Transferrin receptor antigen binding domains and uses thereof

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 369,898, filed on July 29, 2022; U.S. Provisional Application No. 63 / 374,967, filed on September 8, 2022; U.S. Provisional Application No. 63 / 489,693, filed on March 10, 2023; U.S. Provisional Application No. 63 / 495,511, filed on April 11, 2023; and U.S. Provisional Application No. 63 / 513,820, filed on July 14, 2023; each of which is incorporated herein by reference in its entirety.

[0003] Reference to electronically submitted sequence listing

[0004] The contents of the electronically submitted Sequence Listing (Name: 4503_022PC05_SeqListing_ST26.xml; Size: 660,697 bytes; and Creation Date: July 13, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure relates to antigen binding domains that specifically bind to human transferrin receptor (TfR).These antigen binding domains can cross the blood-brain barrier and can transport other agents (e.g., therapeutically active agents) associated with the antigen binding domain across the blood-brain barrier. Background Art

[0006] The passive transfer of substances from the blood to the brain is limited by the blood-brain barrier (BBB). The BBB precisely controls the homeostasis of the central nervous system (CNS), thereby achieving proper neuronal function, and also protects neural tissue from toxins and pathogens. Changes in the BBB are an important part of the pathology and progression of different neurological diseases. However, the BBB has problems in delivering therapeutic agents to the CNS. Although recombinant protein and antibody therapeutic agents have achieved great success outside the CNS, such biologics cannot effectively pass through the BBB. Therefore, the delivery of some therapeutic agents to the CNS depends on the direct injection of therapeutic agents into the CNS. However, such injections are invasive procedures, and their efficacy is limited by the rapid output of cerebrospinal fluid (CSF) containing therapeutic agents from the brain to the blood. Alternatively, therapeutic agents intended for the CNS can be systemically administered at high doses to allow the therapeutic agent to fully penetrate the BBB. However, due to the high doses around or the increased manufacturing and deployment burdens to achieve high doses, this method may produce unexpected effects. Therefore, there is a need for improved products and methods for delivering therapeutic agents across the BBB. Summary of the invention

[0007] Provided herein are antigen binding domains, fusion proteins, antibodies, and multispecific proteins that specifically bind to human transferrin receptor (TfR), and methods of making and using the same.

[0008] In some aspects, provided herein is an antigen binding domain that specifically binds to the apical domain of human transferrin receptor (TfR), wherein the antigen binding domain localizes to the brain parenchyma of a subject following peripheral injection, and wherein the antigen binding domain is not within a modified CH3 domain.

[0009] In some aspects, provided herein is an antigen binding domain that specifically binds to human transferrin receptor (TfR), wherein the antigen binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VHCDR3, and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequence comprising the following amino acid sequences: SEQ ID NOs: 94, 415, 418, 423, 150, and 151, respectively; SEQ ID NOs: 74-76 and 135-137, respectively; SEQ ID NOs: 77-79 and 138-140, respectively; SEQ ID NOs: 80-82 and 135-137, respectively; SEQ ID NOs: 83-85 and 141-143, respectively; SEQ ID NOs: 74, 86, 76, and 135-137, respectively; SEQ ID NOs: 87-89 and 143-145, respectively; SEQ ID NOs: NO:90-92 and 146-148, respectively; SEQ ID NO:83, 93, 85 and 141-143, respectively; SEQ ID NO:94-96 and 149-151, respectively; SEQ ID NO:97, 86, 98 and 135-137, respectively; SEQ ID NO:99, 100, 101 and 152-154, respectively; SEQ ID NO:99, 102, 103 and 155-157, respectively; SEQ ID NO:104-106, 138, 158 and 159, respectively; SEQ ID NO:107-109 and 160-162, respectively; SEQ ID NO:99, 110, 103 and 155-157, respectively; SEQ ID NO:99, 111, 112, 152, 163 and 164, respectively; SEQ ID NO: NO:90, 113, 114, 165, 166 and 157; SEQ ID NO:83, 115, 85, 141, 142 and 167, respectively; SEQ ID NO:116, 110, 103 and 155-157, respectively; SEQ ID NO:117-119 and 168-170, respectively; SEQ ID NO:116, 110, 103 and 168-170, respectively; SEQ ID NO:90, 120, 114, 165, 166 and 157, respectively; SEQ ID NO:90, 113, 114, 171, 166 and 157, respectively; SEQ ID NO:97, 121, 122, 135, 136 and 172, respectively; SEQ ID NO:74, 123, 124, 173, 136 and 137, respectively; SEQ ID NO: NO:74, 125, 126, 174, 136 and 137;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 179-181, 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; NO:99,306,103,307,309 and 157; SEQ ID NO:99,306,103,310,311 and 157, respectively; SEQ ID NO:99,305,103,310,311 and 157, respectively; SEQ ID NO:99,312,103,307,308 and 157, respectively; SEQ ID NO:99,312,103,307,309 and 157, respectively; SEQ ID NO:99,312,103,310,311 and 157, respectively; SEQ ID NO:94,95,418,421,150 and 151, respectively; SEQ ID NO:94,412,419,422,150 and 151, respectively; SEQ ID NO:94, 412, 420, 422, 150 and 151, respectively; SEQ ID NO:94, 413, 418, 422, 150 and 151, respectively; SEQ ID NO:94, 414, 418, 422, 150 and 151, respectively; SEQ ID NO:94, 416, 418, 422, 150 and 151, respectively; SEQ ID NO:94, 417, 418, 424, 150 and 151, respectively; SEQ ID NO:460, 312, 103, 307, 309 and 157, respectively; SEQ ID NO:460, 312, 461, 307, 309 and 157, respectively; SEQ ID NO:460, 312, 462, 307, 309 and 157, respectively; NO:460, 312, 463, 307, 309 and 157; SEQ ID NO:460, 312, 464, 307, 309 and 157, respectively; SEQ ID NO: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, 103, 472, 309 and 157, respectively; SEQ ID NOs: 460, 312, 103, 473, 309 and 157, respectively; NO:460, 312, 103, 474, 309 and 157; SEQ ID NO:460, 312, 103, 475, 309 and 157, respectively; SEQ ID NO:460, 312, 103, 476, 309 and 157, respectively; or SEQ ID NO:460, 312, 103, 471, 309 and 157, respectively. ;

