharmaceutically acceptable carrier (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). In some embodiments, the pharmaceutical compositions described herein are used as medicaments. Compositions used for in vivo administration can be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes.

[0270] Also provided herein are pharmaceutical compositions comprising a polynucleotide encoding an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human TfR as described herein. In some embodiments, the polynucleotide is RNA. In some embodiments, the polynucleotide is synthetic mRNA. In some embodiments, the polynucleotide is modified mRNA. In some embodiments, the pharmaceutical composition comprising the polynucleotide further comprises a lipid-based transfection reagent.

[0271] The pharmaceutical compositions described herein can be used to produce a biological effect(s) in vivo or in vitro, for example, to cross the blood-brain barrier in a subject.

[0272] In some embodiments, the pharmaceutical compositions provided herein are used to treat diseases or conditions such as neurological disorders, neurodegenerative diseases, cancer, ophthalmic disorders, seizure disorders, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, and CNS inflammation. In some embodiments, the pharmaceutical compositions provided herein are used to treat diseases or conditions such as Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, and traumatic brain injury. In some embodiments, the pharmaceutical compositions provided herein are used to treat frontotemporal dementia.

[0273] In some aspects, the pharmaceutical compositions provided herein are formulated for intravenous administration. In some aspects, the pharmaceutical compositions provided herein are formulated for subcutaneous administration.

[0274] Methods of using anti-TfR antigen-binding domains and agents containing same The antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, and multispecific proteins comprising such antigen-binding domains provided herein can advantageously be transported across the blood-brain barrier. Accordingly, provided herein are methods for administering or transporting an antigen-binding protein that specifically binds to human TfR, or a fusion protein comprising an antigen-binding protein that specifically binds to human TfR, an antibody, antigen-binding fragment thereof, or a multispecific protein across the blood-brain barrier in a subject, the methods comprising administering to the subject an antigen-binding protein that specifically binds to human TfR, or a fusion protein comprising an antigen-binding protein that specifically binds to human TfR, an antibody, antigen-binding fragment thereof, or multispecific protein.

[0275] Given the ability of the antigen-binding domains and fusion proteins, antibodies, antigen-binding fragments thereof, and multispecific proteins comprising such antigen-binding domains provided herein to be transported across the blood-brain barrier, they can be used to treat neurological diseases or disorders. In some embodiments, a method of treating a neurological disease or disorder in a subject comprises administering to the subject an antigen-binding protein that specifically binds to human TfR or a fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein comprising an antigen-binding protein that specifically binds to human TfR. In some embodiments, the neurological disease or disorder can be a disease or condition such as, for example, a neuropathic disorder, a neurodegenerative disease, cancer, an ocular disorder, a seizure disorder, a lysosomal storage disorder, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation. Neurological diseases or disorders include, for example, neurodegenerative diseases (Lewy body disease, post-poliomyelitis syndrome, Shy-Drager syndrome, oligocerebellar atrophy, Parkinson's disease, Gaucher disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), tauopathies (such as Alzheimer's disease and supranuclear palsy), prion diseases (bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), ophthalmoplegia, motor neuron diseases, heterogeneous degenerative disorders of the nervous system (e.g., Canavan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette's syndrome, Metformin-resistant Staphylococcus aureus, and others), and neurodegenerative disorders (e.g., encephalopathy ... The cause may be dementia (such as Pick's disease, spinocerebellar ataxia), cancer of the CNS and / or brain (such as glioblastoma or brain metastases resulting from cancer elsewhere in the body), Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), limbic-predominant age-related TDP-43 encephalopathy (LATE), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, or traumatic brain injury.In some aspects, the neurological disease or disorder is dementia. In some aspects, the neurological disease or disorder is frontotemporal dementia. In some embodiments, the neurological disease or disorder is Alzheimer's disease. In some embodiments, the neurological disease or disorder is Parkinson's disease. In some aspects, the neurological disease or disorder is frontotemporal epilepsy. In some embodiments, the neurological disease or disorder is autism. In some aspects, the neurological disease or disorder is lissencephaly.

[0276] In some aspects, provided herein are methods of treating a lysosomal storage disease with the fusion proteins disclosed herein, hi some aspects, the lysosomal storage disease is selected from Gaucher disease, ceroid lipofuscinosis (Batten disease), mucopolysaccharidosis (MPS) type I, MPS type II, and MPS type III.

[0277] The antigen-binding domains and fusion proteins, antibodies, antigen-binding fragments thereof, and multispecific proteins comprising such antigen-binding domains provided herein that specifically bind to human TfR can be used to detect antigens (e.g., CNS antigens or brain antigens). For such purposes, the antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, and multispecific proteins comprising such antigen-binding domains that specifically bind to human TfR can be labeled. Exemplary labels include, for example, radioisotopes (e.g., 64Examples of suitable antibodies include CU) and fluorescent labels. Accordingly, provided are methods of detecting an antigen using an antigen-binding protein that specifically binds to human TfR, or a fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein comprising an antigen-binding protein that specifically binds to human TfR. In some aspects, a method of detecting an antigen in the CNS (e.g., brain) of a subject comprises administering an antigen-binding protein that specifically binds to human TfR, or a fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein comprising an antigen-binding protein that specifically binds to human TfR, to the antigen in the CNS (e.g., brain). Such methods may further comprise, for example, performing positron emission tomography (PET) imaging on the subject. In some aspects, disclosed herein are methods of detecting a CNS antigen in vitro, comprising contacting a sample in vitro with a fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein disclosed herein, and locating an imaging agent in the sample.

[0278] The antigen-binding domains that specifically bind to human TfR, as well as fusion proteins, antibodies, antigen-binding fragments thereof, and multispecific proteins comprising such antigen-binding domains described herein, can be used for prognostic, diagnostic, monitoring, and / or screening applications, including in vivo applications that are well known and standard to those of skill in the art, including applications based on the present disclosure. In some aspects, provided herein are antigen-binding proteins that specifically bind to human TfR, or fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins comprising antigen-binding proteins that specifically bind to human TfR, for use in diagnosis. In some aspects, the antigen-binding proteins that specifically bind to human TfR, or fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins comprising antigen-binding proteins that specifically bind to human TfR, comprise a detectable label.

[0279] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0280] The present disclosure will be better understood by reference to the following examples, which should not be construed as limiting the scope of the disclosure. All citations throughout this disclosure are expressly incorporated herein by reference. [Example]

[0281] Example 1: Generation of Avi-His-tagged variants of the transferrin receptor (TfR) Mammalian expression of human, cynomolgus monkey (cyno), and mouse variants of the transferrin receptor antigen (SEQ ID NOS: 1-7) was achieved by cloning cDNA-based synthetic genes into mammalian expression vectors followed by transient transfection and expression in Expi293 cells. The constructs included a heterologous signal peptide and a C-terminal Avi-His tag to enable purification and biotinylation. Briefly, antigen-encoding plasmids were transfected using the Expifectamine 293 Transfection Kit (ThermoFisher A14524) according to the manufacturer's specifications. Five days after transfection, culture supernatants were harvested, clarified by centrifugation, and purified in a drip column format using HisPur Ni-NTA resin (Thermo Scientific 88223). 200 mL of culture supernatant was filtered using a 0.2 μm filtration unit, and 3 mL of resin slurry in PBS was added to the filtered supernatant. The sample was incubated overnight with shaking at 4°C. The next day, the beads were loaded onto a 20 mL drip column, washed with 10 mL of His-Select Wash Buffer (Millipore Sigma H5288), and eluted with 5 mL of His-Pur Elution Buffer (Millipore Sigma H5413). The eluate was buffer-exchanged into PBS using an Amicon Ultra-15 centrifugal filter unit (Millipore UFC9010). Antibody concentration was quantified by measuring absorbance at 280 nm using a Nanolop 8000 (ThermoFisher). Antigen purity was measured by SDS-PAGE. Antigens were analyzed for aggregation by size-exclusion chromatography (SEC). A portion of the antigen was biotinylated using the BirA Biotin-Protein Ligase Kit (AVIDITY) according to the manufacturer's instructions.

[0282] The apical domain of TfR provides a means for viruses (Helguera, G., et al., Virology 86(7): 4024-4028 (April 2012)) and antibodies (Kariolis M., et al., Sci. Transl. Med. 12(545):eaay1359 (May 2020)) to enter endothelial cells without compromising the receptor's native function. Tagged variants of the apical domain of TfR were generated. These constructs contained full-length human TfR sequence amino acids 69-263 with either a C-terminal Avi-His tag (SEQ ID NO: 4) or a C-terminal myc-V5-His tag (SEQ ID NO: 5) (sequences in Table 4 below). The resulting clones were expressed in Expi293 cells and purified using HisPur Ni-NTA resin as described above.

[0283] DNA fragments encoding permuted forms of the apical domains and N-terminal His / Avi tags of human TfR (huTfR) and cynomolgus monkey TfR (cynoTfR) were ordered from GeneArt and cloned into the pcDNA3.4 vector (SEQ ID NOs: 6 and 7). The resulting clones were expressed in Expi293 cells and purified by Ni-NTA agarose (QIAGEN 30230) using the manufacturer's protocol.

[0284] Table 4. Avi-His-tagged variants of TfR TIFF2025528752000006.tif170165TIFF2025528752000007.tif214165

[0285] Example 2: Generation of a CHO cell line overexpressing TfR Several cell lines were generated to screen the binding of anti-TfR antibodies obtained as described herein. Briefly, CHO cells stably expressing human and mouse TfR were generated using the pLenti-EF1a construct, which expresses full-length human and mouse TfR (SEQ ID NOs: 8 and 9, respectively). Lentivirus constructs (Genecopoia) were used to express human TfR with hygromycin selection and mouse TfR with hygromycin selection. Lentivirus was generated by transfection of 293T cells using the ViraSafe™ Lentiviral Packaging System (CellBiolabs VPK-206). The lentivirus-containing supernatant was then used to transduce Chinese hamster ovary (CHO) cells. Two days after transduction, puromycin or hygromycin was added to the medium as selection pressure for human and mouse TfR. The resulting CHO cells stably expressing human and mouse TfR were analyzed for cell surface expression by flow cytometry.

[0286] Table 5. Full-length TfR sequences TIFF2025528752000008.tif190165

[0287] Example 3: Generation of TfR-humanized mice To humanize the extracellular domain of TfR, a mouse strain was generated at Taconic Biosciences GmbH (Germany). CRISPR was used to replace the mouse ECD with a human one under the control of a mouse promoter, while retaining the mouse intracellular and transmembrane segments. Brain sections from these mice were evaluated by IHC and / or Western blot to confirm the expression and localization of the human ECD in vivo (data not shown).

[0288] Example 4: Production of anti-TfR hybridoma antibodies To obtain antibodies against TfR, hybridomas were generated using the following procedure: BALB / c mice or Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) were immunized twice weekly by subcutaneous or intraperitoneal injection of purified extracellular domain polypeptides of human, cynomolgus monkey, and / or mouse TfR (obtained as described above in Example 1) with or without adjuvant. After a total of 7 to 8 injections and 3 days after the final boost, lymph nodes were harvested from the mice or rats for hybridoma cell line generation.

[0289] Sera from animals were analyzed for reactivity to TfR by FACS against CHO cells overexpressing human or mouse TfR and by ELISA against the Avi-His proteins of human, cynomolgus monkey, and mouse TfR (Example 1). Lymphocytes from animals whose serum showed strong binding to CHO cells overexpressing human or mouse TfR were isolated and fused with SP2 / mIL-6 (CRL-2016, American Type Culture Collection, Rockwill, MD) or Sp2ab (ENZ-70008, Enzo Life Sciences, Farmingdale, NY) mouse myeloma cells by electrofusion (Hybrimune, BTX, Holliston, MA) and incubated overnight at 37°C and 5% CO in Clonacell-HY medium C (Stemcell Technologies, Vancouver, BC, Canada, catalog number 03803).

[0290] The next day, the fused cells were centrifuged and resuspended in Clonacell-HY medium E (Stemcell Technologies, catalog number 03805) with the addition of HAT (Sigma Aldrich, catalog number H0262). Cells were seeded into T225 flasks and grown at 37°C and 5% CO for 7 days, after which the hybridoma library was cryopreserved. The library was then thawed and allowed to recover overnight in Clonacell-HY medium E. The next day, IgG-positive hybridomas were single-cell sorted into Falcon 96-well U-bottom plates using a FACS Aria II cell sorter (BD Biosciences, San Jose, CA). 768 hybridomas were sorted from mouse fused cells, and 768 hybridomas were sorted from rat fused cells. Cells were grown in 200 ul of Clonacell-HY medium E at 37°C under 5% CO for 11 days, after which tissue culture supernatants from each well were screened by FACS against CHO cells overexpressing human or mouse TfR (described below).

[0291] Example 5: Screening of anti-TfR antibody hybridoma supernatants by FACS A total of 1536 anti-TfR hybridoma supernatants were initially screened by FACS for their ability to differentially bind to CHO cells overexpressing human or mouse TfR compared to parental CHO cells. Overexpressing cells were harvested, washed, and labeled with Pacific Blue, FITC, and Pacific Orange dyes (ThermoFisher) to generate uniquely barcoded cell populations. Barcoded cells (5 × 10 of each cell population) were then isolated and labeled with Pacific Blue, FITC, and Pacific Orange dyes (ThermoFisher). 4Cells (1000 cells / ml) were dispensed into a 96-well U-bottom plate and incubated with 50 μl of hybridoma cell culture supernatant or 1 μg / ml of commercially available purified mouse anti-human TfR monoclonal antibody (Sigma Aldrich, Catalog No. SAB4700515) and rat anti-mouse TfR antibody (Invitrogen, Waltham, MA, Catalog No. R17217) on ice for 30 minutes. After this initial incubation, the supernatant was removed by centrifugation, and the cells were washed twice with 175 μl of ice-cold FACS buffer (PBS + 1% FBS + 2 mM EDTA). Then, the cells were incubated with anti-mouse IgG Fc-allophycocyanin (APC) or anti-rat IgG Fc-APC (Jackson Labs, Catalog Nos. 115-136-071 and 112-136-071, respectively) (diluted 1:1000) on ice for 20 minutes. After this secondary antibody incubation, cells were washed twice again with ice-cold FACS buffer and resuspended in FACS buffer to a final volume of 50 μl. Cell binding intensity was analyzed using a FACS Canto system (BD Biosciences), and the ratio of APC mean fluorescence intensity (MFI) for each barcoded cell population was determined for each anti-TfR hybridoma supernatant tested.

[0292] A total of 189 mouse hybridoma and 115 rat hybridoma clones showed a greater than three-fold difference in binding (as measured by MFI) to CHO cells stably overexpressing the human TfR compared to the isotype control. A total of 15 rat hybridomas showed a greater than two-fold difference in binding to CHO cells stably overexpressing the mouse TfR compared to the isotype control (data not shown). None of the hybridoma clones showed cross-reactivity to the human and mouse TfR by FACS.

[0293] Table 6. FACS results of selected mouse hybridoma supernatants (shown as fold increase over CHO parent line) TIFF2025528752000009.tif191165

[0294] Table 7. FACS results of selected rat hybridoma supernatants (shown as fold increase over CHO parent line) TIFF2025528752000010.tif179165

[0295] Example 6: Screening of anti-TfR antibody hybridoma supernatants by recombinant anti-TfR protein binding assay Hybridoma culture supernatants from 319 hybridomas obtained as described above were screened for their ability to bind to Avi-His-tagged huTfR ECD, huTfR apical domain, and muTfR ECD, compared to binding to an irrelevant Avi-His-tagged control protein (as described in Example 1). Briefly, 96-well polystyrene plates were coated overnight at 4°C with 5 μg / ml streptavidin (Thermo Fisher, catalog number PI21125) in coating buffer (0.05 M carbonate buffer, pH 9.6, Sigma, catalog number C3041). The coated plates were then blocked for 1 hour with ELISA diluent (PBS + 0.5% BSA + 0.05% Tween 20). The blocking buffer was removed, and Avi-His-tagged human TfR, human TfR apical domain, mouse TfR, and an unrelated Avi-His-tagged protein were added at 1 μg / ml in ELISA diluent and captured for 1 hour at room temperature. After washing three times with 300 μl of PBST (PBS + 0.05% Tween 20, Thermo 28352), hybridoma cell culture supernatant was added to each well (50 μl / well). After incubation for 30 minutes at room temperature, each plate was washed three times with 300 μl of PBST. Anti-mouse IgG Fc-HRP or anti-rat IgG Fc-HRP (Jackson Immunoresearch, catalog numbers 115-035-071 and 112-036-071, respectively) secondary antibodies were diluted 1:5000 in ELISA diluent and added to each well at 50 μl / well. The plates were incubated for 30 minutes at room temperature with shaking. After one final set of washes (3 × 300 μl in PBST), 50 μl / well of BioFx TMB substrate (Surmodics, catalog no. TMBW-1000-01) was added to the wells. The reaction was then quenched after 5–10 min with 50 μl / well of 2N sulfuric acid. The absorbance of each plate at 450 nm was read on a SpectraMax M5 (Molecular Devices, Sunnyvale, CA) using SoftMax Pro software.