[0010] In some aspects, the antigen binding domain comprises a VH and a VL, wherein the VH and the VL comprise 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 following amino acid sequences: 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, respectively; SEQ ID NOs: 38 and 39, respectively; SEQ ID NOs: 31 and 32, 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; SEQ ID NOs:68 and 69, respectively; 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 320, respectively; NO:319 and 314; SEQ ID NO:320 and 316, respectively; SEQ ID NO:321 and 314, respectively; SEQ ID NO:322 and 316, respectively; SEQ ID NO:323 and 314, respectively; SEQ ID NO:324 and 316, respectively; SEQ ID NO:325 and 326, respectively; SEQ ID NO:327 and 328, respectively; SEQ ID NO: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, respectively; SEQ ID NOs: 345 and 340, respectively; SEQ ID NOs: 345 and 341, respectively; SEQ ID NOs: 345 and 342, respectively; NO:346 and 347; SEQ ID NO:348 and 316, respectively; SEQ ID NO:349 and 347, respectively; SEQ ID NO:349 and 350, respectively; SEQ ID NO:351 and 352, respectively; SEQ ID NO:349 and 353, respectively; SEQ ID NO:346 and 354, respectively; SEQ ID NO:349 and 355, respectively; SEQ ID NO:349 and 356, respectively; SEQ ID NO:357 and 316, respectively; SEQ ID NO:349 and 358, respectively; SEQ ID NO:349 and 316, respectively; SEQ ID NO:359 and 360, respectively; SEQ ID NO:361 and 362, respectively; SEQ ID NO:363 and 316, respectively; SEQ ID NO:364 and 365, respectively; SEQ ID NO:349 and 366, respectively; SEQ ID NO:346 and 316, respectively; SEQ ID NO: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; 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, respectively; SEQ ID NOs: 394 and 395, respectively; SEQ ID NOs: 396 and 395, respectively; NO:397 and 395; SEQ ID NO:398 and 395, respectively; SEQ ID NO:401 and 395, respectively; SEQ ID NO:402 and 403, respectively; SEQ ID NO:443 and 341, respectively; SEQ ID NO:444 and 341, respectively; SEQ ID NO:445 and 341, respectively; SEQ ID NO:446 and 341, respectively; SEQ ID NO:447 and 341, respectively; SEQ ID NO:448 and 341, respectively; SEQ ID NO:449 and 341, respectively; SEQ ID NO:450 and 341, respectively; SEQ ID NO:451 and 341, respectively; SEQ ID NO:452 and 341, respectively; SEQ ID NO:453 and 341, respectively; SEQ ID NO:443 and 454, respectively; SEQ ID NO:443 and 455, respectively; SEQ ID NO:443 and 456, respectively; SEQ ID NO:443 and 457, respectively; SEQ ID NO:458 and 341, respectively; SEQ ID NO:459 and 341, respectively; SEQ ID NO:460 and 341, respectively; SEQ ID NO:461 and 341, respectively; SEQ ID NO:462 and 341, respectively; SEQ ID NO:463 and 341, respectively; SEQ ID NO:464 and 341, respectively; SEQ ID NO:465 and 341, respectively; SEQ ID NO:466 and 341, respectively; SEQ ID NO:467 and 341, respectively; ID NO: 443 and 458; or SEQ ID NO: 443 and 459, respectively.;

[0011] In some aspects, provided herein is an antigen binding domain that specifically binds to human TfR, wherein the antigen binding domain comprises VH and VL, wherein the VH comprises the following amino acid sequence: SEQ ID NO: 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, 334, 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.

[0012] In some aspects, provided herein is an antigen binding domain that specifically binds to human TfR, wherein the antigen binding domain comprises VH and VL, wherein the VL comprises the following amino acid sequence: SEQ ID NO: 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, 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.

[0013] In some aspects, the antigen binding domains provided herein comprise a VH and a VL comprising the following amino acid sequences: 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, 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; NO:46 and 47; SEQ ID NO:48 and 49, respectively; SEQ ID NO:50 and 51, respectively; SEQ ID NO:52 and 53, respectively; SEQ ID NO:54 and 55, respectively; SEQ ID NO:56 and 57, respectively; SEQ ID NO:58 and 59, respectively; SEQ ID NO:60 and 61, respectively; SEQ ID NO:62 and 63, respectively; SEQ ID NO:64 and 65, respectively; SEQ ID NO:66 and 67, respectively; SEQ ID NO:68 and 69, respectively; SEQ ID NO:70 and 71, respectively; SEQ ID NO:72 and 73, respectively; SEQ ID NO:313 and 314, respectively; SEQ ID NO:315 and 316, respectively; SEQ ID NO:317 and 314, respectively; SEQ ID NO:318 and 316, respectively; SEQ ID NO:319 and 314, respectively; SEQ ID NO:320 and 316, respectively; SEQ ID NO:321 and 314, respectively; SEQ ID NO:322 and 323, respectively; NO:322 and 316; SEQ ID NO:323 and 314, respectively; SEQ ID NO:324 and 316, respectively; SEQ ID NO:325 and 326, respectively; SEQ ID NO:327 and 328, respectively; SEQ ID NO:325 and 329, respectively; SEQ ID NO:330 and 331, respectively; SEQ ID NO:325 and 332, respectively; SEQ ID NO: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: 350 and 351, respectively; SEQ ID NOs: 352 and 353, respectively; NO:349 and 350; SEQ ID NO:351 and 352, respectively; SEQ ID NO:349 and 353, respectively; SEQ ID NO:346 and 354, respectively; SEQ ID NO:349 and 355, respectively; SEQ ID NO:349 and 356, respectively; SEQ ID NO:357 and 316, respectively; SEQ ID NO:349 and 358, respectively; SEQ ID NO:349 and 316, respectively; SEQ ID NO:359 and 360, respectively; SEQ ID NO:361 and 362, respectively; SEQ ID NO:363 and 316, respectively; SEQ ID NO:364 and 365, respectively; SEQ ID NO:349 and 366, respectively; SEQ ID NO:346 and 316, respectively; SEQ ID NO:346 and 367, respectively; SEQ ID NO:349 and 368, respectively; SEQ ID NO:369 and 316, respectively; SEQ ID NO:346 and 370, respectively; SEQ ID NO:371 and 372, respectively; SEQ ID NO:373 and 374, 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; 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, respectively; 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 402, respectively; NO:402 and 403; SEQ ID NO:443 and 341, respectively; SEQ ID NO:444 and 341, respectively; SEQ ID NO:445 and 341, respectively; SEQ ID NO:446 and 341, respectively; SEQ ID NO:447 and 341, respectively; SEQ ID NO:448 and 341, respectively; SEQ ID NO:449 and 341, respectively; SEQ ID NO:450 and 341, respectively; SEQ ID NO:451 and 341, respectively; SEQ ID NO:452 and 341, respectively; SEQ ID NO:453 and 341, respectively; SEQ ID NO:443 and 454, respectively; SEQ ID NO:443 and 455, respectively; SEQ ID NO:443 and 456, respectively; SEQ ID NO:443 and 457, respectively; SEQ ID NO:443 and 458, respectively; or SEQ ID NO:443 and 459, respectively. ;