[0296] Screening of this hybridoma supernatant identified a total of 112 anti-TfR hybridoma clones that showed a greater than 5-fold difference in binding to recombinant huTfR Avi-His compared to background, and 86 of these anti-TfR hybridoma clones also bound to the apical domain of huTfR. Binding data (shown as ratios of binding to recombinant huTfR Avi-His relative to an unrelated protein) for selected mouse and rat anti-TfR hybridoma clones are shown in Tables 8 and 9, respectively.

[0297] Table 8. ELISA (OD450) results of selected mouse hybridoma supernatants (shown as fold increase over an unrelated protein) TIFF2025528752000011.tif192165

[0298] Table 9: ELISA (OD450) results of selected rat hybridoma supernatants (shown as fold increase over an unrelated protein) TIFF2025528752000012.tif179165

[0299] Example 7: In vitro internalization of anti-TfR antibodies into blood-brain barrier endothelial cell lines Anti-TfR hybridoma antibodies were purified and screened for their ability to internalize into the hCMEC / D3 cell line.

[0300] Supernatants from anti-TfR hybridoma clones were purified using a ProPlus Phylip column (Biotage, Uppsala, Sweden, catalog number PTH 91-20-07) on a Hamilton STAR platform (Hamilton Company, Reno, NV). Briefly, antibodies from the supernatants were captured by Protein A coupled to a resin-packed tip, washed twice with PBS, eluted with Pierce IgG elution buffer (ThermoFisher, catalog number 21004), and neutralized to a final pH of 6.0 with 1 M Tris-HCl pH 8. The concentration of the purified antibodies was determined by measuring absorbance at 280 nm using a Nanolop 8000 (ThermoFisher). Hybridoma-purified antibodies were then tested for their ability to internalize.

[0301] Internalization into blood-brain barrier endothelial cells is the first step in transcytosis, leading to translocation across the BBB into the brain. To identify anti-TfR antibodies with internalization ability, HCMEC / D3 cells were seeded at 2.5 × 10^4 cells / well in black-walled, clear-bottom 96-well plates (#3904, Corning). The next day, cells were treated with 6 μg / ml of anti-TfR antibodies pre-bound to an equal concentration of pHrodo-Red labeling reagent (Z25612, Invitrogen) in 100 μl of culture medium (EBM2, Lonza). Anti-TfR antibodies of human IgG isotype and known internalization ability were included in the assay as negative and positive controls, respectively.

[0302] Each plate was then placed in an IncuCyte machine (live cell analysis system) and images were taken every 2 hours for 24 hours. Images were then processed and analyzed using IncuCyte software. Internalization data (pHrodo Red-positive area μm) at the 24 hour time point were 2 / image) are summarized in Table 10 below as relative fold change to isotype hIgG1.

[0303] Data were obtained as total red area per well and are presented in Table 10 as fold change relative to an irrelevant mouse IgG antibody used as a negative control. The assay also included an anti-TfR antibody used as a positive control.

[0304] Table 10. Internalization of purified anti-TfR hybridoma antibodies TIFF2025528752000013.tif231165

[0305] Example 8: Molecular cloning of anti-TfR antibodies The anti-TfR antibodies obtained from the hybridomas described above were cloned as follows: 1–2 × 10 5 Hybridoma cells were harvested, washed with PBS, and resuspended in 200 μl of RNAlater (Invitrogen, catalog no. AM7021). Samples were stored at -80°C and sent to Abterra Biosciences (San Diego, CA) for sequencing. Briefly, RNA was extracted and cDNA synthesis was performed. The variable regions of IgG / IgM, IgK, and IgL were amplified using proprietary primers in a 5' RACE assay. Hybridoma variable region amplicons were sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA). Reads from the hybridomas were processed through Abterra's Reptor analysis pipeline.

[0306] A total of 32 unique sequences were identified from 72 apical domain-positive anti-TfR hybridoma clones. The amino acid sequences of the variable heavy and light chains of unique mouse-derived apical domain-binding anti-TfR antibodies are shown in Table 11 below. An additional 21 unique sequences were identified from 24 rat-derived apical domain-positive anti-TfR hybridoma clones.

[0307] Table 11. Sequences of anti-huTfR apical domain antibodies derived from mouse hybridomas TIFF2025528752000014.tif137165TIFF2025528752000015.tif238165TIFF20255287520 00016.tif238165TIFF2025528752000017.tif238165TIFF2025528752000018.tif228165

[0308] Table 12. Sequences of anti-huTfR apical domain antibodies from mouse hybridomas (Kabat) TIFF2025528752000019.tif215165TIFF2025528752000020.tif232165TIFF2025528752000021.tif226165TIFF2025528752000022.tif135165

[0309] Table 13. Sequences of anti-huTfR apical domain antibodies from mouse hybridomas (Kabat) TIFF2025528752000023.tif215165TIFF2025528752000024.tif232165TIFF2025528752000025.tif232165TIFF2025528752000026.tif135165

[0310] Example 9: Reformatting of hybridoma antibodies as 2+1 bispecific antibodies Antibodies for formatting into a 2+1 bispecific format were selected based on several criteria: 1) antibodies covering a wide range of affinities based on ELISA and FACS binding assays, 2) antibodies covering a wide range of internalization activities, 3) no particularly high-risk modification sites identified within the CDRs, and 4) antibodies phylogenetically diverse from each other within the resulting hybridoma sequences.

[0311] A non-target specific IgG isotype antibody ("inactive isotype control antibody") with knob-and-hole mutations in the heavy chain constant domain was used to format a 2+1 bispecific antibody. The anti-TfR scFv (VH and VL) was attached to the IgG isotype antibody via a linker at the C-terminus of the constant domain with the "hole" mutation (the "hole" side of the IgG isotype antibody). The IgG isotype antibody conjugated to the scFv is referred to as a 2+1 bispecific antibody, as shown in Figure 1A and Figure 1D (i). The 20-amino acid linker connecting the VH and VL domains within the scFv was GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 183) (Bird et al., Science 1988. Oct 21;242(4877):423-6), and the linker connecting the C-terminus of the Fc "hole" domain to the scFv was (GGGGS)x3 (SEQ ID NO: 184). DNA encoding the 2+1 bispecific antibody was prepared by gene synthesis and cloned into the expression vector pcDNA3.4 (ThermoFisher).

[0312] The scFv (VH and VL) sequences were formatted into expression constructs for IgG heavy chains with "hole" mutations ("heavy chain hole" constructs) using the framework of SEQ ID NO: 185. In some cases, the "heavy chain hole" constructs contained a series of mutations (H435R, Y436F) to minimize binding to Protein A (Tustian et al., MAbs. May-Jun 2016;8(4):828-38) (SEQ ID NO: 186). Corresponding heavy chain knob and light chain expression constructs were also generated (SEQ ID NOs: 187-188). In some cases, the heavy chain hole and heavy chain knob also contained mutations to reduce effector function (SEQ ID NOs: 189-194).

[0313] An exemplary structure of the 2+1 bispecific antibody included the following elements: (1) an isotype control hIgG1 wild-type antibody with a knob (T366W) mutation and a hole (T366S_L368A_Y407V) mutation in the constant region, (2) a (G4S) x 3 linker between the "hole" side of the hIgG1 antibody and the scFv, (3) the VH sequence of the scFv, (4) a 20 amino acid linker sequence between the VH and VL of the scFv, and (5) the VL sequence of the scFv.

[0314] Table 14: Sequences of 2+1 bispecific antibodies TIFF2025528752000027.tif204165TIFF2025528752000028.tif237165TIFF2025528752000029.tif234165 TIFF2025528752000030.tif237165TIFF2025528752000031.tif178165TIFF2025528752000032.tif213165

[0315] To generate the 2+1 bispecific antibody, transient transfection of Expi293 cells (Invitrogen) was performed according to the manufacturer's instructions. For a 20 mL culture, 20 μg of total DNA consisting of three expression plasmids (heavy chain-hole-scFv sequence, heavy chain-knob sequence, and light chain sequence) was used. The molar ratio of heavy chain-knob, heavy chain-hole-scFv, and light chain plasmids was optimized to 1:3:6 to obtain high purity of the 2+1 bispecific antibody. Five days after transfection, cell culture supernatant was collected. The clarified supernatant was purified using a drip column containing MabSelect Sure resin (Cytiva), washed with PBS, eluted with pH 3.5 elution buffer, and neutralized with Tris-HCl to a final pH of 5.5-6.0. The neutralized eluate containing the antibody was dialyzed into PBS. Quantification of antibody concentration was determined by measuring absorbance at 280 nm using a Nanodrop 8000 (ThermoFisher) or Lunatic (Unchained Labs). Purity of the 2+1 bispecific antibodies was determined by SDS-PAGE. The 2+1 bispecific antibodies were analyzed for aggregation by size exclusion chromatography (SEC). Antibodies were then produced as 2+1 bispecific antibodies, purified, and analyzed by ELISA, FACS, and internalization to confirm retention of binding in the 2+1 format.

[0316] Example 10: Generation and characterization of 2+1 anti-TfR bispecific antibodies Anti-TfR hybridoma antibodies were converted to a 2+1 format, expressed, and purified as described in Example 9. Retention of binding was confirmed by ELISA and FACS.

[0317] To measure the binding retention of the reformatted scFv2+1 anti-TfR antibody by ELISA, high-binding ELISA plates (Thermo Fisher) were coated with streptavidin (1 μg / ml, Thermo Fisher) overnight at 4°C. Plates were then washed three times with phosphate-buffered saline containing 0.05% Tween-20 (PBST) and incubated with blocking buffer (PBS + 1% BSA, 1 h, RT). Plates were washed three times again with PBST and incubated with biotinylated human TfR, cynomolgus monkey TfR, and mouse TfR, as well as the irrelevant antigen, human transferrin receptor (1 μg / ml, 1 h, RT). Plates were washed three times again with PBST and incubated with anti-TfR antibody (1 μg / ml, 1 h, RT). Plates were washed again three times with PBST and incubated with a secondary detection antibody, anti-human horseradish peroxidase-conjugated antibody (dilution 1:5000, 30 min, room temperature). Plates were washed three times for the final time and incubated with TMB-ELISA substrate solution (Thermo Fisher) followed by quenching with H2SO4. Anti-TfR binding was then monitored by optical density (OD) at 450 nm using a SpectraMax M5 (Molecular Devices). 450 The binding characteristics of scFv2+1 anti-TfR antibodies to TfR are summarized in Table 15 below as fold changes compared to isotype hIgG.

[0318] To determine the binding retention of the reformatted 2+1 scFv anti-TfR antibody by FACS, the antibody was evaluated for binding to HCMEC / D3 cells (a cell line derived from human brain endothelial cells), CHO cell lines overexpressing human and mouse TfR, and CHO cells for nonspecific binding. Cells were seeded (50,000 cells / well) in tissue culture plates, washed, and resuspended in FACS buffer (PBS + 2% BSA + 1 mM EDTA). The anti-TfR antibody was then incubated with the cells (5 μg / ml, 1 hour on ice) and washed twice with FACS buffer. The cells were then incubated with allophycocyanin-conjugated anti-mouse secondary antibody (Jackson Immunoresearch) (1:1000, 30 minutes on ice) and then washed twice with FACS buffer. The cells were then resuspended in FACS buffer supplemented with 0.5% propidium iodide (Thermo Fisher). Anti-TfR binding was then analyzed by measuring mean fluorescence intensity (MFI) on an iQue flow cytometer (IntelliCyt). FACS data are summarized in Table 15 as fold change in MFI compared to isotype hIgG1.

[0319] In total, 28 of the 32 reformatted antibodies retained binding affinity to TfR in the 2+1 format (Table 15). Importantly, only 6 of the 32 antibodies (TfR6, TfR9, TfR12, TfR15, TfR19, TfR27) showed strong cynomolgus monkey cross-reactivity and therefore only these were considered for further maturation.

[0320] Table 15. Binding properties of anti-TfR antibodies reformatted in a 2+1 bispecific format TIFF2025528752000033.tif92165TIFF2025528752000034.tif160165

[0321] Example 11: In vitro internalization of 2+1 anti-TfR specific antibodies into BBB endothelial cell lines Internalization of the 2+1 anti-TfR bispecific antibodies into the hCMEC / D3 cell line was performed as previously described (Example 7). Each 2+1 anti-TfR bispecific antibody demonstrated broad internalization capacity, with many demonstrating significantly higher internalization than an anti-TfR control antibody.

[0322] Table 16. Internalization properties (fold change in integrated intensity / phase area per well) of anti-TfR antibodies reformatted in a 2+1 bispecific format TIFF2025528752000035.tif228165

[0323] Example 12: Humanization of anti-TfR mouse antibodies The scFv portions of TfR9, TfR12, and TfR15 were selected for humanization. These antibodies were selected for their function (internalization), functional stability (ability to be internalized after heat stress), and sequence diversity, as well as cross-reactivity with cynomolgus monkeys.

[0324] Humanization was performed using the CDR grafting method. One of the most common methods for humanizing non-human antibodies is to graft the CDRs of the non-human antibody into a human antibody acceptor framework. Often, such CDR grafting leads to a weakening or complete loss of affinity of the humanized antibody due to perturbation of the framework. Therefore, to restore the weakened or lost affinity, it may be necessary to retain certain residues in the mouse framework and replace them with human residues at the corresponding positions (backmutation). Therefore, it is important to accurately predict the residues to be retained in the context of the germline acceptor framework of the selected human antibody, which can preserve the function and paratope of the humanized antibody. Furthermore, maintaining or improving thermal stability and solubility is desirable for good manufacturability and downstream development.

[0325] Structure-based antibody modeling using the BioMOE module of MOE (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada) was applied to the humanization process of anti-TfR mouse monoclonal antibodies (mAbs). Briefly, the VH and VL sequences of the mouse mAb to be humanized were compared with functional human VL, VH, LJ, and HJ germline sequences obtained from IMGT (http: / / www.imgt.org / ). Pseudogenes and ORFs were excluded. For each mouse mAb (query), the five most similar VL germline sequences and the five most similar VH germline sequences were selected and combined with the most similar VJ and HJ genes to generate 25 humanized sequences. CDRs embedded in the human frameworks were defined according to the AbM definition (http: / / www.bioinf.org.uk / abs / #cdrdef).

[0326] Two humanized sequences were selected based on the frequency of their VH and VL frameworks in the human repertoire. An Fv homology model was constructed using the query and humanized sequences. The Fv homology model was constructed using the BioMOE or Antibody Modeler modules of MOE (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada). The AMBER10:EHT force field was used for energy minimization throughout the antibody homology modeling process. Based on the Fv homology model, molecular descriptors, such as the interaction energy between the VL and VH, coordinate-based isoelectric point (3DpI), hydrophobic patches, and charged surface area, were calculated and analyzed and sorted using the scoring matrix provided by MOE. These molecular descriptors were used to prioritize humanized mAbs for downstream experimental procedures, such as protein expression, purification, and binding affinity testing, as well as functional assays.

[0327] The BioMOE module of MOE provides Mutation Site Properties, a tool to visualize and classify potential backmutation residues. A backmutation is defined as an amino acid substitution that replaces the humanized sequence back to the original query sequence. Using this tool, the original query (reference) was individually compared with the selected humanized variants in both the primary amino acid sequence and the 3D structure of the 3D Fv homology model.

[0328] Changes between the reference and humanized variants were classified based on differences in amino acid type, potential interactions with residues in the CDRs, potential effects on VL / VH pairing, and potential changes in hydrophobic and charged surface area in and near the CDRs.

[0329] Mutations close to the CDRs or VL / VH interface, or with large charge differences, or involving strong hydrogen-bonding interactions were evaluated individually, and significant perturbing mutations were reverted to the original query residues. As a result, the humanized sequence may contain up to five backmutations.