[0014] In some aspects, the antigen binding domain specifically binds to the apical domain of human transferrin receptor (TfR), the antigen binding domain is localized to the brain parenchyma of the subject after peripheral injection, and the antigen binding domain is not within the modified CH3 domain.

[0015] In some aspects, the antigen binding domain can pass through the blood-brain barrier (BBB). In some aspects, the antigen binding domain is bound to cynomolgus monkey TfR. In some aspects, the antigen binding domain is internalized in blood-brain barrier epithelial cells. In some aspects, the blood-brain barrier epithelial cells are HCMEC / D3 cells.

[0016] In some aspects, the antigen binding domain binds to human TfR with an affinity between 500nM and 10μM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 2μM and 8μM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 2μM and 5μM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 750nM and 2μM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 50nM and 500nM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 100nM and 250nM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 1nM and 50nM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 6.7nM and 3.5μM. In some aspects, the antigen binding domain binds to cynomolgus monkey TfR with an affinity of 38nM to 2.3μM. In some aspects, the antigen binding domain binds to human TfR with an affinity of about 3.5μM. In some aspects, the antigen binding domain binds to cynomolgus monkey TfR with an affinity of about 1.5μM. In some aspects, affinity is measured by high-throughput surface plasmon resonance (SPR) detection.

[0017] In some aspects, relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, antigen binding domains will not reduce the cell surface expression of TfR on HCMEC / D3 cells by more than 60%. In some aspects, relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, antigen binding domains will not reduce the cell surface expression of TfR on HCMEC / D3 cells by more than 40%. In some aspects, relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, antigen binding domains will not significantly increase the cell surface expression of TfR on HCMEC / D3 cells. In some aspects, in the mouse brain of vascular depletion, the accumulation of antigen binding domains is at least 4 times or at least 5 times that of isotype controls.

[0018] In some aspects, the specific binding of the antigen binding domain to human TfR is at least 5 times that of the binding to unrelated proteins. In some aspects, the specific binding of the antigen binding domain to cynomolgus monkey TfR is at least 5 times that of the binding to unrelated proteins. In some aspects, the binding of the antigen binding domain to human TfR is at least 5 times that of the binding to unrelated proteins and / or the specific binding to cynomolgus monkey TfR is at least 5 times that of the binding to unrelated proteins. In some aspects, after intravenous administration of the antigen binding domain, the antigen binding domain does not significantly reduce the TfR expression level in the primate brain.

[0019] In some aspects, the antigen-binding domain comprises VH and VL on a single polypeptide chain. In some aspects, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some aspects, scFv is in the orientation of VH-joint-VL. In some aspects, scFv is in the orientation of VL-joint-VH. In some aspects, the joint 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 aspects, the joint comprises GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 183) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 288) amino acid sequence. In some aspects, the antigen-binding domain comprises VH on the first polypeptide and VL on the second polypeptide.

[0020] In some aspects, 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.

[0021] In some aspects, the antigen binding domain is a murine, chimeric, humanized or human antigen binding domain, optionally wherein the antigen binding domain is a humanized antigen binding domain.

[0022] 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) the VH CDR1, VH CDR2, and VHCDR3 of the antigen binding domain provided herein, or (ii) the VH of the antigen binding domain provided herein, optionally wherein the VHH is capable of crossing the blood-brain barrier (BBB).

[0023] In some aspects, a fusion protein is provided herein, which comprises an antigen binding domain and a heterologous protein or peptide provided herein. In some aspects, a heterologous protein or peptide provided herein comprises the following amino acid sequence: β-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), prosphingolipid activator protein (PSAP), glycoprotein non-transfer protein white 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 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 A 6A (MS4A6A) or transmembrane protein 106B (TMEM106b), clusterin (APOJ), reelin, ubiquitin protein ligase E3A (UBE3A), tripeptidyl peptidase 1 (CLN2 / TPP1), α-L-iduronidase (IDUA), iduronate 2-sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-α-glucosaminide N-acetyltransferase (HGSNAT), N-acetyl-α-glucosaminidase (NAGLU), or N-sulfoglucosamine sulfohydrolase (SGSH), or a portion thereof.

[0024] In some aspects, the present invention provides a fusion protein further comprising an Fc domain. In some aspects, the Fc domain is capable of binding to FcRn.

[0025] In some aspects, a fusion protein is provided herein, comprising (i) a single scFv or VHH or Fab antigen binding domain that is bound to human TfR, and (ii) two copies of a heterologous protein or peptide. In some aspects, the fusion protein comprising a single scFv, Fab or VHH antigen binding domain that is bound to human TfR is connected to the C-terminal of one of the two copies of a heterologous protein or peptide. In some aspects, two copies of a heterologous protein or peptide are connected to the N-terminal of the Fc domain. In some aspects, a single scFv, Fab or VHH antigen binding domain that is bound to human TfR is connected to the N-terminal of the Fc domain of the fusion protein provided herein. In some aspects, two copies of a heterologous protein or peptide are connected to the C-terminal of the Fc domain of the fusion protein provided herein.

[0026] In some aspects, a fusion protein is provided herein, comprising (i) an antibody that binds to human TfR, wherein the antibody comprises 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 connected to the C-terminus of one of the two antibody heavy chains. In some aspects, a fusion protein is provided herein, 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 connected to the C-terminus of the Fc domain, and the two copies of the heterologous protein or peptide are connected to the N-terminus of the Fc domain. In some aspects, a fusion protein is provided herein, 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 connected to the C-terminus of the Fc domain, and the heterologous protein or polypeptide is connected to the N-terminus of the Fc domain. In some aspects, a fusion protein is provided herein, 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 connected to the N-terminus of the Fc domain, and the heterologous protein or polypeptide is connected to the C-terminus of the Fc domain. In some aspects, 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 and the heterologous protein or peptide are both connected to the N-terminus of the Fc domain.