[0330] For TfR9, TfR12, and TfR15, the humanized sequences are listed in Table 17 below.

[0331] Table 17. Humanized variants of TfR9, TfR12, and TfR15 The heavy and light chain sequences of the scFv are underlined. TIFF2025528752000036.tif201165TIFF2025528752000037.tif238165TIFF20255287520 00038.tif207165TIFF2025528752000039.tif240165TIFF2025528752000040.tif230165

[0332] Table 18. Variable heavy and light chain domains of humanized variants of TfR9, TfR12, and TfR15 TIFF2025528752000041.tif197165TIFF2025528752000042.tif240165TIFF20255287520 00043.tif239165TIFF2025528752000044.tif239165TIFF2025528752000045.tif210165

[0333] Example 13: Effect of 2+1 anti-TfR bispecific antibodies on TfR recycling and degradation in hCEMC / D3 cells An ideal BBB transport molecule would efficiently transport to the brain without affecting the expression level or localization of its target receptor, in this case, TfR. To determine the effect of the 2+1 anti-TfR bispecific antibody on transferrin receptor recycling, we used both FACS and Western blotting.

[0334] FACS analysis: HCMEC / D3 cells were seeded in 100 μL of medium (EBM2, Lonza) at 1.5 × 10^5 cells / well in 24-well plates. Cells were then treated with 20 mg / ml of the 2+1 anti-TfR bispecific antibody for 24 hours and then processed using a FACS Canto system (BD Biosciences). Human IgG1 isotype was included in the assay as a negative control. Because the 2+1 anti-TfR bispecific antibody is specific for the TfR apical domain, staining the cells with the b3 / 25 Ab (Luria-Perez R., et al.; Bol Med Hosp Invant Mex. 2016;73(6):372-379), which does not bind to this domain, avoids antibody competition for receptor binding. To determine the effect of 2+1 anti-TfR bispecific antibodies on TfR recycling, the mean fluorescence intensity (MFI) of cells treated with individual 2+1 anti-TfR bispecific antibodies was normalized to the MFI obtained for the human IgG isotype. Treatment of HCMEC / D3 cells with 2+1 anti-TfR bispecific antibodies reduced cell surface expression of TfR by 40–80% (Figure 2).

[0335] Two of these clones, TfR9 and TfR12, were selected for further manipulation to generate a panel of humanized antibodies (Examples 8 and 12). Interestingly, certain antibody clones derived from TfR9 (i.e., TfR9.1B and TfR9.5B) did not exhibit any adverse effects on receptor recycling. In contrast, all clones generated from TfR12 strongly interfered with receptor recycling (Figure 3). Using the same approach, we generated a panel of humanized antibodies based on TfR15 (Examples 8 and 12), and identified another antibody clone, TfR15.WH8, that did not exhibit any adverse effects on TfR recycling (data not shown).

[0336] To validate the FACS data, the total amount of TfR protein in cell lysates obtained from HCMEC / D3 after antibody treatment was quantified by Western blot.

[0337] Western blotting: HCMEC / D3 cells were seeded at 3 x 10^5 cells per well of a 12-well plate in 1 mL of medium (EBM2, Lonza). Cells were then exposed to 20 mg / mL of 2+1 anti-TfR bispecific antibody for 24 hours before Western blotting (WB). As controls, additional wells were treated with either a human IgG1 isotype control (20 mg / mL) or PBS. After washing twice with ice-cold PBS, cells were collected into 1.5 mL microcentrifuge tubes using a scraper. After centrifugation (2000 g for 5 minutes), the supernatant was removed from the tube, and the cells were lysed in RIPA buffer (R0278, Sigma-Aldrich) containing a protease inhibitor cocktail (cOmplete™ Mini Protase Inhibitor Cocktail, Roche, product code 11836153001). The cells were then incubated on a nutator at 4°C, followed by centrifugation at maximum speed in a benchtop centrifuge for 20 minutes at 4°C. The supernatant was then transferred to a new tube, mixed with LDS sample buffer (Invitrogen, no. NP0007), and incubated on a heat block (95°C) for 5 minutes. The samples were then subjected to protein gel electrophoresis, and proteins were transferred to nitrocellulose membranes. The membranes were treated with blocking buffer (TBST + 5% milk powder) for 1 hour at room temperature and then probed with 1 mg / ml of 2+1 anti-TfR bispecific antibody (R&D Systems Inc., no. MAB2474) for 24 hours at 4°C. The membranes were then washed four times with PBST and then incubated with a 1:3000 dilution of HRP-conjugated goat anti-mouse IgG1 antibody (Jackson Immunoresearch, no. 115-035-003) for 1 hour at room temperature. After washing four times with PBST, the membranes were developed with Supersignal West Pico reagent (ThermoFisher, no. 34580), imaged, and analyzed using an iBright instrument.The membranes were then incubated with stripping buffer (ThermoFisher, no. 21059) to remove bound antibodies and then probed with anti-human b-actin (Abcam, no. ab8227) for quantification. The TfR band density for each antibody was normalized to the corresponding b-actin band density. These values ​​were then normalized to those obtained from cells treated with the human IgG1 isotype.

[0338] Consistent with the FACS data, treatment of HCMEC / D3 cells with the 2+1 anti-TfR bispecific antibody had little to no effect on total TfR protein levels, particularly TfR9.1B. In contrast, total TfR protein in HCMEC / D3 was significantly reduced by exposure to the humanized antibody generated from TfR12 (Figure 4).

[0339] Example 14: Engineering humanized variants of TfR9.1B Humanized TfR9.1B was selected for further affinity optimization. The anti-TfR scFv portion of TR9.1B was cloned into the pComb3X phagemid vector (Addgene, Waterown MA), and overlap extension polymerase chain reaction (PCR) mutagenesis was used to generate a randomized VH and VL library consisting of different sequences with a theoretical library size of 9E+07. These were packaged into bacteriophage, each carrying and expressing a unique VH / VL combination. This phage library was subjected to three rounds of panning with increasing stringency using soluble biotinylated TfR Avi-his protein. Individual colonies were picked after the third round, and scFv from periplasmic extracts (PPE) were screened for binding by ELISA. After initial ELISA screening, 46 of the 9_1B scFv variants were selected for cloning into the 2+1 "hole" vector (Example 9 above) between the BamHI and NotI sites 3' to the 3x(G4S) linker connecting the Fc and scFv portions. These 46 TfR9.1B 2+1 bispecific antibodies were expressed and purified in the Expi293 expression system as described above (Example 9) for further characterization and are shown in Table 19 below.

[0340] Table 19. Affinity-optimized TfR9.1B variant sequences TIFF2025528752000046.tif206165TIFF2025528752000047.tif205165TIFF2025528752000048.tif20616 5TIFF2025528752000049.tif206165TIFF2025528752000050.tif206165TIFF2025528752000051.tif20616 5TIFF2025528752000052.tif206165TIFF2025528752000053.tif206165TIFF2025528752000054.tif20616 5TIFF2025528752000055.tif206165TIFF2025528752000056.tif205165TIFF2025528752000057.tif62165

[0341] Table 20. Variable heavy and variable light domains of affinity-optimized TfR9.1B variants TIFF2025528752000058.tif202165TIFF2025528752000059.tif238165TIFF2025528752000060.tif238165TIFF20255287520 00061.tif238165TIFF2025528752000062.tif238165TIFF2025528752000063.tif238165TIFF2025528752000064.tif142165

[0342] Clone TfR9.1B.39 was selected as a template for further rounds of random mutagenesis to remove two potential isomerization sites (D34 in VL and D62 in VH). Using biotinylated human and cynomolgus TfR Avi-His proteins as bait for panning and ELISA for screening, seven mutants in which both potential isomerization sites were fixed were selected and cloned into the same vector in a 2+1 bispecific format for further characterization (see Table 21).

[0343] Table 21: TfR9.1B.39 variant sequences (VH and VL sequences are underlined) TIFF2025528752000065.tif200165TIFF2025528752000066.tif78165

[0344] Table 22. Variable heavy and light chain domains of TfR9.1B.39 variants TIFF2025528752000067.tif200165TIFF2025528752000068.tif78165

[0345] Table 23. CDR sequences of affinity-optimized TfR9.1B.39 variants (Kabat definition) TIFF2025528752000069.tif203165

[0346] Example 15: Engineering a humanized TfR15 antibody Humanized TfR15 was engineered to optimize target affinity and remove potential oxidation sites in VH (Trp38). DNA fragments of WH7, WH8, and WH12, in which Trp38 was replaced with Tyr, were ordered from GeneArt and cloned into the same vectors described above. Proteins were expressed and purified from the Expi293 expression system for further characterization. The sequences of these engineered variants are shown in Table 24 below. TfR15.WH8.1.1 was optimized (by alanine scanning of the CDRs) to generate variants with a wide range of binding affinities (see Examples 16 and 17 below).

[0347] Table 24. TfR15 variants with removed or reduced affinity liability sites TIFF2025528752000070.tif164165

[0348] Example 16: Engineering a humanized TfR.15.WH8.1 variant Humanized TfR.15.WH8.1 variants were engineered to optimize target affinity. The first step involved alanine scanning of the CDRs, followed by amino acid substitutions at selected positions within the CDRs. To perform alanine scanning of the CDRs, DNA fragments containing the variants were ordered from Integrated DNA Technologies (IDT) and cloned into expression vectors. Proteins were expressed and purified from Expi293 cells for further characterization. Based on the results of the alanine scanning (data not shown), key amino acid positions were selected for further amino acid substitutions from alanine (A) to glycine (G), aspartic acid (D), asparagine (N), histidine (H), lysine (K), glutamine (Q), glutamic acid (E), or serine (S). In addition to amino acid substitutions, three additional variants exist in which the orientation of the VL and VH domains is swapped or the linker is modified. In the WH8.1.42A.LH variant, the VH and VL orientation was swapped to VL-linker-VH. In the WH8.1.42A.3×(G4S) variant, the linker was changed to (GGGGS)×3 (SEQ ID NO: 184). In the WH8.1.42A.L1 variant, the linker was changed to SPNSASHSGSAPQTSSAPGSQ (SEQ ID NO: 442) (Hennecke et al., (1998) PEDS, 11:405-410). DNA fragments containing the variants were ordered from IDT and cloned into expression vectors in a 2+2 antibody format. The 2+2 antibodies were expressed and purified from the Expi293 expression system as described above for further characterization. The sequences are shown in Tables 25-27 below.

[0349] Table 25. Affinity-optimized TfR.15.WH8.1 variant sequences The heavy and light chain sequences are underlined. TIFF2025528752000071.tif209165TIFF2025528752000072.tif202165TIFF2025528752000073.tif175165

[0350] Table 26. Variable heavy and variable light domains of affinity-optimized TfR.15.WH8.1 variants TIFF2025528752000074.tif197165TIFF2025528752000075.tif202165TIFF2025528752000076.tif175165

[0351] Table 27. CDR sequences of affinity-optimized TfR.15.WH8.1 variants (Kabat definition) TIFF2025528752000077.tif202165TIFF2025528752000078.tif238165TIFF2025528752000079.tif86165

[0352] Example 17: Binding kinetics of TfR.15.WH8.1 variants The binding kinetics of the 2+2 TfR.15.WH8.1 variant (an isotype IgG containing two anti-TfR scFvs) to Avi-His-tagged human and cynomolgus monkey TfR apical domains was assessed using a Carterra LSA instrument (Carterra, Salt Lake City, UT). Capture lawns were prepared using a goat anti-human Fcγ polyclonal antibody (Jackson ImmunoResearch) on an HC30M sensor chip (Carterra) according to the instrument manufacturer's instructions. Two independent experiments were performed as follows, with N values ​​between one and two determinations for each antibody. Spots with test article binding less than 20 RU were excluded from further analysis.

[0353] 2+2 anti-TfR bispecific antibodies were prepared by diluting them in running buffer (HBS-EP+, Teknova) and adding 0.5 mg / mL BSA (MP Biomedicals) to a final concentration of 15 μg / mL and capturing them as an array on a goat anti-human Fcγ capture surface. The captured antibodies were tested for their ability to bind to recombinant human and cynomolgus monkey orthologues of TfR apical Avi-His. Affinity estimates were generated by injecting each test compound across the antibody array using a single-channel flow cell. TfR apical Avi-His analytes were diluted in running buffer and subjected to eight 3-fold serial dilutions starting with 2500 nM human and cynomolgus monkey TfR apical Avi-His, followed by two 10-fold dilutions. Test compounds were injected for 5 min and dissociation was followed for 15 min. After each series of test compound injections, the antibodies were regenerated with 10 mM glycine pH 2.0 buffer (Carterra). Data were processed and analyzed using NextGenKIT high-throughput kinetic analysis software (Carterra).

[0354] The equilibrium dissociation constants (K) were calculated from the fitted on- and off-rate constants (k-on and k-off) for the 2+2 anti-TfR bispecific antibodies of the present disclosure that bind to human and cynomolgus monkey TfR apical Avi-His. D For N>1 measurements, the mean and standard deviation were calculated, and measurements from all samples were combined and graphed using GraphPad Prism. D The values ​​are summarized below in Table 28. As shown in the table, some antibodies exhibited heterogeneous binding profiles that did not fit the 1:1 binding model, and therefore their rate constants could not be determined in this set of experiments.

[0355] Table 28. Equilibrium dissociation constants (K D ) TIFF2025528752000080.tif145165

[0356] These results demonstrate that the 2+2 anti-TfR bispecific antibodies exhibit a wide range of affinities for TfR apical domain binding, ranging from approximately 3 nM to greater than 2.5 μM. Specifically, the affinities of the 2+2 anti-TfR bispecific antibodies binding to human TfR Avi-His ranged from 6 nM to greater than 2.5 μM, while the affinities of the anti-TfR antibodies of the present disclosure binding to cynomolgus monkey TfR Avi-His ranged from 3 nM to greater than 2.5 μM.

[0357] Example 18: Binding kinetics of 2+1 anti-TfR bispecific antibodies The binding kinetics of a humanized 2+1 anti-TfR bispecific antibody to human and cynomolgus monkey TfR Avi-His was evaluated using a Carterra LSA instrument (Carterra, Salt Lake City, UT). Briefly, the 2+1 anti-TfR bispecific antibody was prepared in duplicate by diluting 20-180 times in 10 mM acetic acid, pH 4.5 (Carterra) to a final concentration of 30 μg / mL. Using a single-channel flow cell, an HC30M sensor chip (Carterra) was activated with a 7-minute injection of a 1:1:1 mixture of 100 mM MES pH 5.5, 100 mM sulfo-NHS, and 400 mM EDC (all reconstituted in MES pH 5.5; 100 μl of each was mixed in a vial immediately before the assay). After switching to the multichannel array flow cell, each antibody was injected for 15 minutes over four 96-spot arrays of activated chips. The remaining unbound active groups on the chip were then blocked by injecting 1 M ethanolamine, pH 8.5 (Carterra) for 7 min using a single-channel flow cell. The resulting sensor chip contained eight spots for each antibody at four different densities. Two independent experiments were performed as follows, yielding an N of 1 to 8 determinations for each antibody. Spots with less than 20 RU of binding of the test substance were excluded from further analysis.

[0358] Running buffer HBS-EP containing 0.5 mg / ml BSA (MP Biomedicals) +After priming with ELISA kits (Teknova), immobilized anti-TfR2+1 antibodies were examined for their ability to bind to the two forms of TfR Avi-His, the human and cynomolgus monkey orthologs described above. Affinity estimates were generated by injecting each test article across the antibody array using a single-channel flow cell. TfR Avi-His test articles were diluted in running buffer in eight 3-fold serial dilutions starting at 1 μM for human and cynomolgus monkey TfR Avi-His. Test articles were injected for 5 min, and dissociation was followed for 10 min. After each test article injection, the antibodies were regenerated with Pierce™ IgG Elution Buffer (ThermoScientific). Data were processed and analyzed using NextGenKIT high-throughput kinetic analysis software (Carterra).

[0359] The equilibrium dissociation constant (K D These values ​​were combined, the mean and standard deviation were calculated, and graphs were generated using GraphPad Prism. D The values ​​are summarized below in Table 29. As shown in the table, some antibodies exhibited heterogeneous binding profiles that did not fit the 1:1 binding model, and therefore their rate constants could not be determined.