[0027] In some aspects, the Fc domain is a heterodimeric Fc, optionally comprising a knob and hole mutation. In some aspects, Fc is a single chain monovalent Fc. In some aspects, Fc is a modified Fc with one or more modifications listed in Table 1 or 2. In some aspects, Fc comprises 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.

[0028] In some aspects, an antibody is provided herein, comprising an antigen binding domain provided herein. In some aspects, an antibody or antigen binding fragment thereof is provided herein, which binds to the same human TfR epitope as the antigen binding domain provided herein. In some aspects, an antibody or antigen binding fragment thereof is provided herein, which competitively inhibits the binding of the antigen binding domain provided herein to human TfR.

[0029] In some aspects, a multispecific protein is provided herein, comprising a first antigen binding domain, the first antigen binding domain being an antigen binding domain provided herein connected to a second antigen binding domain. In some aspects, the second antigen binding domain specifically binds to a CNS antigen. In some aspects, a multispecific protein is provided herein, comprising an antigen binding domain provided herein connected to an antibody or its antigen binding fragment. In some aspects, an antibody or its antigen binding fragment specifically binds to a CNS antigen. In some aspects, an antibody or its antigen binding fragment comprises a heavy chain constant region. In some aspects, the antigen binding domain provided herein is optionally connected to the C-terminus of the heavy chain constant region via an amino acid linker. In some aspects, the multispecific protein is bispecific. In some aspects, the multispecific protein is divalent, trivalent or tetravalent. In some aspects, the multispecific protein is divalent. In some aspects, the multispecific protein is trivalent, optionally wherein the trivalent protein comprises an antigen binding domain bound to human TfR and two antigen binding domains bound to CNS antigens. In some aspects, the multispecific protein is tetravalent, optionally wherein the tetravalent protein comprises two antigen binding domains that bind to human TfR and two antigen binding domains that bind to a CNS antigen.

[0030] In some aspects, provided herein is a multispecific protein that is trivalent and bispecific and comprises 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 a scFv linked to the C-terminus of one of the two antibody heavy chains, optionally via an amino acid linker.

[0031] In some aspects, provided herein is a multispecific protein that is trivalent and bispecific and comprises 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 a scFv linked to the N-terminus of one of the two antibody heavy chains, optionally via an amino acid linker.

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

[0033] In some aspects, provided herein is a multispecific protein that is tetravalent and bispecific and comprises 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, wherein each of the two antigen binding domains is a scFv, Fab or VHH, wherein one of the two antigen binding domains is optionally linked to the N-terminus of one of the antibody heavy chains via an amino acid linker, and wherein the other antigen binding domain is optionally linked to the N-terminus of the other antibody heavy chain via an amino acid linker.

[0034] In some aspects, provided herein is a multispecific protein that is tetravalent and bispecific and comprises 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, wherein each of the two antigen binding domains is a scFv, Fab or VHH, wherein one of the two antigen binding domains is optionally linked to the N-terminus of one of the antibody heavy chains via an amino acid linker, and wherein the other antigen binding domain is optionally linked to the C-terminus of the other antibody heavy chain via an amino acid linker.

[0035] In some aspects, the two antigen binding domains provided herein are two copies of the same antigen binding domain.

[0036] In some aspects, provided herein is a multispecific protein that is bivalent and bispecific, comprising: (i) an Fc domain, (ii) a single antigen binding domain provided herein, wherein the antigen binding domain is a single scFv, VHH or Fab antigen binding domain that binds to human TfR and is connected to the N-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 connected to the C-terminus of the Fc domain.

[0037] In some aspects, provided herein is a multispecific protein that is bivalent and bispecific, comprising: (i) an Fc domain, (ii) a single antigen binding domain provided herein, wherein the antigen binding domain is a single scFv, VHH or Fab antigen binding domain that binds to human TfR and is 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 N-terminus of the Fc domain.

[0038] In some aspects, the Fc domain is a heterodimeric Fc, optionally comprising knob and hole mutations. In some aspects, the Fc is a modified Fc with one or more modifications listed in Table 1 or 2. In some aspects, the Fc comprises 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. In some aspects, the antibody or its antigen-binding fragment comprises 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. In some aspects, 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. In some aspects, the antibody or antigen-binding fragment thereof comprises a constant region comprising a hole 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.

[0039] In some aspects, the antibody or antigen-binding fragment thereof comprises a constant region containing a knob mutation and a constant region containing a hole mutation. In some aspects, the antigen binding domain is optionally linked to the constant region containing the hole mutation via an amino acid linker. In some aspects, the antigen binding domain is optionally linked to the constant region containing the knob mutation via an amino acid linker.

[0040] In some aspects, the amino acid linker is a glycine-serine linker. In some aspects, the 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).

[0041] In some aspects, the CNS antigen is a brain antigen. In some aspects, the CNS antigen is not TfR.

[0042] In some aspects, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof. In some aspects, 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.

[0043] In some aspects, the multispecific protein binds human TfR with an equilibrium dissociation constant (KD) of about 65 nM to about 4 μM and / or binds cynomolgus monkey TfR with a KD of about 37 nM to about 1.3 μM.

[0044] In some aspects, the internalization of multispecific proteins in blood-brain barrier epithelial cells is greater than 10 times or greater than 40 times compared with the internalization of isotype control, and optionally wherein blood-brain barrier epithelial cells are HCMEC / D3 cells. In some aspects, relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, multispecific proteins will not reduce the cell surface expression of TfR on HCMEC / D3 cells by more than 60%. In some aspects, relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, multispecific proteins will not reduce the cell surface expression of TfR on HCMEC / D3 cells by more than 40%. In some aspects, relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, multispecific proteins will not significantly increase the cell surface expression of TfR on HCMEC / D3 cells.

[0045] In some aspects, in the vascular depleted mouse brain, the accumulation of multispecific proteins is at least 4 times or 5 times that of the isotype control. In some aspects, the specific binding of the second antigen binding domain to human TfR is at least 5 times the binding to unrelated proteins. In some aspects, the specific binding of the second antigen binding domain to cynomolgus monkey TfR is at least 5 times the binding to unrelated proteins. In some aspects, the binding of the second antigen binding domain to human TfR is at least 5 times the binding to unrelated proteins and / or the specific binding to cynomolgus monkey TfR is at least 5 times the binding to unrelated proteins.

[0046] In some aspects, 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-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 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 A 6A (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 aspects, the CNS antigen is MS4A4A, optionally wherein (i) the antigen binding domain, antibody or antigen binding domain that binds to MS4A4A comprises VH and / or VL, the VH comprises the MS4A4A binding VH sequence of SEQ ID NO: 406, the VL comprises 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 SEQID NO: 406.

[0047] In some aspects, the multispecific protein comprises the amino acid sequence of SEQ ID NOs: 405-407.

[0048] In some aspects, the multispecific protein is capable of binding FcRn.

[0049] In some aspects, the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein is linked to an imaging agent.

[0050] In some aspects, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are capable of crossing the BBB.

[0051] In some aspects, provided herein is 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 a multi-specific 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.

[0052] In some aspects, provided herein is 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 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, optionally wherein the first antigen binding domain and the second antigen binding domain that bind to human TfR comprise the same amino acid sequence.

[0053] In some aspects, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation.

[0054] In some aspects, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation. In some aspects, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0055] In some aspects, the ratio of the first polynucleotide, the second polynucleotide, and the third polynucleotide is about 1:3:6.

[0056] In some aspects, provided herein is a composition comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide 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 wherein the second polynucleotide encodes a light chain.

[0057] In some aspects, provided herein is a host cell comprising a composition provided herein.

[0058] In some aspects, a kind of isolated polynucleotide is provided herein, which comprises a nucleic acid molecule encoding the heavy chain of the antigen binding domain provided herein. In some aspects, a kind of isolated polynucleotide is provided herein, which comprises a nucleic acid molecule encoding the light chain variable region of the antigen binding domain provided herein.

[0059] In some aspects, provided herein is an isolated vector comprising a polynucleotide provided herein.

[0060] 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 an antigen binding domain.

[0061] In some respects, a host cell is provided herein, which comprises a polynucleotide provided herein or a carrier provided herein. In some respects, the host cell is selected from the group consisting of: Escherichia coli (E.coli), Pseudomonas (Pseudomonas), Bacillus (Bacillus), Streptomyces (Streptomyces), Saccharomyces, CHO, YB / 20, NSO, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells and human cells in tissue culture.

[0062] In some aspects, provided herein is a method of producing an antigen binding domain or a multispecific protein, comprising culturing a host cell provided herein so as to produce the antigen binding domain or the multispecific protein, optionally wherein the method further comprises isolating the antigen binding domain or the multispecific protein from the culture.

[0063] In some aspects, provided herein is an isolated antigen binding domain or a multispecific protein thereof produced by the methods provided herein.

[0064] In some aspects, a pharmaceutical composition is provided herein, comprising (i) an antigen binding domain, a fusion protein, an antibody or its antigen binding fragment, or a multi-specific protein provided herein, and (ii) a pharmaceutically acceptable carrier. In some aspects, compared with an isotype control, the concentration of the fusion protein, antibody or its antigen binding fragment, or multi-specific protein in the brain increases after administration to the subject. In some aspects, compared with an isotype control, the administration increases the delivery of the fusion protein, antibody or its antigen binding fragment, multi-specific protein, or pharmaceutical composition to the brain by at least 50%, at least 100%, at least 200%, at least 500%, or at least 1000%.

[0065] In some aspects, there is provided herein a method for treating a neurological disease or disorder of a subject, comprising administering to the subject a fusion protein, an antibody or its antigen-binding fragment, a multi-specific protein, or a pharmaceutical composition provided herein. In some aspects, compared with an isotype control, the administration increases the delivery of a fusion protein, an antibody or its antigen-binding fragment, a multi-specific protein, or a pharmaceutical composition to the brain by at least 50%, at least 100%, at least 200%, at least 500%, or at least 1000%. In some aspects, the administration increases the delivery of a fusion protein, an antibody or its antigen-binding fragment, a multi-specific protein, or a pharmaceutical composition to the frontal cortex, the entorhinal cortex, and / or the hippocampus.

[0066] In some aspects, the nervous system disease or disorder is selected from a neuropathic disorder, a neurodegenerative disease, cancer, an ocular disease disorder, epileptic seizures, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, and CNS inflammation. In some aspects, the nervous system disease or disorder is selected from Alzheimer's disease (AD), Huntington's disease, dystonia, ataxia, Bell's palsy, stroke, dementia, Lewy body dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman's 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 aspects, the neurological disease or disorder is Alzheimer's disease. In some aspects, 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 aspects, the neurological disease or disorder is Parkinson's disease. In some aspects, the neurological disease or disorder is frontotemporal lobe epilepsy. In some aspects, the neurological disease or disorder is autism. In some aspects, the neurological disease or disorder is lissencephaly.

[0067] In some aspects, a method for treating a lysosomal storage disease in a subject is provided herein, comprising administering to the subject a fusion protein, an antibody or its antigen-binding fragment, a multispecific protein, or a 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.

[0068] In some aspects, provided herein is a method of transporting a fusion protein, an antibody or antigen-binding fragment thereof, or a multispecific protein across the BBB of a subject, comprising administering to the subject a fusion protein, an antibody or antigen-binding fragment thereof, a multispecific protein, or a pharmaceutical composition provided herein.

[0069] In some aspects, the concentration of the fusion protein, antibody or antigen binding fragment thereof, or multispecific protein in the brain is increased after administration compared to an isotype control. In some aspects, 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.

[0070] In some aspects, administration of the fusion protein, antibody or antigen binding fragment thereof, or multispecific protein does not result in a greater than 10% decrease in reticulocyte count in a subject compared to administration of an isotype control. In some aspects, administration of the fusion protein, antibody or antigen binding fragment thereof, or multispecific protein does not result in a decrease in reticulocyte count in a subject compared to an isotype control.

[0071] In some aspects, provided herein is 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 provided herein, wherein the concentration of the CNS-binding antigen is increased compared to administering the CNS-binding antigen alone to the subject.