[0360] Table 29. Equilibrium dissociation constants (K D ) TIFF2025528752000081.tif206165

[0361] These results demonstrate that the 2+1 anti-TfR bispecific antibodies exhibit a wide range of affinities for TfR Avi-His, ranging from approximately 6 nM to 900 nM. Specifically, the affinities of the 2+1 anti-TfR bispecific antibodies of the present disclosure for binding to huTfR Avi-His ranged from 6.7 nM to 340 nM. The affinities of the anti-TfR antibodies of the present disclosure for binding to cynoTfR Avi-His ranged from 18 nM to 870 nM.

[0362] Example 19: PK of 2+1 anti-TfR bispecific antibodies in huTfR ECD KI mice Generation of 2+1 anti-TfR bispecific antibodies: 2+1 anti-TfR bispecific antibodies for in vivo PK studies were expressed and purified in a 2+1 backbone with an isotype control (non-binding) Fab arm.

[0363] Antibodies for in vivo PK studies were produced by transient transfection of ExpiCHO cells (Invitrogen) according to the manufacturer's instructions. 320 μg of total DNA consisting of three expression plasmids (heavy chain-hole-scFv, heavy chain-knob, and light chain sequences) was used in a 400 mL culture. The molar ratio of the three plasmids was optimized to obtain a highly pure 2+1 anti-Tfr bispecific antibody. Cell culture supernatants were harvested 10 days after transfection. Protein A affinity chromatography was followed by ion exchange chromatography on an AKTA Avant 25 (Cytiva) to purify the antibody and remove product-related impurities. The purified antibody was dialyzed into PBS. Analytical characterization was performed by absorbance at 280 nm, CE-SDS, size exclusion chromatography, and endotoxin measurement.

[0364] Study Design: To establish in vivo proof-of-concept for brain penetration of a 2+1 anti-TfR bispecific antibody, heterozygous knock-in (KI) mice were generated in which the ectodomain region of the mouse TfR was replaced with a human allele. This allows the 2+1 anti-TfR bispecific antibody to specifically bind to the primate TfR but not to the mouse TfR. Notably, the resulting total TfR protein levels in TfRmu / huKI mice were indistinguishable from those observed in wild-type (WT) animals (data not shown). A two-dose approach was used to determine the time course of brain uptake and peripheral clearance of the injected Ab. Groups of WT and hu-TfRmu / hu mice were administered 10 mg / kg of either isotype IgG or the 2+1 anti-TfR bispecific antibody on days 1 and 14. Blood samples from antibody-treated animals were collected at different time points into serum separator tubes, which were then allowed to clot at room temperature (RT) and then centrifuged. The resulting supernatant, which corresponds to the serum, was then transferred to a new tube and stored at −80° C. until analysis.

[0365] Sample preparation: Brain tissue was collected from antibody-treated mice on day 15 (i.e., 24 hours after secondary antibody injection). Prior to this, mice were anesthetized and cardiac perfused with 15 ml of PBS to flush the blood vessels. The brain tissue was then crushed and homogenized in 1 ml of HBSS buffer (MilliporeSigma, No. 55037C) containing 10 mM HEPES (Gibco, No. 15630130) using a manual grinder. The sample was then centrifuged at 1000 g for 5 minutes to pellet the blood vessels. The supernatant, corresponding to the parenchyma, was then transferred to a new tube and mixed with 10x RIPA buffer (final concentration 1x) containing a protease and phosphatase inhibitor cocktail (cOmplete™ Mini Protase Inhibitor Cocktail, Roche, No. 11836153001). After 20 minutes of incubation at 4°C on a nutator, the samples were frozen on dry ice and stored at -80°C until analysis. After centrifugation through 18% dextran (70 kDa, product code 31390, Sigma-Aldrich), the vascular fraction was cleared from myelin debris by washing twice in HBSS buffer. The samples were then lysed in RIPA buffer (R0278, Sigma-Aldrich) and stored at -80°C until analysis. The validity of this method was confirmed using WB analysis, which revealed the absence of brain endothelial cell markers (e.g., CD31 and claudin-5) in the brain parenchyma (data not shown).

[0366] Pharmacokinetic (PK) analysis: Antibody concentrations in serum and brain samples were measured using the MSD (Meso Scale Discovery) method. Briefly, 50 μl / well of goat anti-human IgG1 Ab (Jackson ImmunoResearch, no. 109-005-097) was added to MSD plates at 1 μg / ml in PBS and incubated overnight at 4°C. Each plate was then washed three times with wash buffer (1x PBS, 0.05% Tween-20) and then incubated with blocking buffer (wash buffer containing 3% BSA) for 1 hour at room temperature. Serum (diluted 1:10,000 in PBS) and brain (diluted 1:2 in PBS) samples were then added in duplicate to each plate and incubated for 1 hour on a shaker at 500 RPM. Known concentrations of human IgG1 isotype Ab were included in the assay to generate a standard curve. Next, each plate was washed three times with washing buffer, and then 30 μl / well of 0.2 μg / ml Sulfo-Tag labeled goat anti-human Ab (R32AJ-5, Meso Scale Discovery) in PBS was added and incubated for 1 hour on a shaker at 500 RPM. Finally, after washing three times with washing buffer, 150 μl / well of 1× READ buffer (R92PC-2, Meso Scale Discovery) was added to each plate, and then they were read using a Sector Imager S600 instrument.

[0367] When antibody concentrations were measured in the vascular-depleted brain, the levels of the 2+1 anti-TfR bispecific antibody in hu-TfRmu / hu mice were 4-5 times higher than those of isotype IgG (Figure 5). In contrast, brain uptake of the 2+1 anti-TfR bispecific antibody in WT animals, which do not express the target receptor, was similar to that observed with isotype IgG. Taken together, these results indicate that the 2+1 anti-TfR bispecific antibody enhances transferrin receptor-mediated brain entry. Peripheral PK was determined by measuring antibody concentrations in plasma samples from antibody-treated mice. In WT mice, plasma levels of the 2+1 anti-TfR bispecific antibody were characterized by a slow half-life and were indistinguishable from those of the isotype antibody (Figure 6). In contrast, both 2+1 anti-TfR bispecific antibodies increased brain uptake by hu-TfR. mu / hu It exhibits higher plasma clearance in mice compared to isotype IgG, consistent with TfR-mediated behavior in the periphery.

[0368] Example 20: Effect of 2+1 anti-TfR bispecific antibodies on TfR expression on brain epithelial cells in vivo To determine whether the 2+1 anti-Tfr bispecific antibodies cause dysregulation of receptor recycling in target cells in vivo, the total amount of TfR protein was quantified in the vascular portion of brain tissue harvested from antibody-treated animals. For this purpose, isolated blood vessels were lysed in RIPA buffer and processed by WB as described in Example 13. Using quantitative analysis, the total amount of TfR protein in hu-TfR cells injected with anti-TfR antibodies (i.e., TfR-9.1B.39 and TfR-15.WH8) was significantly increased. + / -No significant differences in TfR band density were observed between samples obtained from KI mice compared with isotype-treated animals (Figure 7). Of note, in this assay, membranes were probed with an anti-human TfR antibody that does not contain cross-reactivity to mouse TfR (R&D Systems Inc, no. MAB2474). As a result, no TfR bands were detected in vessels obtained from WT animals. Taken together, these observations indicate that the 2+1 anti-TfR bispecific antibody does not alter TfR recycling at the blood-brain barrier (BBB) ​​in vivo.

[0369] Example 21: In vivo reticulocyte depletion upon 2+1 anti-TfR bispecific antibody treatment in mice Anti-TfR antibodies are known to induce cell death in reticulocyte populations in vivo. + / -Groups of KI mice were injected with either a 2+1 anti-TfR bispecific antibody (TfR-9.1B.39 and TfR-15.WH8), a human IgG1 isotype, or the previously published anti-ms TfR antibody 8D3 (Boado et al., Biotechnol Bioeng. 2009 Mar 1;102(4):1251-1258), both of which are known to induce robust reticulocyte depletion in mice. The latter two antibodies were included in the experiment as negative and positive controls, respectively. All tested antibodies were on a WT human IgG1 backbone, which can induce ADCC, ADCP, and CDC responses in vivo. Blood samples were collected from antibody-treated animals 24 hours after injection and analyzed by FACS. Reticulocytes can be distinguished from mature red blood cells (RBCs) by staining cells with anti-CD71 (Invitrogen, Catalog No. 17-0719-42), thiazole orange (MilliporeSigma, Catalog No. 390062), and anti-mouse CD45 (Invitrogen, Catalog No. 12-0451-83) to exclude circulating leukocytes. Thiazole orange binds to residual ribosomal RNA molecules present in reticulocytes but absent from mature RBCs. Compared to RBCs, reticulocytes express high levels of the transferrin receptor (CD71). As expected, injection of the 8D3 antibody resulted in a complete loss of reticulocytes in WT animals (data not shown). In contrast, this cell population remained unchanged in isotype-treated animals, regardless of genotype. Similarly, no changes in reticulocyte counts were observed in WT mice treated with anti-TfR2+1 bispecific antibodies, which makes sense since these animals do not express the target receptor (Figure 8). In contrast, hu-TfR treated with TfR-9.1B.39 and TfR-15.WH8 + / -KI mice showed approximately 80%–90% reduction in reticulocyte counts, respectively (Figure 8). Based on published results (Yamamoto et al., Molecular Genetics and Metabolism Reports, Volume 27, June 2021, 100758), the reticulocyte depletion observed here may be mediated through the induction of an ADCC (antibody-dependent cellular cytotoxicity) response, which was further evaluated in the Examples below.

[0370] Example 22: Evaluation of in vitro effector functions (CDC and ADCC) To explore the mechanism of reticulocyte depletion observed with 2+1 anti-TfR bispecific antibodies, we assessed the ability of these same antibodies to induce in vitro effector responses, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Furthermore, since anti-TfR antibodies on a WT huIgG1 backbone were shown to induce ADCC responses, we tested alternative Fc formats, such as N325S / L328F (NSLF) and L234A / L235A / P331S (LALAPS), which have reduced binding to activating FcgRs (particularly FcgRIIIa, the primary inducer of ADCC).

[0371] Methods and Results for CDC: The ability of 2+1 anti-TfR bispecific antibodies to induce complement deposition was measured in the hCMEC / D3 cell line, which expresses high levels of both receptors. Target cells were detached, washed once in PBS, and cultured at 2 × 10 in RPMI 1640 medium. 6Target cells were diluted to 1000 cells / mL. 50 μL of target cells (1 x 10 cells per well) were dispensed per well into a round-bottom 96-well plate (Falcon #351177). 25 μL of 4x antibody diluted in the same medium was added to these cells. After incubating the cells and antibody for 15 minutes at 37°C, 25 μL of pooled complement human serum (Innovative Research, IPLA-CSER) was added per well as a complement source, and each plate was incubated for an additional 2 hours at 37°C. Cells were then washed twice with FACS buffer (PBS + 2% FBS + 1 mM EDTA) and resuspended in 50 μL of FACS buffer + 0.25 μL / well of propidium iodide (Fischer Scientific, BD556463) before analysis on an iQue flow cytometer (IntelliCyt). CDC was analyzed as the percentage of PI+ single cells in each well. In this assay, none of the 2+1 anti-TfR bispecific antibodies induced a significant CDC response (data not shown).

[0372] ADCC Methods and Results: The ability of the 2+1 bispecific antibodies to induce antibody-dependent cellular cytotoxicity (ADCC) was assessed using the Promega ADCC Reporter Bioassay system (number G7010). This system relies on an engineered Jurkat T cell line stably expressing the FcgRIIIa receptor (V158 variant) and an NFAT response element to drive the expression of firefly luciferase. Target cells were cultured at 1.2 x 10 per mL in assay buffer (RPMI + 4% low IgG serum). 6 The target cells were diluted to a concentration of 1000, and 25 μL of cells (30,000 per well) were dispensed into the appropriate wells of a 96-well white assay plate. 25 μL of 3× antibody, also diluted in assay buffer, was added to the wells containing the cells. After addition of the antibody to the target cells, the provided effector cells (2×10 7The target cells (frozen at 1000 / mL) were thawed at 37°C, and 630 μL was added to 3.6 mL of warmed (37°C) assay buffer. After gentle mixing, 25 μL of effector cells (75,000 cells per well, for an E:T ratio of 2.5) were immediately added to the wells containing the target cells and antibody. The plate was then incubated at 37°C and 5% CO2 for 6 hours to activate the receptor cells and allow luciferase expression. After 6 hours, the plate was equilibrated to room temperature (15 minutes) while Bio-Glo Luciferase Assay Reagent was prepared. 75 μL of luciferase assay reagent was added to each well, the plate was incubated for 10 minutes, and luminescence was measured using a BioTek plate reader. In the wild-type human IgG1 format, some 2+1 bispecific antibodies against TfR were able to induce significant ADCC signals (Figure 9). Further experiments were performed to test whether these responses could be improved by reducing binding to distinct FcRs.

[0373] Fc Engineering and ADCC Results: Two of the 2+1 anti-TfR bispecific antibodies that elicited ADCC signals (TfR9.1B.39 and TfR15.WH8) were re-expressed in various Fc formats, including wild-type hIgG1, hIgG1 NSLF, and hIgG1 LALAPS, and re-tested in the same ADCC assay format described above. Both NSLF and LALAPS have previously been reported to significantly reduce binding to FcγRIIIA, a major inducer of ADCC activity (Leoh et al., Molecular Immunology, 29 Jul 2015, 67(2Pt B):407-415; Shang et al., J Biol Chem. 2014;289:15309-15318). In this assay, both NSLF and LALAPS were able to significantly reduce the ADCC signal, with NSLF showing a greater than 90% reduction in signal at the highest concentrations tested for both 2+1 anti-Tfr bispecific antibodies tested (Figure 10).

[0374] Example 23: Binding kinetics of affinity- and liability-optimized clones of 2+1 anti-TfR bispecific antibodies The binding kinetics of the 2+1 anti-TfR bispecific antibody to human TfR Avi-His was assessed using a GatorBio BLI instrument (GatorBio, Palo Alto, CA). Briefly, each 2+1 anti-TfR bispecific antibody was diluted in 7-, 5-, and 3-fold serial dilutions in running buffer (HBS-EP+, Teknova) containing 0.5 mg / ml BSA (MP Biomedicals), followed by two 10-fold dilutions starting at a concentration of 4 μM for Tf9.1B.39.36, TfR9.1B.39.37, TfR9.1B.39.38, and WH8 parent antibodies, and at a concentration of 2 μM for TfR9.1B.39, TfR9.1B.39.05, TfR9.1B.39.39, TfR9.1B.39.40, TfR15.WH7.1, TfR15.WH8.1, and TfR15.WH12.1. Biotinylated TfR Avi-His was diluted to 5 μg / mL in running buffer. A streptavidin probe (Gator) was immersed in the biotinylated TfR Avi-His solution for 5 seconds to load the protein onto the tip. Each probe was then immersed in the respective antibody concentration for 3 minutes of binding, followed by 3 minutes of dissociation in running buffer. A wash step in running buffer was performed between the loading and binding steps. The probes bearing biotinylated TfR Avi-His were regenerated with 10 mM glycine-HCl, pH 2.5. Biotinylated TfR Avi-His was replenished for 5 seconds at the start of each binding cycle. Data were double-referenced with a reference probe and a buffer (blank) well and processed and analyzed using Gator Analysis Software (GatorBio).

[0375] The equilibrium dissociation constants (KD) were calculated from the fitted on- and off-rate constants (k-on and k-off) for the 2+1 anti-Tfr bispecific antibodies. These values ​​were combined, the mean and standard deviation calculated, and graphs were generated using GraphPad Prism. As shown in the table, some antibodies exhibited heterogeneous binding profiles that did not fit the 1:1 binding model, and therefore their rate constants could not be determined. K D The values ​​are summarized in Table 30 below.

[0376] Table 30. Equilibrium dissociation constants (K D ) TIFF2025528752000082.tif79165

[0377] These results demonstrate that the 2+1 anti-TfR bispecific antibody exhibits a wide range of affinities for TfR Avi-His, ranging from approximately 30 nM to 4 μM. The affinities of the 2+1 anti-TfR bispecific antibody for binding to human TfR Avi-His ranged from 65 nM to 3.5 μM, and the affinities of the 2+1 anti-TfR bispecific antibody for binding to cynomolgus monkey TfR Avi-His ranged from 38 nM to 2.3 μM.