[0072] In some aspects, provided herein is a method of imaging a CNS antigen in a subject, comprising administering to the subject a fusion protein, an antibody or antigen-binding fragment thereof, or a multispecific protein provided herein, and localizing an imaging agent in the subject.

[0073] In some aspects, provided herein is a method for 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 localizing an imaging agent within the sample.

[0074] In some aspects, provided herein is a 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.

[0075] In some aspects, provided herein is a fusion protein, an antibody or antigen-binding fragment thereof, a multispecific protein, or a pharmaceutical composition provided herein for use in a method provided herein.

[0076] It should be understood that one, some or all of the characteristics 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 become apparent to those skilled in the art. These and other aspects of the present disclosure are further described by the following specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1A An example of a 2+1 bispecific antibody is shown.

[0078] Figure 1B An example of a 2+2 bispecific antibody is shown.

[0079] Figure 1C An example of a 2+2 bispecific antibody with two different scFvs is shown.

[0080] Figure 1D Exemplary formats comprising a TfR antigen binding domain are shown. (i) an exemplary 2+1 bispecific antibody format is shown; (ii) an exemplary 1+1 antibody format is shown; and (iii) another exemplary 1+1 antibody format is shown, having two different VHH domains.

[0081] Figure 1E Exemplary formats of fusion proteins comprising a TfR antigen binding domain and a heterologous protein or polypeptide are shown. The circular "pacman" shape represents a 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.

[0082] Figure 2 Shown are TfR surface levels on hCMEC / D3 cells after treatment with 2+1 anti-TfR bispecific antibodies, evaluated via FACS. (See Example 13.)

[0083] Figure 3 Shown are TfR surface levels on hCMEC / D3 cells after treatment with humanized 2+1 anti-TfR bispecific antibodies, evaluated via FACS. (See Example 13.)

[0084] Figure 4 Total TfR protein levels in hCMEC / D3 cells after treatment with 2+1 anti-TfR bispecific antibodies are shown, as assessed by Western blot. (See Example 13.)

[0085] Figure 5 The fold change in brain PK of the 2+1 anti-TfR bispecific antibody relative to the isotype control is shown. (See Example 17.)

[0086] Figure 6 The 2+1 anti-TfR bispecific antibody is shown to be active against wild-type and hTfR ECD + / - Serum PK in knock-in mice. (See Example 19.)

[0087] Figure 7 Compared with total vascular TfR levels from isotype-treated animals, the expression of hu-TfR in+ / - Total vascular TfR levels in KI mice after injection of anti-TfR antibodies (i.e., TfR-9.1B.39 and TfR-15.WH8). (See Example 20.)

[0088] Figure 8 Blood reticulocyte levels after 2+1 anti-TfR bispecific antibody treatment are shown. (See Example 21.)

[0089] Fig. 9 A modest antibody-dependent cellular cytotoxicity (ADCC) response of the 2+1 anti-TfR bispecific antibody against a BBB cell line was shown. (See Example 22.)

[0090] Fig.10 It was shown that NSLF and LALAPS strongly improved the ADCC signal from the 2+1 anti-TfR bispecific antibody. (See Example 22.)

[0091] Fig.11 The activity of the 2+1 anti-TfR bispecific antibody measured by sTREM2 levels in an in vitro assay is shown. (See Example 24.)

[0092] Fig.12 Absolute reticulocyte counts in non-human primates (NHPs) after administration of 2+1 anti-TfR bispecific antibodies are shown. (See Example 26.)

[0093] Fig.13 Serum and CSF levels of 2+1 anti-TfR bispecific antibodies after the first and second doses are shown. (See Example 27.)

[0094] Fig.14 Antibody concentrations in NHP brain fractions after administration of 2+1 anti-TfR bispecific antibodies are shown. (See Example 28.)

[0095] Fig.15 Soluble TREM2 (sTREM2) levels in serum and CSF of NHPs after administration of 2+1 anti-TfR bispecific antibodies are shown. (See Example 29.)

[0096] Fig.16 CSF-1 levels in CSF of NHPs after administration of 2+1 anti-TfR bispecific antibodies are shown. (See Example 29.)

[0097] Fig.17Relative TfR protein levels (determined by sandwich Meso Scale Discovery method) in cynomolgus monkey brain sections 48 hours after the second dose of 2+1 anti-TfR antibody are shown. The Tfr protein concentration of each animal has been normalized and is shown as a percentage of the TfR level in the mean of control treated animals (hIgG1 Iso). (See Example 30.) DETAILED DESCRIPTION

[0098] The present disclosure relates to antigen-binding domains that specifically bind to human transferrin receptor (TfR); antibodies and antigen-binding fragments thereof comprising such antigen-binding domains; methods for preparing 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.

[0099] The techniques and procedures described or referred to herein are generally well understood by those skilled in the art and are routinely used using conventional methods, such as widely used methods, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel et al., eds., (2003); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000).

[0100] definition

[0101] The terms "central nervous system" and "CNS" refer to the complex of nervous tissue that controls body functions and includes the brain and spinal cord.

[0102] The terms "blood-brain barrier" and "BBB" refer to the network of endothelial cells of brain capillaries that are tightly sealed by tight junctions.

[0103] "Central nervous system antigens" or "CNS antigens" are antigens expressed in the CNS (including the brain) that can be targeted with antibodies or small molecules. 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, beta-glucocerebrosidase (Gcase or GBA), progranulin (PGRN), prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75), and β-secretase. 5NTR), interleukin 6 receptor (IL6R), 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 non-metastatic melanoma protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4 domain A4A (MS4A4A), transmembrane 4 domain A 6A (MS4A6A), ubiquitin protein ligase E3A (UBE3A) or transmembrane protein 106B (TMEM106b).

[0104] A "brain antigen" is a CNS antigen expressed in the brain.

[0105] Unless otherwise indicated, the terms "transferrin receptor", "TfR", "TfR polypeptide" or "TfR protein" are used interchangeably herein to 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 known as transferrin receptor protein 1, TR, tfR1, Trfr, T9 and p90. In some aspects, the terms encompass wild-type sequences and naturally occurring variant sequences, such as splice variants or allelic variants. In some aspects, the terms encompass "full-length" unprocessed TfR as well as any form of TfR produced by processing in a cell. In some aspects, TfR is human TfR. As used herein, the term "human TfR" refers to a polypeptide having the amino acid sequence of SEQ ID NO:8.