[0378] Example 24: In vitro sTREM2 assay of TD1-TfR bispecific antibodies To evaluate the pharmacokinetics and pharmacodynamics of a 2+1 anti-TfR antibody, a 2+1 anti-TfR bispecific antibody (termed "TD1-TfR") (described in Example 25) was generated containing (i) the TfR9.1B.39.38 scFv and (ii) a monoclonal antibody specific for human MS4A4A (termed "TD1") in a 2+1 (whole) format. To confirm that TD1-TfR retains the functionality of the parent TD1 antibody, a soluble TREM2 (sTREM2) assay was performed using human monocyte-derived macrophages. These cells are expected to show increased production of sTREM2 upon antibody treatment with TD1.

[0379] Human monocytes were isolated from whole blood using RosetteSep Human Monocyte Enrichment Cocktail (Stemcell Technologies) and Ficoll centrifugation according to the manufacturer's protocol. After erythrocyte lysis with ACK lysis buffer, monocytes were resuspended in complete medium (RPMI, 10% FBS, penicillin / streptomycin, L-glutamine, HEPES, non-essential amino acids, and sodium pyruvate). To obtain macrophages from these isolated monocytes, 50 ng / ml human M-CSF and 8% v / v human serum were added to the cells for 5–7 days.

[0380] 10 per well 5 Macrophages were stimulated with 1 μg / mL of test antibody (huIgG1, TD1, iso-TfR (isotype control IgG antibody linked to TfR9.1B.39.38), or TD1-TfR) for 48 hours. Duplicate wells were processed as technical replicates. Each supernatant was tested for sTREM2 using the MSD platform.

[0381] As shown in Figure 11, TD1-TfR demonstrated activity by increasing sTREM2 levels in in vitro assays compared to hIgG1 and Iso-TfR. TD-1 and TD1-TfR showed similar increases in sTREM2 levels in in vitro assays. This data indicates that adding the anti-TfR scFv to the TD1 antibody does not impair TD1 function.

[0382] Example 25: TD1-TfR bispecific antibody and method for intravenous administration to non-human primates To evaluate the pharmacokinetics and pharmacodynamics of the 2+1 anti-TfR antibodies disclosed herein, a 2+1 anti-TfR bispecific antibody (termed "TD1-TfR") (described in Example 25) was generated containing (i) TfR9.1B.39.38 scFv and (ii) a monoclonal antibody specific for human MS4A4A (termed "TD1") in a 2+1 (whole) format. TD1 has been shown to increase several biomarkers in the brain of non-human primates, including soluble TREM2 (sTREM2) and colony-stimulating factor 1 (CSF1) (data not shown). The complete amino acid sequence of TD1-TfR is as follows:

[0383] TD1 heavy chain hIgG1 NSLF (N325S, L328F) knob (T366W) QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGVPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 405)

[0384] TD1 heavy chain hIgG1 NSLF (N325S, L328F) with mutations (H435R, Y436F) and whole scFv (T366S, L368A, Y407V) - (G4S)3 linker - VH - 20AA linker - VL (SEQ ID NO: 406)

[0385] TD1 light chain DVVMTQSPLSLPVTLGQPASISCKSSRSLLYSAGKTYLSWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGIDFHQTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 407)

[0386] Additionally, for control purposes, a bispecific antibody containing (i) a TfR-specific scFv and (ii) an IgG1 NSLF in a 2+1 (whole) format was generated (referred to as "Iso-TfR"). The complete amino acid sequence of Iso-TfR is as follows:

[0387] Isotype control heavy chain hIgG1 NSLF (N325S, L328F) knob (T366W) EVRLLESGGGLVQPGGSLRLSCAASGFTFSNYAMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTTSRDDSKNALYLQMNSLRAEDTAVYYCARGPGPGWYAADVWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 408)

[0388] Isotype control heavy chain hIgG1 NSLF (N325S, L328F) with mutations (H435R, Y436F) whole scFv (T366S, L368A, Y407V) - (G4S)3 linker - VH - 20AA linker - VL (SEQ ID NO: 409)

[0389] Isotype Control Light Chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQADLPAFAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 410)

[0390] The complete amino acid sequence of an antibody (TD1) specific for human MSA4A alone is as follows:

[0391] TD1 heavy chain hIgG1 NSLF (N325S, L328F) QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGVPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 411)

[0392] TD1 light chain DVVMTQSPLSLPVTLGQPASISCKSSRSLLYSAGKTYLSWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGIDFHQTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 407)

[0393] PK / PD studies were conducted in naive male cynomolgus monkeys (n=12) after intravenous (iv) administration of 20 mg / kg of each of the four antibodies in Table 31 (two doses total) (n=3) on days 1 and 29. This dose level of TD1 has been shown to produce incomplete PD responses in NHPs due to poor brain penetration of the antibody. The objective of this study was to examine increases in antibody levels in the brain and concomitant increases in relevant PD biomarkers.

[0394] Table 31. In vivo administration of anti-TfR bispecific antibodies TIFF2025528752000083.tif55165

[0395] The intravenous dose was administered via the saphenous vein as a slow push injection. The target dose level (mg / kg), target dose concentration (mg / mL), and target dose volume (mL / kg) were consistent across groups 1–4.

[0396] Example 26: Hematological parameters after intravenous administration of TD1-TfR bispecific antibodies to non-human primates Blood samples (approximately 2 mL) for hematological analysis were collected from the femoral vein into tubes containing potassium EDTA before dosing and after dose administration on days 3, 8, 15, 29, and 31.

[0397] Table 32. Hematological parameters tested after intravenous administration of anti-TfR bispecific antibodies TIFF2025528752000084.tif48165

[0398] Figure 12 shows absolute reticulocyte counts over the course of the study. Normal ranges are indicated by dashed lines. Overall, no significant changes were observed in any of the hematological parameters, and no consistent decrease in reticulocytes was observed throughout the study.

[0399] Example 27: CSF and serum levels of TD1-TfR bispecific antibody after IV administration in non-human primates Cerebrospinal fluid (CSF) and serum were collected from NHPs at different times after the first and second doses as described in Example 25. Serum and CSF were tested for antibody levels as described below.

[0400] Sandwich ELISA: 100 μL / well of goat anti-human IgG monkey-adsorbed biotin (Southern Biotech; catalog no. 2049-08) working solution (0.5 μg / mL) was added to a 96-well Pierce™ streptavidin-coated high-binding plate with SuperBlock™ blocking buffer (ThermoScientific, reference no. 15500) and incubated for 1 hour ± 10 minutes at room temperature (RT) on a plate shaker at 350 revolutions per minute (RPM). Serum or CSF quality control (QC) and test samples were diluted to the minimum required dilution (MRD) in assay buffer (1× TBS, 0.05% Tween-20, and 0.1% BSA) and then loaded onto the assay plate. Standard calibration curves and assay buffer QCs (AB-QC) were added directly to the assay plate. After incubation was complete, the plate was washed three times with wash buffer (1x PBS and 0.05% Tween-20) using a plate washer and 100 μL / well of calibration standards, QCs, and MRD-diluted serum or CSF QCs. Test samples were then added to the plate and incubated for 2 hours ± 15 minutes on a plate shaker at 350 RPM at room temperature. The plate was then washed, and 100 μL / well of goat anti-human IgG monkey-adsorbed-HRP (Southern Biotech, catalog no. 2049-05) working solution (0.08 μg / mL) was added to the plate and incubated for 1 hour ± 10 minutes on a plate shaker at 350 RPM at room temperature. After the HRP incubation, the plate was washed, and 100 μL / well of tetramethylbenzidine (TMB) substrate solution (Surmodics Product No. TMBS-1000-01) was added and incubated for 15±10 minutes at room temperature (RT), protected from light by covering with aluminum foil or placing in a drawer. After the incubation was complete, the reaction was stopped by adding 100 μL / well of stop solution (Surmodics Product No. NSTP-1000-01) to the plate. The plate was then placed on a plate shaker at 350 RPM for at least 2 minutes at room temperature.Plates were then read on a Molecular Devices SpectraMax M5 instrument, and raw optical density (OD450-540) signals were generated from each well. The concentration of analyte in each unknown sample was determined by interpolation of concentrations using a calibration standard curve, using a weighting factor of 1 / Y, by SoftMax Pro (Molecular Devices / version 7.1). 2 Standard regression was performed using a four-parameter logistic (4-PL) model with:

[0401] Figure 13 shows that TD1-TfR showed increased CSF Cmax compared to TD1. Iso-TfR also showed increased CSF Cmax compared to the isotype control. Furthermore, as shown in Figure 13, TD1-TfR showed faster serum clearance in NHPs compared to Iso-TfR or TD1 alone.

[0402] Example 28: Effect of TfR binding arms on brain uptake and brain pharmacodynamics in non-human primates Antibody levels were also assessed directly in multiple NHP brain regions (frontal cortex, endocrine cortex, and hippocampus) using blood vessel-depleted brain lysates.

[0403] Brain sample preparation: Brain tissue was collected from antibody-treated cynomolgus monkeys on day 30 (i.e., 48 hours after secondary antibody administration). Prior to this, the animals were anesthetized and cardiac perfused with PBS to wash out the blood vessels. For administration details, see Example 25. The brain tissue was then crushed and homogenized using a manual grinder in HBSS buffer (MilliporeSigma, No. 55037C) containing 10 mM HEPES (No. 15630130, Gibco). The sample was then centrifuged at 1000 g for 5 minutes to pellet the blood vessels. The supernatant, corresponding to the parenchyma, was then transferred to a new tube and mixed with 10x RIPA buffer (final concentration 1x) containing a protease and phosphatase inhibitor cocktail (cOmplete™, Mini Protase Inhibitor Cocktail, Roche, No. 11836153001). After incubation on a nutator at 4°C for 45 minutes, the samples were frozen on dry ice and stored at -80°C until analysis. After centrifugation through 18% dextran (70 kDa, product number 31390, Sigma-Aldrich), the vascular fraction was cleared from myelin debris by washing twice in HBSS buffer. The samples were then lysed in RIPA buffer (R0278, Sigma-Aldrich) and stored at -80°C until analysis. The validity of this method was previously confirmed using Western blot analysis, which showed the absence of brain endothelial cell markers (e.g., CD31 and claudin-5) in the brain parenchyma (data not shown).

[0404] Pharmacokinetic (PK) analysis: Antibody concentrations in brain samples were measured using the MSD (Meso Scale Discovery) method. Briefly, 50 μl / well of goat anti-human IgG1 (monkey absorbed) antibody was added to MSD plates at 1 μg / ml in PBS and incubated overnight at 4°C on a 500 RPM shaker. Each plate was then washed three times with wash buffer (0.05% Tween-20 in 1x PBS) and then incubated with blocking buffer (wash buffer containing 3% BSA) at room temperature for 1 hour. Brain samples (diluted in PBS) were then added in duplicate to each plate and incubated for 2 hours at room temperature on a 500 RPM shaker. Next, each plate was washed three times with wash buffer, followed by the addition of 40 μl / well of 0.5 μg / ml sulfo-tagged goat anti-human Ab (R32AJ-5, Meso Scale Discovery) in PBS and incubation for 1 hour on a 500 RPM shaker. Finally, after three washes with wash buffer, 150 μl / well of 1x READ buffer (R92PC-2, Meso Scale Discovery) was added to each plate, which was then read using a Sector Imager S600 instrument. Eleven two-fold serial dilutions of each antibody (100–0.019 ng / ml) were used to generate standard curves for each treatment. These curves were fitted using a four-parameter logistic regression to calculate the antibody concentrations in the samples.

[0405] Figure 14 shows that the TfR BBB-targeting arm was able to increase the brain uptake of (i) TD1-TfR compared to TD1 in all three brain regions tested (increases ranging from 2- to 11-fold), and (ii) Iso-TfR compared to the isotype parent antibody in all three brain regions tested (increases ranging from 8- to 64-fold).

[0406] Example 29: Pharmacodynamic analysis of TD1-TfR bispecific antibodies in NHP samples CSF and serum from NHP test subjects described in Example 25, and NHP brain lysates described in Example 28, were further tested for downstream biomarkers of TD1 function, including sTREM2 and CSF1R, as described below.

[0407] Summary of CSF1R Method (Brain Lysate) Colony-stimulating factor 1 receptor (CSF1R) concentrations in cynomolgus monkey brain lysates were measured using an enzyme-linked immunosorbent assay (ELISA) kit (catalog no. DY329) from R&D Systems. Mouse anti-human M-CSF R capture antibody (R&D Systems, part no. 841246) was diluted to a working concentration of 4 μg / mL in 1x PBS (Corning, reference no. 21-040-CM) and coated onto a 96-well microplate (Nunc-Immuno Maxisorp, ThermoScientific, catalog no. 446612) at 100 μL / well and incubated overnight at room temperature without shaking. The next day, the plate was washed three times with wash buffer (1x PBS and 0.05% Tween-20), blocked with 300 μL of reagent diluent (R&D Systems, part no. 841380), and incubated for at least 1 hour without shakin...

Claims

1. An antigen-binding domain that specifically binds to the apical domain of the human transferrin receptor (TfR), wherein the antigen-binding domain is localized to the brain parenchyma of a subject after peripheral injection and is not located within a modified CH3 domain.

2. An antigen-binding domain that specifically binds to the human transferrin receptor (TfR), (i) SEQ ID NOs: 94, 415, 418, 423, 150, and 151, respectively; (ii) SEQ ID NOs: 74-76 and 135-137, respectively; (iii) SEQ ID NOs: 77-79 and 138-140, respectively; (iv) SEQ ID NOs: 80-82 and 135-137, respectively; (v) SEQ ID NOs: 83-85 and 141-143, respectively; (vi) SEQ ID NOs: 74, 86, 76, and 135-137, respectively; (vii) SEQ ID NOs: 87-89 and 143-145, respectively; (viii) SEQ ID NOs: 90-92 and 146-148, respectively; (ix) SEQ ID NOs: 83, 93, 85, and 141-143, respectively; (x) SEQ ID NOs: 94 to 96 and 149 to 151, respectively; (xi) SEQ ID NOs: 97, 86, 98, and 135-137, respectively; (xii) SEQ ID NOs: 99, 100, 101, and 152-154, respectively; (xiii) SEQ ID NOs: 99, 102, 103, and 155-157, respectively; (xiv) SEQ ID NOs: 104-106, 138, 158, and 159, respectively; (xv) SEQ ID NOs: 107-109 and 160-162, respectively; (xvi) SEQ ID NOs: 99, 110, 103, and 155-157, respectively; (xvii) SEQ ID NOs: 99, 111, 112, 152, 163, and 164, respectively; (xviii) SEQ ID NOs: 90, 113, 114, 165, 166, and 157, respectively; (xix) SEQ ID NOs: 83, 115, 85, 141, 142, and 167, respectively; (xx) SEQ ID NOs: 116, 110, 103, and 155-157, respectively; (xxi) SEQ ID NOs: 117-119 and 168-170, respectively; (xxii) SEQ ID NOs: 116, 110, 103, and 168-170, respectively; (xxiii) SEQ ID NOs: 90, 120, 114, 165, 166, and 157, respectively; (xxiv) SEQ ID NOs: 90, 113, 114, 171, 166, and 157, respectively; (xxv) SEQ ID NOs: 97, 121, 122, 135, 136, and 172, respectively; (xxvi) SEQ ID NOs: 74, 123, 124, 173, 136, and 137, respectively; (xxvii) SEQ ID NOs: 74, 125, 126, 174, 136, and 137, respectively; (xxviii) SEQ ID NOs: 99, 127, 128, 155, 175, and 176, respectively; (xxix) SEQ ID NOs: 90, 129, 130, 165, 166, and 157, respectively; (xxx) SEQ ID NOs: 131, 132, 133, 177, 166, and 178, respectively; (xxxi) SEQ ID NOs: 131, 132, 133, and 179-181, respectively; (xxxii) SEQ ID NOs: 90, 134, 114, 165, 166, and 157, respectively; (xxxiii) SEQ ID NOs: 83, 302, 303, 141, 142, and 182, respectively; (xxxiv) SEQ ID NOs: 99, 305, 103, 307, 308, and 157, respectively; (xxxv) SEQ ID NOs: 99, 306, 103, 307, 308, and 157, respectively; (xxxvi) SEQ ID NOs: 99, 306, 103, 307, 309, and 157, respectively; (xxxvii) SEQ ID NOs: 99, 306, 103, 310, 311, and 157, respectively; (xxxviii) SEQ ID NOs: 99, 305, 103, 310, 311, and 157, respectively; (xxxix) SEQ ID NOs: 99, 312, 103, 307, 308, and 157, respectively; (xl) SEQ ID NOs: 99, 312, 103, 307, 309, and 157, respectively; (xli) SEQ ID NOs: 99, 312, 103, 310, 311, and 157, respectively; (xlii) SEQ ID NOs: 94, 95, 418, 421, 150, and 151, respectively; (xliii) SEQ ID NOs: 94, 412, 419, 422, 150, and 151, respectively; (xliv) SEQ ID NOs: 94, 412, 420, 422, 150, and 151, respectively; (xlv) SEQ ID NOs: 94, 413, 418, 422, 150, and 151, respectively; (xlvi) SEQ ID NOs: 94, 414, 418, 422, 150, and 151, respectively; (xlvii) SEQ ID NOs: 94, 416, 418, 422, 150, and 151, respectively; (xlviii) SEQ ID NOs: 94, 417, 418, 424, 150, and 151, respectively; (xlix) SEQ ID NOs: 460, 312, 103, 307, 309, and 157, respectively; (l) SEQ ID NOs: 460, 312, 461, 307, 309, and 157, respectively; (li) SEQ ID NOs: 460, 312, 462, 307, 309, and 157, respectively; (lii) SEQ ID NOs: 460, 312, 463, 307, 309, and 157, respectively; (liii) SEQ ID NOs: 460, 312, 464, 307, 309, and 157, respectively; (liv) SEQ ID NOs: 460, 312, 465, 307, 309, and 157, respectively; (lv) SEQ ID NOs: 460, 312, 466, 307, 309, and 157, respectively; (lvi) SEQ ID NOs: 460, 312, 467, 307, 309, and 157, respectively; (lvii) SEQ ID NOs: 460, 312, 468, 307, 309, and 157, respectively; (lviii) SEQ ID NOs: 460, 312, 469, 307, 309, and 157, respectively; (lix) SEQ ID NOs: 460, 312, 470, 307, 309, and 157, respectively; (lx) SEQ ID NOs: 460, 312, 103, 471, 309, and 157, respectively; (lxi) SEQ ID NOs: 460, 312, 103, 472, 309, and 157, respectively; (lxii) SEQ ID NOs: 460, 312, 103, 473, 309, and 157, respectively; (lxiii) SEQ ID NOs: 460, 312, 103, 474, 309, and 157, respectively; (lxiv) SEQ ID NOs: 460, 312, 103, 475, 309, and 157, respectively; (lxv) SEQ ID NOs: 460, 312, 103, 476, 309, and 157, respectively; or (lxvi) SEQ ID NOs: 460, 312, 103, 471, 309, and 157, respectively. heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of The antigen-binding domain comprising:

3. VH and VL, wherein the VH and VL are: (i) SEQ ID NOs: 399 and 400, respectively; (ii) SEQ ID NOs: 10 and 11, respectively; (iii) SEQ ID NOs: 12 and 13, respectively; (iv) SEQ ID NOs: 14 and 15, respectively; (v) SEQ ID NOs: 16 and 17, respectively; (vi) SEQ ID NOs: 18 and 19, respectively; (vii) SEQ ID NOs: 20 and 21, respectively; (viii) SEQ ID NOs: 22 and 23, respectively; (ix) SEQ ID NOs: 24 and 25, respectively; (x) SEQ ID NOs: 26 and 27, respectively; (xi) SEQ ID NOs: 28 and 29, respectively; (xii) SEQ ID NOs: 30 and 31, respectively; (xiii) SEQ ID NOs: 32 and 33, respectively; (xiv) SEQ ID NOs: 34 and 35, respectively; (xv) SEQ ID NOs: 36 and 37, respectively; (xvi) SEQ ID NOs: 38 and 39, respectively; (xvii) SEQ ID NOs: 40 and 41, respectively; (xviii) SEQ ID NOs: 42 and 43, respectively; (xix) SEQ ID NOs: 44 and 45, respectively; (xx) SEQ ID NOs: 46 and 47, respectively; (xxi) SEQ ID NOs: 48 and 49, respectively; (xxii) SEQ ID NOs: 50 and 51, respectively; (xxiii) SEQ ID NOs: 52 and 53, respectively; (xxiv) SEQ ID NOs: 54 and 55, respectively; (xxv) SEQ ID NOs: 56 and 57, respectively; (xxvi) SEQ ID NOs: 58 and 59, respectively; (xxvii) SEQ ID NOs: 60 and 61, respectively; (xxviii) SEQ ID NOs: 62 and 63, respectively; (xxix) SEQ ID NOs: 64 and 65, respectively; (xxx) SEQ ID NOs: 66 and 67, respectively; (xxxi) SEQ ID NOs: 68 and 69, respectively; (xxxii) SEQ ID NOs: 70 and 71, respectively; (xxxiii) SEQ ID NOs: 72 and 73, respectively; (xxxiv) SEQ ID NOs: 313 and 314, respectively; (xxxv) SEQ ID NOs: 315 and 316, respectively; (xxxvi) SEQ ID NOs: 317 and 314, respectively; (xxxvii) SEQ ID NOs: 318 and 316, respectively; (xxxviii) SEQ ID NOs: 319 and 314, respectively; (xxxix) SEQ ID NOs: 320 and 316, respectively; (xl) SEQ ID NOs: 321 and 314, respectively; (xli) SEQ ID NOs: 322 and 316, respectively; (xlii) SEQ ID NOs: 323 and 314, respectively; (xliii) SEQ ID NOs: 324 and 316, respectively; (xliv) SEQ ID NOs: 325 and 326, respectively; (xlv) SEQ ID NOs: 327 and 328, respectively; (xlvi) SEQ ID NOs: 325 and 329, respectively; (xlvii) SEQ ID NOs: 330 and 331, respectively; (xlviii) SEQ ID NOs: 325 and 332, respectively; (xlix) SEQ ID NOs: 330 and 333, respectively; (l) SEQ ID NOs: 334 and 332, respectively; (li) SEQ ID NOs: 335 and 336, respectively; (lii) SEQ ID NOs: 337 and 338, respectively; (liii) SEQ ID NOs: 38 and 33, respectively; (liv) SEQ ID NOs: 339 and 340, respectively; (lv) SEQ ID NOs: 339 and 341, respectively; (lvi) SEQ ID NOs: 339 and 342, respectively; (lvii) SEQ ID NOs: 343 and 340, respectively; (lviii) SEQ ID NOs: 343 and 342, respectively; (lix) SEQ ID NOs: 344 and 340, respectively; (lx) SEQ ID NOs: 344 and 341, respectively; (lxi) SEQ ID NOs: 344 and 342, respectively; (lxii) SEQ ID NOs: 345 and 340, respectively; (lxiii) SEQ ID NOs: 345 and 341, respectively; (lxiv) SEQ ID NOs: 345 and 342, respectively; (lxv) SEQ ID NOs: 346 and 347, respectively; (lxvi) SEQ ID NOs: 348 and 316, respectively; (lxvii) SEQ ID NOs: 349 and 347, respectively; (lxviii) SEQ ID NOs: 349 and 350, respectively; (lxix) SEQ ID NOs: 351 and 352, respectively; (lxx) SEQ ID NOs: 349 and 353, respectively; (lxxi) SEQ ID NOs: 346 and 354, respectively; (lxxii) SEQ ID NOs: 349 and 355, respectively; (lxxiii) SEQ ID NOs: 349 and 356, respectively; (lxxiv) SEQ ID NOs: 357 and 316, respectively; (lxxv) SEQ ID NOs: 349 and 358, respectively; (lxxvi) SEQ ID NOs: 349 and 316, respectively; (lxxvii) SEQ ID NOs: 359 and 360, respectively; (lxxviii) SEQ ID NOs: 361 and 362, respectively; (lxxix) SEQ ID NOs: 363 and 316, respectively; (lxxx) SEQ ID NOs: 364 and 365, respectively; (lxxxi) SEQ ID NOs: 349 and 366, respectively; (lxxxii) SEQ ID NOs: 346 and 316, respectively; (lxxxiii) SEQ ID NOs: 346 and 367, respectively; (lxxxiv) SEQ ID NOs: 349 and 368, respectively; (lxxxv) SEQ ID NOs: 369 and 316, respectively; (lxxxvi) SEQ ID NOs: 346 and 370, respectively; (lxxxvii) SEQ ID NOs: 371 and 316, respectively; (lxxxviii) SEQ ID NOs: 372 and 356, respectively; (lxxxix) SEQ ID NOs: 357 and 358, respectively; (xc) SEQ ID NOs: 349 and 373, respectively; (xci) SEQ ID NOs: 346 and 374, respectively; (xcii) SEQ ID NOs: 375 and 316, respectively; (xciii) SEQ ID NOs: 376 and 316, respectively; (xciv) SEQ ID NOs: 346 and 377, respectively; (xcv) SEQ ID NOs: 378 and 379, respectively; (xcvi) SEQ ID NOs: 380 and 381, respectively; (xcvii) SEQ ID NOs: 349 and 382, ​​respectively; (xcviii) SEQ ID NOs: 357 and 383, respectively; (xcix) SEQ ID NOs: 349 and 358, respectively; (c) SEQ ID NOs: 384 and 316, respectively; (ci) SEQ ID NOs: 385 and 316, respectively; (cii) SEQ ID NOs: 357 and 386, respectively; (ciii) SEQ ID NOs: 387 and 388, respectively; (civ) SEQ ID NOs: 359 and 316, respectively; (cv) SEQ ID NOs: 389 and 316, respectively; (cvi) SEQ ID NOs: 390 and 316, respectively; (cvii) SEQ ID NOs: 391 and 392, respectively; (cviii) SEQ ID NOs: 393 and 356, respectively; (cix) SEQ ID NOs: 390 and 392, respectively; (cx) SEQ ID NOs: 357 and 386, respectively; (cxi) SEQ ID NOs: 394 and 395, respectively; (cxii) SEQ ID NOs: 396 and 395, respectively; (cxiii) SEQ ID NOs: 397 and 395, respectively; (cxiv) SEQ ID NOs: 398 and 395, respectively; (cxv) SEQ ID NOs: 401 and 395, respectively; (cxvi) SEQ ID NOs: 402 and 403, respectively; (cxvii) SEQ ID NOs: 443 and 341, respectively; (cxviii) SEQ ID NOs: 444 and 341, respectively; (cxix) SEQ ID NOs: 445 and 341, respectively; (cxx) SEQ ID NOs: 446 and 341, respectively; (cxxi) SEQ ID NOs: 447 and 341, respectively; (cxxii) SEQ ID NOs: 448 and 341, respectively; (cxxiii) SEQ ID NOs: 449 and 341, respectively; (cxxiv) SEQ ID NOs: 450 and 341, respectively; (cxxv) SEQ ID NOs: 451 and 341, respectively; (cxxvi) SEQ ID NOs: 452 and 341, respectively; (cxxvii) SEQ ID NOs: 453 and 341, respectively; (cxxviii) SEQ ID NOs: 443 and 454, respectively; (cxxix) SEQ ID NOs: 443 and 455, respectively; (cxxx) SEQ ID NOs: 443 and 456, respectively; (cxxxi) SEQ ID NOs: 443 and 457, respectively; (cxxxii) SEQ ID NOs: 443 and 458, respectively; or (cxxxiii) SEQ ID NOs: 443 and 459, respectively 3. The antigen-binding domain of claim 1 or 2, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of

4. An antigen-binding domain that specifically binds to human TfR, comprising a VH and a VL, wherein the VH is selected from the group consisting of: The antigen-binding domain comprises the amino acid sequence of 337, 38, 339, 343, 344, 345, 346, 348, 349, 351, 357, 359, 361, 363, 364, 369, 371, 372, 375, 376, 378, 380, 384, 385, 387, 389, 390, 391, 393, 394, 396, 397, 398, 401, 402, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, or 453.

5. 1. An antigen binding domain that specifically binds to human TfR, comprising a VH and a VL, wherein the VL is selected from the group consisting of SEQ ID NOs: 400, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 314, 316, 326, 328, 329, 331, 332, 333 370, 373, 374, 377, 379, 381, 382, ​​383, 386, 388, 392, 395, 403, 454, 455, 456, 457, 458, or 459.