[0106] (SEQ ID NO:8)

[0107] As used herein, the terms "antibody" and "immunoglobulin" are used interchangeably and refer to antibody molecules that recognize and specifically bind to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing (e.g., glycoprotein) through 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 molecules, as long as the antibody exhibits the desired biological activity. Based on the identity of the antibody heavy chain constant region, which is referred to as α, δ, ε, γ, and μ, respectively, an antibody may be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or its subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Different classes of antibodies have different and well-known subunit structures and three-dimensional configurations. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6.

[0108] The terms "anti-TfR antibody," "antibody that binds to TfR," and "antibody that specifically binds to TfR" refer to an antibody that is capable of binding to TfR with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent targeting TfR. In one aspect, the extent of binding of the anti-TfR antibody to unrelated non-TfR polypeptides is less than about 10% of the binding of the antibody to TfR, as measured, for example, by radioimmunoassay (RIA). In certain aspects, the antibody that binds to TfR has the following dissociation constant (KD): <20 μM, <15 μM, <12 μM, <10 μM, <7.5 μM, <5 μM, <, 2.5 μM, <1 μΜ, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 M or lower, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M). In certain aspects, the anti-TfR antibody binds to a TfR epitope that is conserved among TfRs from different species.

[0109] The term "antibody fragment" refers to a part of an antibody. An "antigen-binding fragment" of an antibody refers to a part of an antibody that binds to an antigen. An antigen-binding fragment of an antibody may include an antigen-determining region (e.g., a complementary determining region (CDR)) of an antibody. Examples of antigen-binding fragments of an antibody 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 a rodent (e.g., a mouse, a rat or a hamster) and a human, or may be artificially produced.

[0110] An "antigen binding domain" 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 (e.g., a complementarity determining region (CDR)) of an antibody. An antibody or antigen binding fragment thereof (including monospecific and multispecific (e.g., bispecific) antibodies or antigen binding fragments thereof) may comprise an antigen binding domain. In some aspects, the antigen binding domain is not present in the context of an antibody.

[0111] 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 can be used as a diagnostic and / or therapeutic agent targeting TfR. In one aspect, the extent of binding of the anti-TfR antigen binding domain to unrelated non-TfR polypeptides is less than about 10% of the binding of the antigen binding domain to TfR, as measured, for example, by radioimmunoassay (RIA). In certain aspects, the antibody that binds to TfR has a dissociation constant (KD) 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 lower, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M). In certain aspects, the anti-TfR antigen binding domain binds to an epitope of TfR that is conserved among TfRs from different species.

[0112] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably and 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 that include an Fc region. The constant region may be a native sequence constant region (e.g., a human native sequence constant region) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

[0113] "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, while 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 has a variable domain (V H ), followed by a number of constant domains. Each light chain has a variable domain (V L ) and have a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain.

[0114] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment (the name reflects the ability to crystallize readily). The Fab fragment consists of an intact light chain together with the variable region domain (V H ) and the first constant domain of a heavy chain (C H 1) composition. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen binding site. Pepsin treatment of an antibody produces 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 differs from the Fab fragment in that the C H The carboxyl terminus of the constant domain has several additional residues, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains carry a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0115] The Fc fragment comprises the carboxyl terminal portions of two heavy chains held together by disulfide bonds.The effector functions of antibodies are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain types of cells.

[0116] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain in tight, non-covalent association. The folding of the two domains thus produces six hypervariable loops (3 loops each from the H chain and the L chain), which contribute amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or a half Fv containing only three CDRs specific for an antigen) can recognize and bind to an antigen, but with a lower affinity than a complete binding site.

[0117] "Single-chain Fv" is also abbreviated as "sFv" or "scFv", which is an antibody fragment comprising a VH antibody domain and a VL antibody domain connected in a single polypeptide chain. In some aspects, the scFv polypeptide further comprises a VH antibody domain and a VL antibody domain. H Domain and V L A polypeptide linker between the domains that enables the scFv to form the desired structure for antigen binding.

[0118] The term "diabodies" refers to small antibody fragments prepared by: H Domain and V L scFv fragments (see previous paragraph) are constructed with short linkers (about 5-10 residues) between domains, allowing interchain rather than intrachain pairing of variable domains, thereby generating bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" scFv fragments, where the V H Domain and V L Domains are present on different polypeptide chains.

[0119] As used herein, the "2+1 antibody format" of 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, wherein the antibody comprises two heavy chains and two light chains; wherein the single antigen binding domain that binds to human TfR is attached to the C-terminus of one of the two antibody heavy chains. This format is Figure 1A and 1D (i) is an example.

[0120] As used herein, the "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 antigen binding domain that binds to human TfR is connected to the C-terminus of one of the two antibody heavy chains, and the other antigen binding domain that binds to human TfR is connected 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 two antigen binding domains that bind to human TfR may comprise different amino acid sequences. This format is Figure 1B and 1C Examples are given in the following sections.

[0121] As used herein, a "1+1 antibody format" of a TfR antibody refers to a bivalent, bispecific antibody format comprising (i) one antigen binding domain that binds to human TfR; and (ii) an antigen binding domain that binds to an antigen other than human TfR, optionally comprising an Fc domain. Figure 1D An exemplary 1+1 fusion protein format is shown in Figure 1E Examples are given in the following sections.