6. (i) SEQ ID NOs: 399 and 400, respectively; (ii) SEQ ID NOs: 10 and 11, respectively; (iii) SEQ ID NOs: 12 and 13, respectively; (iv) SEQ ID NOs: 14 and 15, respectively; (v) SEQ ID NOs: 16 and 17, respectively; (vi) SEQ ID NOs: 18 and 19, respectively; (vii) SEQ ID NOs: 20 and 21, respectively; (viii) SEQ ID NOs: 22 and 23, respectively; (ix) SEQ ID NOs: 24 and 25, respectively; (x) SEQ ID NOs: 26 and 27, respectively; (xi) SEQ ID NOs: 28 and 29, respectively; (xii) SEQ ID NOs: 30 and 31, respectively; (xiii) SEQ ID NOs: 32 and 33, respectively; (xiv) SEQ ID NOs: 34 and 35, respectively; (xv) SEQ ID NOs: 36 and 37, respectively; (xvi) SEQ ID NOs: 38 and 39, respectively; (xvii) SEQ ID NOs: 40 and 41, respectively; (xviii) SEQ ID NOs: 42 and 43, respectively; (xix) SEQ ID NOs: 44 and 45, respectively; (xx) SEQ ID NOs: 46 and 47, respectively; (xxi) SEQ ID NOs: 48 and 49, respectively; (xxii) SEQ ID NOs: 50 and 51, respectively; (xxiii) SEQ ID NOs: 52 and 53, respectively; (xxiv) SEQ ID NOs: 54 and 55, respectively; (xxv) SEQ ID NOs: 56 and 57, respectively; (xxvi) SEQ ID NOs: 58 and 59, respectively; (xxvii) SEQ ID NOs: 60 and 61, respectively; (xxviii) SEQ ID NOs: 62 and 63, respectively; (xxix) SEQ ID NOs: 64 and 65, respectively; (xxx) SEQ ID NOs: 66 and 67, respectively; (xxxi) SEQ ID NOs: 68 and 69, respectively; (xxxii) SEQ ID NOs: 70 and 71, respectively; (xxxiii) SEQ ID NOs: 72 and 73, respectively; (xxxiv) SEQ ID NOs: 313 and 314, respectively; (xxxv) SEQ ID NOs: 315 and 316, respectively; (xxxvi) SEQ ID NOs: 317 and 314, respectively; (xxxvii) SEQ ID NOs: 318 and 316, respectively; (xxxviii) SEQ ID NOs: 319 and 314, respectively; (xxxix) SEQ ID NOs: 320 and 316, respectively; (xl) SEQ ID NOs: 321 and 314, respectively; (xli) SEQ ID NOs: 322 and 316, respectively; (xlii) SEQ ID NOs: 323 and 314, respectively; (xliii) SEQ ID NOs: 324 and 316, respectively; (xliv) SEQ ID NOs: 325 and 326, respectively; (xlv) SEQ ID NOs: 327 and 328, respectively; (xlvi) SEQ ID NOs: 325 and 329, respectively; (xlvii) SEQ ID NOs: 330 and 331, respectively; (xlviii) SEQ ID NOs: 325 and 332, respectively; (xlix) SEQ ID NOs: 330 and 333, respectively; (l) SEQ ID NOs: 334 and 332, respectively; (li) SEQ ID NOs: 335 and 336, respectively; (lii) SEQ ID NOs: 337 and 338, respectively; (liii) SEQ ID NOs: 38 and 33, respectively; (liv) SEQ ID NOs: 339 and 340, respectively; (lv) SEQ ID NOs: 339 and 341, respectively; (lvi) SEQ ID NOs: 339 and 342, respectively; (lvii) SEQ ID NOs: 343 and 340, respectively; (lviii) SEQ ID NOs: 343 and 342, respectively; (lix) SEQ ID NOs: 344 and 340, respectively; (lx) SEQ ID NOs: 344 and 341, respectively; (lxi) SEQ ID NOs: 344 and 342, respectively; (lxii) SEQ ID NOs: 345 and 340, respectively; (lxiii) SEQ ID NOs: 345 and 341, respectively; (lxiv) SEQ ID NOs: 345 and 342, respectively; (lxv) SEQ ID NOs: 346 and 347, respectively; (lxvi) SEQ ID NOs: 348 and 316, respectively; (lxvii) SEQ ID NOs: 349 and 347, respectively; (lxviii) SEQ ID NOs: 349 and 350, respectively; (lxix) SEQ ID NOs: 351 and 352, respectively; (lxx) SEQ ID NOs: 349 and 353, respectively; (lxxi) SEQ ID NOs: 346 and 354, respectively; (lxxii) SEQ ID NOs: 349 and 355, respectively; (lxxiii) SEQ ID NOs: 349 and 356, respectively; (lxxiv) SEQ ID NOs: 357 and 316, respectively; (lxxv) SEQ ID NOs: 349 and 358, respectively; (lxxvi) SEQ ID NOs: 349 and 316, respectively; (lxxvii) SEQ ID NOs: 359 and 360, respectively; (lxxviii) SEQ ID NOs: 361 and 362, respectively; (lxxix) SEQ ID NOs: 363 and 316, respectively; (lxxx) SEQ ID NOs: 364 and 365, respectively; (lxxxi) SEQ ID NOs: 349 and 366, respectively; (lxxxii) SEQ ID NOs: 346 and 316, respectively; (lxxxiii) SEQ ID NOs: 346 and 367, respectively; (lxxxiv) SEQ ID NOs: 349 and 368, respectively; (lxxxv) SEQ ID NOs: 369 and 316, respectively; (lxxxvi) SEQ ID NOs: 346 and 370, respectively; (lxxxvii) SEQ ID NOs: 371 and 316, respectively; (lxxxviii) SEQ ID NOs: 372 and 356, respectively; (lxxxix) SEQ ID NOs: 357 and 358, respectively; (xc) SEQ ID NOs: 349 and 373, respectively; (xci) SEQ ID NOs: 346 and 374, respectively; (xcii) SEQ ID NOs: 375 and 316, respectively; (xciii) SEQ ID NOs: 376 and 316, respectively; (xciv) SEQ ID NOs: 346 and 377, respectively; (xcv) SEQ ID NOs: 378 and 379, respectively; (xcvi) SEQ ID NOs: 380 and 381, respectively; (xcvii) SEQ ID NOs: 349 and 382, ​​respectively; (xcviii) SEQ ID NOs: 357 and 383, respectively; (xcix) SEQ ID NOs: 349 and 358, respectively; (c) SEQ ID NOs: 384 and 316, respectively; (ci) SEQ ID NOs: 385 and 316, respectively; (cii) SEQ ID NOs: 357 and 386, respectively; (ciii) SEQ ID NOs: 387 and 388, respectively; (civ) SEQ ID NOs: 359 and 316, respectively; (cv) SEQ ID NOs: 389 and 316, respectively; (cvi) SEQ ID NOs: 390 and 316, respectively; (cvii) SEQ ID NOs: 391 and 392, respectively; (cviii) SEQ ID NOs: 393 and 356, respectively; (cix) SEQ ID NOs: 390 and 392, respectively; (cx) SEQ ID NOs: 357 and 386, respectively; (cxi) SEQ ID NOs: 394 and 395, respectively; (cxii) SEQ ID NOs: 396 and 395, respectively; (cxiii) SEQ ID NOs: 397 and 395, respectively; (cxiv) SEQ ID NOs: 398 and 395, respectively; (cxv) SEQ ID NOs: 401 and 395, respectively; (cxvi) SEQ ID NOs: 402 and 403, respectively; (cxxxiv) SEQ ID NOs: 443 and 341, respectively; (cxxxv) SEQ ID NOs: 444 and 341, respectively; (cxxxvi) SEQ ID NOs: 445 and 341, respectively; (cxxxvii) SEQ ID NOs: 446 and 341, respectively; (cxxxviii) SEQ ID NOs: 447 and 341, respectively; (cxxxix) SEQ ID NOs: 448 and 341, respectively; (cxl) SEQ ID NOs: 449 and 341, respectively; (cxli) SEQ ID NOs: 450 and 341, respectively; (cxlii) SEQ ID NOs: 451 and 341, respectively; (cxliii) SEQ ID NOs: 452 and 341, respectively; (cxliv) SEQ ID NOs: 453 and 341, respectively; (cxlv) SEQ ID NOs: 443 and 454, respectively; (cxlvi) SEQ ID NOs: 443 and 455, respectively; (cxlvii) SEQ ID NOs: 443 and 456, respectively; (cxlviii) SEQ ID NOs: 443 and 457, respectively; (cxlix) SEQ ID NOs: 443 and 458, respectively; or (cxvii) SEQ ID NOs: 443 and 459, respectively.

6. The antigen-binding domain of claim 1 , comprising a VH and a VL comprising the amino acid sequence:

7. The antigen-binding domain of any one of claims 1 to 6, which is capable of crossing the blood-brain barrier (BBB).

8. The antigen-binding domain of any one of claims 1 to 7, which binds to cynomolgus monkey TfR.

9. The antigen-binding domain of any one of claims 1 to 8, which binds to human TfR with an affinity of 500 nM to 10 μM.

10. The antigen-binding domain of any one of claims 1 to 8, which binds to human TfR with an affinity of 2 μM to 8 μM.

11. The antigen-binding domain of any one of claims 1 to 8, which binds to human TfR with an affinity of 2 μM to 5 μM.

12. The antigen-binding domain of any one of claims 1 to 8, which binds to human TfR with an affinity of 750 nM to 2 μM.

13. The antigen-binding domain of any one of claims 1 to 8, which binds to human TfR with an affinity of 50 nM to 500 nM.

14. The antigen-binding domain of any one of claims 1 to 8, which binds to human TfR with an affinity of 100 nM to 250 nM.

15. The antigen-binding domain of any one of claims 1 to 8, which binds to human TfR with an affinity of 1 nM to 50 nM.

16. The antigen-binding domain of any one of claims 1 to 8, which binds to human TfR with an affinity of 6.7 nM to 3.5 μM.

17. The antigen-binding domain of any one of claims 1 to 8, which binds to cynomolgus monkey TfR with an affinity of 38 nM to 2.3 μM.

18. The antigen-binding domain of any one of claims 1 to 8, which binds to human TfR with an affinity of about 3.5 μM.

19. 20. The antigen-binding domain of any one of claims 1 to 19, which binds to cynomolgus monkey TfR with an affinity of about 1.5 μM.

20. The antigen-binding domain of any one of claims 9 to 19, wherein the affinity is measured by high-throughput surface plasmon resonance (SPR) detection.

21. The antigen-binding domain of any one of claims 1 to 20, wherein the antigen-binding domain is internalized into blood-brain barrier epithelial cells, and optionally the blood-brain barrier epithelial cells are HCMEC / D3 cells.

22. The antigen-binding domain of any one of claims 1 to 21, which does not reduce cell surface expression of TfR on HCMEC / D3 cells by more than 60% compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control.

23. The antigen-binding domain of any one of claims 1 to 22, which does not reduce cell surface expression of TfR on HCMEC / D3 cells by more than 40% compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control.

24. The antigen-binding domain of any one of claims 1 to 23, which does not significantly increase cell surface expression of TfR on HCMEC / D3 cells compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control.

25. 25. The antigen-binding domain of any one of claims 1 to 24, which accumulates in the brain of vascular-depleted mice at least 4-fold or at least 5-fold more than an isotype control.

26. 26. An antigen-binding domain according to any one of claims 1 to 25, which specifically binds to human TfR at least 5 times more than it binds to an unrelated protein.

27. 27. An antigen-binding domain according to any one of claims 1 to 26, which specifically binds to cynomolgus monkey TfR at least 5 times more than it binds to an unrelated protein.

28. 28. An antigen-binding domain according to claim 26 or 27, which binds to human TfR at least 5-fold more than its binding to an unrelated protein and / or which specifically binds to cynomolgus monkey TfR at least 5-fold more than its binding to an unrelated protein.

29. The antigen-binding domain of any one of claims 1 to 28, which does not significantly reduce TfR expression levels in the brain of a primate following intravenous administration of the antigen-binding domain.

30. The antigen-binding domain according to any one of claims 1 to 29, comprising a VH and a VL on a single polypeptide chain.

31. The antigen-binding domain of any one of claims 1 to 30, comprising a single-chain variable region fragment (scFv).

32. The antigen-binding domain of claim 31, wherein the scFv is in a VH-linker-VL orientation.

33. The antigen-binding domain of claim 31, wherein the scFv is in a VL-linker-VH orientation.

34. 34. The antigen-binding domain of claim 32 or 33, wherein the linker is (i) about 5 to about 25 amino acids, or about 5 to about 20 amino acids, or about 10 to about 25 amino acids, or about 10 to about 20 amino acids, and / or (ii) comprises the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 183) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 288).

35. 35. The antigen-binding domain of any one of claims 2 to 34, which binds to the apical domain of the human transferrin receptor (TfR), localizes to the brain parenchyma of a subject after peripheral injection, and is not located within a modified CH3 domain.

36. The antigen-binding domain of any one of claims 1 to 33, comprising the amino acid sequence of any one of SEQ ID NOs: 404, 185, 186, 189, 190, 192, 193, 195-259, and 261-284.

37. 30. The antigen-binding domain of any one of claims 1 to 29, comprising a VH on a first polypeptide and a VL on a second polypeptide.

38. 38. The antigen-binding domain of any one of claims 1 to 37, which is a murine, chimeric, humanized, or human antigen-binding domain, optionally a humanized antigen-binding domain.

39. An antigen-binding domain that specifically binds to human TfR, the antigen-binding domain comprising: (i) a VH CDR1, a VH CDR2, and a VH CDR3 of the antigen-binding domain of any one of claims 1 to 36 and 38; or (ii) A VHH comprising the VH of the antigen-binding domain of any one of claims 1 to 36 and 38, wherein the VHH is optionally capable of passing through the blood-brain barrier (BBB).

40. A fusion protein comprising the antigen-binding domain of any one of claims 1 to 39 and a heterologous protein or peptide.

41. the heterologous protein or peptide is beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebrosidase (GC ase or GBA), progranulin (PGRN), prosaposin (PSAP), glycoprotein non-transferase protein B (GPNMB), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 3 (Siglec3), Sialic acid-binding Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4-domain A4A (MS4A4A), transmembrane 4-domain A6A (MS4A6A), or transmembrane protein 106B (TMEM106b), clusterin (APOJ), reelin, ubiquitin protein ligase E3A (U BE3A), tripeptidyl peptidase 1 (CLN2 / TPP1), alpha-L-iduronidase (IDUA), iduronate 2-sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosamide N-acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), N-sulfoglucosamine sulfohydrolase (SGSH), or portions thereof 41. The fusion protein of claim 40, comprising the amino acid sequence:

42. 42. The fusion protein of claim 40 or 41, further comprising an Fc domain.

43. 43. The fusion protein of claim 42, wherein the Fc domain is capable of binding to FcRn.

44. 44. The fusion protein of claim 42 or 43, comprising (i) a single scFv or VHH or Fab antigen-binding domain that binds to human TfR, and (ii) two copies of the heterologous protein or peptide.

45. The fusion protein of claim 44, wherein the single scFv, Fab, or VHH antigen-binding domain that binds to human TfR is linked to the C-terminus of one of the two copies of the heterologous protein or peptide.

46. 45. The fusion protein of claim 44, wherein the two copies of the heterologous protein or peptide are linked to the N-terminus of the Fc domain.

47. 45. The fusion protein of claim 44, wherein the single scFv, Fab, or VHH antigen-binding domain that binds to human TfR is linked to the N-terminus of the Fc domain.

48. 45. The fusion protein of claim 44, wherein the two copies of the heterologous protein or peptide are linked to the C-terminus of the Fc domain.

49. 44. The fusion protein of any one of claims 40 to 43, comprising: (i) an antibody that binds to human TfR, the antibody comprising two heavy chains and two light chains; and (ii) two copies of the heterologous protein or peptide, each copy of the heterologous protein or peptide being linked to the C-terminus of one of the two antibody heavy chains.

50. 44. The fusion protein of any one of claims 40 to 43, comprising: (i) two scFv, Fab, or VHH antigen-binding domains that bind to human TfR; (ii) an Fc domain; and (iii) two copies of the heterologous protein or peptide, wherein the two scFv, Fab, or VHH antigen-binding domains that bind to human TfR are linked to the C-terminus of the Fc domain, and the two copies of the heterologous protein or peptide are linked to the N-terminus of the Fc domain.

51. 44. The fusion protein of any one of claims 40 to 43, comprising: (i) a single scFv, VHH, or Fab antigen-binding domain that binds to human TfR; (ii) an Fc domain; and (iii) a single copy of the heterologous protein or peptide, wherein the single scFv, VHH, or Fab antigen-binding domain that binds to human TfR is linked to the C-terminus of the Fc domain, and the heterologous protein or peptide is linked to the N-terminus of the Fc domain.

52. 44. The fusion protein of any one of claims 40 to 43, comprising: (i) a single scFv, VHH, or Fab antigen-binding domain that binds to human TfR; (ii) an Fc domain; and (iii) a single copy of the heterologous protein or peptide, wherein the single scFv, VHH, or Fab antigen-binding domain that binds to human TfR is linked to the N-terminus of the Fc domain, and the heterologous protein or peptide is linked to the C-terminus of the Fc domain.

53. 44. The fusion protein of any one of claims 40 to 43, comprising: (i) a single scFv, VHH, or Fab antigen-binding domain that binds to human TfR; (ii) an Fc domain; and (iii) a single copy of the heterologous protein or peptide, wherein the single scFv, VHH, or Fab antigen-binding domain that binds to human TfR and the heterologous protein or peptide are both linked to the N-terminus of the Fc domain.

54. 54. The fusion protein of any one of claims 42 to 53, wherein the Fc domain is a heterodimeric Fc and optionally comprises a knob-and-hole mutation.

55. 55. The fusion protein of any one of claims 42 to 54, wherein the Fc is a single-chain monovalent Fc.

56. 56. The fusion protein of any one of claims 42 to 55, wherein the Fc is a modified Fc having a modification as set forth in Table 1 or 2.

57. 57. The fusion protein of any one of claims 40 to 56, wherein the Fc comprises mutations that reduce effector function, and optionally the mutations that reduce effector function include (i) L234A, L235A, and / or P331S, and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S.

58. An antibody comprising an antigen-binding domain according to any one of claims 1 to 39.

59. An antibody or antigen-binding fragment thereof that binds to the same human TfR epitope as the antigen-binding domain of any one of claims 1 to 39.

60. An antibody or antigen-binding fragment thereof that competitively inhibits the binding of the antigen-binding domain of any one of claims 1 to 39 to human TfR.

61. 40. A multispecific protein comprising a first antigen-binding domain that is the antigen-binding domain of any one of claims 1 to 39 linked to a second antigen-binding domain, optionally wherein said second antigen-binding domain specifically binds to a CNS antigen.

62. 40. A multispecific protein comprising the antigen-binding domain of any one of claims 1 to 39 linked to an antibody or antigen-binding fragment thereof, optionally wherein the antibody or antigen-binding fragment thereof specifically binds to a CNS antigen.

63. 63. The multispecific protein of claim 62, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region.

64. 64. The multispecific protein of claim 63, wherein the antigen-binding domain of any one of claims 1 to 39 is linked, optionally via an amino acid linker, to the C-terminus of the heavy chain constant region.

65. 65. The multispecific protein of any one of claims 61 to 64, which is bispecific.

66. 66. The multispecific protein of any one of claims 61 to 65, which is bivalent, trivalent, or tetravalent.

67. 67. The multispecific protein of claim 66, which is bivalent.

68. 67. The multispecific protein of claim 66, wherein the multispecific protein is trivalent, optionally wherein the trivalent protein comprises one antigen binding domain that binds to human TfR and two antigen binding domains that bind to CNS antigens.

69. 67. The multispecific protein of claim 66, wherein the multispecific protein is tetravalent, optionally wherein the tetravalent protein comprises two of the antigen binding domains that bind to human TfR and two antigen binding domains that bind to a CNS antigen.

70. 40. A multispecific protein comprising the antigen-binding domain of any one of claims 1 to 36 and 38 linked to an antibody that is trivalent and bispecific and binds to a CNS antigen, said antibody comprising two heavy chains and two light chains, said antigen-binding domain being an scFv, and said scFv being linked to the C-terminus of one of the two antibody heavy chains, optionally via an amino acid linker.

71. 40. A multispecific protein comprising the antigen-binding domain of any one of claims 1 to 36 and 38 linked to an antibody that is trivalent and bispecific and binds to a CNS antigen, said antibody comprising two heavy chains and two light chains, said antigen-binding domain being an scFv, and said scFv being linked to the N-terminus of one of the two antibody heavy chains, optionally via an amino acid linker.

72. 49. A tetravalent and bispecific multispecific protein comprising two antigen-binding domains according to any one of claims 1 to 36 and 38 and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, each of the two antigen-binding domains being an scFv, Fab or VHH, wherein one of the two antigen-binding domains is linked to the C-terminus of one of the antibody heavy chains, optionally via an amino acid linker, and the other of the antigen-binding domains is linked to the C-terminus of the other of the antibody heavy chains, optionally via an amino acid linker.

73. 49. A tetravalent and bispecific multispecific protein comprising two antigen-binding domains according to any one of claims 1 to 36 and 38 and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, each of the two antigen-binding domains being an scFv, Fab or VHH, wherein one of the two antigen-binding domains is linked to the N-terminus of one of the antibody heavy chains, optionally via an amino acid linker, and the other of the antigen-binding domains is linked to the N-terminus of the other of the antibody heavy chains, optionally via an amino acid linker.

74. 49. A tetravalent and bispecific multispecific protein comprising two antigen-binding domains according to any one of claims 1 to 36 and 38 and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, each of the two antigen-binding domains being an scFv, Fab or VHH, wherein one of the two antigen-binding domains is linked to the N-terminus of one of the antibody heavy chains, optionally via an amino acid linker, and the other of the antigen-binding domains is linked to the C-terminus of the other of the antibody heavy chains, optionally via an amino acid linker.

75. 75. The multispecific protein of claim 73 or 74, wherein the two antigen-binding domains of any one of claims 1 to 36 and 38 are two copies of the same antigen-binding domain.

76. 40. A multispecific protein that is bivalent and bispecific, comprising: (i) an Fc domain; (ii) a single antigen-binding domain of any one of claims 1 to 39 that binds to human TfR and is a single scFv, VHH, or Fab antigen-binding domain linked to the N-terminus of the Fc domain; and (iii) a second antigen-binding domain that specifically binds to a CNS antigen, which is a single scFv, VHH, or Fab linked to the C-terminus of the Fc domain.

77. 40. A multispecific protein that is bivalent and bispecific, comprising: (i) an Fc domain; (ii) a single antigen-binding domain of any one of claims 1 to 39 that binds to human TfR and is a single scFv, VHH, or Fab antigen-binding domain linked to the C-terminus of the Fc domain; and (iii) a second antigen-binding domain that specifically binds to a CNS antigen, which is a single scFv, VHH, or Fab linked to the N-terminus of the Fc domain.

78. 78. The fusion protein of claim 76 or 77, wherein the Fc domain is a heterodimeric Fc and optionally comprises a knob-and-hole mutation.

79. 79. The multispecific protein of any one of claims 76 to 78, wherein the Fc is a modified Fc having one or more modifications according to Table 1 or 2.

80. 80. The multispecific protein of any one of claims 76 to 79, wherein the Fc comprises mutations that reduce effector function, optionally wherein the mutations that reduce effector function include (i) L234A, L235A, and / or P331S, and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S.

81. 76. The multispecific protein of any one of claims 62 to 75, wherein the antibody or antigen-binding fragment thereof comprises a constant region comprising a knob mutation and a constant region comprising a hole mutation.

82. 82. The multispecific protein of claim 81 , wherein said antigen binding domain is linked to said constant region comprising a hole mutation, optionally via an amino acid linker.

83. 82. The multispecific protein of claim 81 , wherein said antigen binding domain is linked, optionally via an amino acid linker, to said constant region comprising a knob mutation.

84. 84. The multispecific protein of claim 82 or 83, wherein the amino acid linker is a glycine-serine linker.

85. 85. The multispecific protein of claim 84, wherein said glycine-serine linker comprises the amino acid sequence (GGGGS) x 3 (SEQ ID NO: 184) or the amino acid sequence (GGSGG) x 3 (SEQ ID NO: 289).

86. 86. The multispecific protein of any one of claims 61 to 85, wherein the CNS antigen is a brain antigen.

87. 87. The multispecific protein of any one of claims 61 to 86, wherein the CNS antigen is not TfR.

88. 88. The multispecific protein of any one of claims 62 to 75 and 81 to 87, wherein the antibody or antigen-binding fragment thereof comprises mutations that reduce effector function, optionally wherein the mutations that reduce effector function include (i) L234A, L235A, and / or P331S, and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S.

89. 89. The multispecific protein of any one of claims 62 to 75 and claims 81 to 88, wherein the antibody or antigen-binding fragment thereof comprises a constant region comprising a knob mutation and a mutation that reduces effector function, optionally wherein the mutation that reduces effector function comprises: (i) L234A, L235A, and / or P331S, and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S.

90. 89. The multispecific protein of any one of claims 62 to 75 and 81 to 88, wherein the antibody or antigen-binding fragment thereof comprises a constant region comprising hole mutations and mutations that reduce effector function, and optionally the mutations that reduce effector function comprise: (i) L234A, L235A, and / or P331S, and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S.

91. 91. The multispecific protein of any one of claims 62 to 75 and 81 to 90, wherein the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

92. 92. The multispecific protein of claim 91 , wherein the IgG antibody or antigen-binding fragment thereof is an IgG1 antibody or antigen-binding fragment thereof or an IgG4 antibody or antigen-binding fragment thereof.

93. Equilibrium dissociation constant (K Ｄ ) and / or binds to human TfR with a K of about 37 nM to about 1.3 μM Ｄ 93. The multispecific protein of any one of claims 61 to 92, which binds to cynomolgus monkey TfR at

94. 94. The multispecific protein of any one of claims 61 to 93, wherein the multispecific protein is internalized into blood-brain barrier epithelial cells at greater than 10-fold or greater than 40-fold compared to internalization by an isotype control, and optionally the blood-brain barrier epithelial cells are HCMEC / D3 cells.

95. 95. The multispecific protein of any one of claims 61 to 94, which does not reduce cell surface expression of TfR on HCMEC / D3 cells by more than 60% compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control.

96. 96. The multispecific protein of any one of claims 61 to 95, which does not reduce cell surface expression of TfR on HCMEC / D3 cells by more than 40% compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control.

97. 97. The multispecific protein of any one of claims 61 to 96, which does not significantly increase cell surface expression of TfR on HCMEC / D3 cells compared to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control.

98. 98. The multispecific protein of any one of claims 61 to 97, which accumulates in the vascular-depleted mouse brain at least 4-fold or at least 5-fold more than an isotype control.

99. 98. The multispecific protein of any one of claims 61, 65-69, 76-80 and 93-97, wherein said second antigen-binding domain specifically binds to human TfR at least 5-fold more than it binds to an unrelated protein.

100. 99. The multispecific protein of any one of claims 61, 65-69, 76-80 and 93-98, wherein said second antigen-binding domain specifically binds to cyno TfR at least 5-fold more than it binds to an unrelated protein.

101. 101. The multispecific protein of claim 99 or 100, which binds to human TfR at least 5-fold greater than its binding to an unrelated protein and / or specifically binds to cynomolgus monkey TfR at least 5-fold greater than its binding to an unrelated protein.

102. The CNS antigen is selected from the group consisting of beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), sialic acid-binding I Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), sialic acid-binding Ig-like lectin 11 (Siglec11), glycoprotein non-metastatic melanoma protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4-domain A4 102. The multispecific protein of any one of claims 61 to 101, wherein the multispecific protein is selected from the group consisting of: MS4A4A, transmembrane 4-domain A6A (MS4A6A), MS4A4E, transmembrane protein 106B (TMEM106b), ubiquitin protein ligase E3A (UBE3A), CR1, ABCA1, ABCA7, HLA-DR1, HLA-DR5, IL1RAP, TREML2, IL-34, SORL1, and ADAM1.

103. 103. The multispecific protein of claim 102, wherein the CNS antigen is MS4A4A, and optionally (i) the antigen binding domain, antibody, or antigen binding domain that binds to MS4A4A comprises a VH comprising the MS4A4A-binding VH sequence of SEQ ID NO: 406 and / or a VL comprising the VL sequence of SEQ ID NO: 407, and / or (ii) the antigen binding domain that binds to human TfR comprises the scFv sequence of SEQ ID NO:

406.

104. 104. The multispecific protein of claim 103, comprising the amino acid sequence of SEQ ID NOs: 405-407.

105. 105. The multispecific protein of any one of claims 61 to 104, which is capable of binding to FcRn.

106. 60. The fusion protein of any one of claims 40 to 57, the antibody or antigen-binding fragment thereof of any one of claims 58 to 60, or the multispecific protein of any one of claims 61 to 105, linked to an imaging agent.

107. A fusion protein according to any one of claims 40 to 57, an antibody or antigen-binding fragment thereof according to any one of claims 58 to 60, or a multispecific protein according to any one of claims 61 to 105, which is capable of crossing the BBB.

108. 106. A composition comprising a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein said first polynucleotide, second polynucleotide, and third polynucleotide encode the multispecific protein of any one of claims 61 to 105, wherein said first polynucleotide encodes a first heavy chain, said second polynucleotide encodes a second heavy chain and the antigen-binding domain that specifically binds to human TfR, and said third polynucleotide encodes a light chain.

109. 106. A composition comprising a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first polynucleotide, the second polynucleotide, and the third polynucleotide encode the multispecific protein of any one of claims 61 to 105, wherein the first polynucleotide encodes a first heavy chain and a first antigen-binding domain that specifically binds to human TfR, the second polynucleotide encodes a second heavy chain and a second antigen-binding domain that specifically binds to human TfR, and the third polynucleotide encodes a light chain, and optionally the first antigen-binding domain that binds to human TfR and the second antigen-binding domain comprise the same amino acid sequence.

110. 110. The composition of claim 108 or 109, wherein the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation.

111. 111. The composition of any one of claims 108 to 110, wherein the ratio of the first polynucleotide, the second polynucleotide, and the third polynucleotide is about 1:3:

6.

112. 110. The composition of claim 108 or 109, wherein the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

113. 106. A composition comprising a first polynucleotide and a second polynucleotide, wherein said first polynucleotide and second polynucleotide encode the multispecific protein of any one of claims 61 to 105, wherein said first polynucleotide encodes a heavy chain and the antigen-binding domain that binds to human TfR, and said second polynucleotide encodes a light chain.

114. A host cell comprising the composition of any one of claims 108 to 113.

115. An isolated polynucleotide molecule comprising a nucleic acid molecule encoding the heavy chain of the antigen-binding domain of any one of claims 1 to 39.

116. An isolated polynucleotide molecule comprising a nucleic acid molecule encoding the light chain variable region of the antigen-binding domain of any one of claims 1 to 39.

117. 117. An isolated vector comprising the polynucleotide of claim 115 and / or the polynucleotide of claim 116.

118. An isolated vector comprising a nucleic acid molecule encoding a heavy chain variable region of the antigen-binding domain of any one of claims 1 to 39, and a nucleic acid molecule encoding a light chain variable region of the antigen-binding domain.

119. A host cell comprising a polynucleotide according to claim 115 or 116 or a vector according to claim 117 or 118.

120. 120. The host cell of any one of claims 114-119, selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture.

121. 121. A method for producing an antigen-binding domain or a multispecific protein, comprising culturing the host cell of any one of claims 114, 119, and 120 so that the antigen-binding domain or multispecific protein is produced, and optionally further comprising isolating the antigen-binding domain or multispecific protein from the culture.

122. 122. An isolated antigen-binding domain or multispecific protein thereof produced by the method of claim 121.

123. 108. A pharmaceutical composition comprising: (i) the antigen-binding domain, fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein of any one of claims 1 to 107; and (ii) a pharmaceutically acceptable carrier.

124. The pharmaceutical composition of claim 123, wherein the concentration of the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein is increased in the brain after administration to a subject compared to an isotype control.

125. 125. The pharmaceutical composition of claim 123 or 124, wherein administration increases delivery of the fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition into the brain by at least 50%, at least 100%, at least 200%, at least 500%, or at least 1000% compared to an isotype control.

126. 126. A method of treating a neurological disease or disorder in a subject, comprising administering to the subject a fusion protein, an antibody or antigen-binding fragment thereof, a multispecific protein, or a pharmaceutical composition according to any one of claims 40 to 107 and claims 123 to 125.

127. 127. The method of claim 126, wherein administration increases delivery of the fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition into the brain by at least 50%, at least 100%, at least 200%, at least 500%, or at least 1000% compared to an isotype control.

128. 128. The method of claim 126 or 127, wherein administration increases delivery of the fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition to the frontal cortex, entorhinal cortex, and / or hippocampus.

129. 129. The method of any one of claims 126 to 128, wherein the neurological disease or disorder is selected from a neuropathic disorder, a neurodegenerative disease, a cancer, an ocular disorder, a seizure disorder, a lysosomal storage disorder, an amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation.

130. 130. The method of claim 129, wherein the neurological disease or disorder is selected from Alzheimer's disease (AD), Huntington's disease, dystonia, ataxia, Bell's palsy, stroke, dementia, dementia with Lewy bodies, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, encephalitis, traumatic brain injury, and limbic-predominant age-related TDP-43 encephalopathy (LATE).

131. 131. The method of claim 130, wherein the dementia is frontotemporal dementia (FTD).

132. 131. The method of claim 130, wherein the neurological disease or disorder is Alzheimer's disease.

133. 133. The method of claim 132, wherein the Alzheimer's disease is early-onset Alzheimer's disease, prodromal Alzheimer's disease, mild Alzheimer's disease, or late-onset Alzheimer's disease.

134. 131. The method of claim 130, wherein the neurological disease or disorder is Parkinson's disease.

135. 127. The method of claim 126, wherein the neurological disease or disorder is frontotemporal epilepsy.

136. 127. The method of claim 126, wherein the neurological disease or disorder is autism.

137. 127. The method of claim 126, wherein the neurological disease or disorder is lissencephaly.

138. 126. A method of treating a lysosomal storage disease in a subject, comprising administering to the subject a fusion protein, an antibody or antigen-binding fragment thereof, a multispecific protein, or a pharmaceutical composition according to any one of claims 40 to 107 and claims 123 to 125.

139. 139. The method of claim 138, wherein the lysosomal storage disease is selected from Gaucher disease, ceroid lipofuscinosis (Batten disease), mucopolysaccharidosis (MPS) type I, MPS type II, and MPS type III.

140. 126. A method for transporting a fusion protein, an antibody or antigen-binding fragment thereof, or a multispecific protein across the BBB in a subject, the method comprising administering to the subject a fusion protein, an antibody or antigen-binding fragment thereof, a multispecific protein, or a pharmaceutical composition according to any one of claims 40 to 107 and 123 to 125.

141. 141. The method of claim 140, wherein the concentration of the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein is increased in the brain after administration compared to an isotype control.

142. 142. The method of claim 140 or 141, wherein the concentration of the fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition in the brain is increased by at least 50%, at least 100%, at least 200%, at least 500%, or at least 1000% compared to an isotype control.

143. 143. The method of any one of claims 140 to 142, wherein administration of the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein does not result in more than a 10% decrease in reticulocyte count in the subject compared to administration of an isotype control.

144. 144. The method of claim 143, wherein administration of the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein does not result in a decrease in reticulocyte count in the subject compared to an isotype control.

145. 106. A method of increasing the concentration of a CNS-binding antigen in the CSF of a subject, comprising administering to the subject a multispecific protein of any one of claims 61 to 105, wherein the concentration of the CNS-binding antigen is increased compared to administering the CNS-binding antigen alone to the subject.

146. 107. A method for imaging a CNS antigen in a subject, comprising administering to the subject a fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein described in claim 106, and determining the location of the imaging agent within the subject.

147. A method for detecting a CNS antigen in vitro, comprising contacting an in vitro sample with the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein described in claim 106, and determining the location of the imaging agent in the sample.

148. 148. Use of a fusion protein, an antibody or antigen-binding fragment thereof, a multispecific protein, or a pharmaceutical composition according to any one of claims 40 to 107 and 123 to 125 in a method according to any one of claims 126 to 147.

149. 148. A fusion protein, an antibody or antigen-binding fragment thereof, a multispecific protein, or a pharmaceutical composition according to any one of claims 40 to 107 and 123 to 125 for use in a method according to any one of claims 126 to 147.

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