[0122] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. A variable region generally refers to a part of an antibody, generally a part of a light chain or a heavy chain, generally about 110 to 120 amino acids or 110 to 125 amino acids at the amino terminus in a mature heavy chain and about 90 to 115 amino acids in a mature light chain, whose sequence is very different between antibodies, and is used for the binding and specificity of a particular antibody to its specific antigen. The variability of the sequence is concentrated in those regions referred to as complementary determining regions (CDRs), while the more highly conserved regions in the variable domains are referred to as framework regions (FRs). It is not desirable to be bound by any particular mechanism or theory, and it is believed that the CDRs of light and heavy chains are primarily responsible for the interaction and specificity of antibodies to antigens. In some aspects, a variable region is a human variable region. In some aspects, a variable region comprises rodent or mouse CDRs and human framework regions (FRs). In some aspects, a variable region is a primate (e.g., non-human primate) variable region. In some aspects, the variable region comprises rodent or mouse CDR and primate (e.g., non-human primate) framework region (FR). The term "Kabat numbering" and similar terms are recognized in the art and refer to the numbering system of amino acid residues in the heavy chain variable region and light chain variable region of an antibody or its antigen-binding fragment. In certain aspects, CDR can be determined according to the Kabat numbering system (see, e.g., Kabat EA and 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 within an antibody heavy chain molecule are generally present at amino acid positions 31 to 35, which may optionally include one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDRH1); amino acid positions 50 to 65 (CDRH2) and amino acid positions 95 to 102 (CDRH3). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are generally present at amino acid positions 24 to 34 (CDRL1), amino acid positions 50 to 56 (CDRL2) and amino acid positions 89 to 97 (CDRL3). In some aspects, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme. Chothia refers to the position of the structural loop (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)).When using the Kabat numbering convention, the ends of the Chothia CDRH1 loops vary between H32 and H34 depending on the loop length (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; 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 aspects, the CDRs may be "contact" CDRs. "Contact" CDRs are based on analysis of available complex crystal structures. The residues from each of these CDRs are noted below.

[0123]

[0124] The CDRs may comprise "extended CDRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these extended HVR definitions.

[0125] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0126] As used herein, the term "heavy chain" when used to refer to an antibody can refer to any of the different types based on the amino acid sequence of the constant region, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which produce the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4. Heavy chain amino acid sequences are well known in the art. In some aspects, the heavy chain is a human heavy chain.

[0127] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0128] As used herein, the term "light chain" when used in reference to an antibody can refer to any of the different types based on the amino acid sequence of the constant region, such as kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.

[0129] As used herein, the term "constant region" is a region of a non-antibody variable region in an antibody, such as a carboxyl terminal portion of a light chain and / or heavy chain that is not directly involved in the binding of an antibody to an antigen, but which may exhibit various effector functions, such as interactions with Fc receptors. The constant region of an immunoglobulin molecule typically has an amino acid sequence that is more conservative relative to an immunoglobulin variable domain. In certain aspects, an antibody or antigen-binding fragment comprises a constant region or portion thereof sufficient to achieve antibody-dependent cell-mediated cytotoxicity (ADCC).

[0130] "Constant domain" refers to a domain within the constant region that is capable of forming an immunoglobulin fold. The constant domain includes a CH1 domain, a CH2 domain, a CH3 domain, and a CL domain.

[0131] When referring to an antibody or its antigen binding fragment, the term "monoclonal" refers to a homogeneous antibody or antigen binding fragment population that participates in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies for different antigenic determinants. The term "monoclonal" antibody or its antigen binding fragment encompasses complete and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising antibodies or antibody portions, and any other modified immunoglobulin molecules comprising antigen recognition sites. In addition, "monoclonal" antibodies or their antigen binding fragments refer to such antibodies and their antigen binding fragments made in a variety of ways, including but not limited to by hybridomas, phage selection, recombinant expression, and transgenic animals.

[0132] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof in which the amino acid sequence is 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 with the desired specificity, affinity and capacity derived from one mammalian species (e.g., mouse, rat, rabbit, etc.), while the constant regions are homologous to sequences in an antibody or antigen-binding fragment thereof derived from another species (usually human) to avoid eliciting an immune response in the species.

[0133] The term "humanized" antibody or antigen-binding fragment thereof refers to a form of a non-human (e.g., mouse) antibody or antigen-binding fragment that is a specific immunoglobulin chain, a chimeric immunoglobulin, or a fragment thereof that contains minimal non-human (e.g., mouse) sequences. Typically, a humanized antibody or antigen-binding fragment thereof is a human immunoglobulin in which residues from a complementary determining region (CDR) are replaced ("CDR-grafted") with residues from a CDR of a molecule derived from a non-human species (e.g., mouse, rat, rabbit, hamster) with the desired specificity, affinity, and ability (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 their antigen-binding fragments can be further modified by substitution of additional residues in the Fv framework region and / or in the replaced non-human residues to improve and optimize the specificity, affinity and / or ability of the antibody or its antigen-binding fragment. In general, a humanized antibody or its antigen-binding fragment will comprise VH and VL, which comprise substantially all of at least one and usually two or three CDR regions corresponding to a non-human immunoglobulin, and all or substantially all FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody or its antigen-binding fragment may also comprise an immunoglobulin constant region or Fc region, typically at least a portion of a constant region or Fc region of a human immunoglobulin. Examples of methods for generating 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 aspects, a "humanized antibody" is a resurfaced antibody.

[0134] 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, wherein such antibody or antigen-binding fragment is made using any technique known in the art. This definition of human antibodies or antigen-binding fragments thereof includes complete or full-length antibodies and fragments thereof.

[0135] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as herein defined.

[0136] As used herein, an "acceptor human framework" is a human immunoglobulin framework or a human consensus framework. L or V HThe framework of the amino acid sequence of the framework. The acceptor human framework "derived from" a human immunoglobulin framework or a human common framework may comprise the same amino acid sequence thereof, or it may comprise pre-existing amino acid sequence changes. In some aspects, 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. When pre-existing amino acid changes are present in VH, in some aspects, those changes occur only at three, two, or one of positions 71H, 73H, and 78H; for example, the amino acid residues at those positions may become 71A, 73T, and / or 78A. In some aspects, the sequence of the VL acceptor human framework is identical to that of the VH. L The human immunoglobulin framework sequences or human consensus framework sequences are identical.

[0137] "Human consensus framework" refers to the selection of human immunoglobulin V L or V H The framework sequence is the framework of the most frequently occurring amino acid residues. L or V H The sequence is selected from a subset of variable domain sequences. Generally, the subset of sequences is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include sequences for V L , the subgroup may be subgroup κI, κII, κIII or κIV as in Kabat et al., supra. H , the subgroup may be subgroup I, subgroup II or subgroup III as in Kabat et al., supra.

[0138] For example, an "amino acid modification" at a specified position of an antibody of the present disclosure refers to a substitution or deletion of a specified residue, or the insertion of at least one amino acid residue adjacent to a specified residue. An insertion "adjacent" to a specified residue means an insertion within one to two residues of the specified residue. The insertion may be at the N-terminus or C-terminus of the specified residue. In some aspects, the amino acid modification is a substitution.

[0139] 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 with the antibody isotype.

[0140] The term "Fc region" or "fragment crystallizable region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, a human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to its carboxyl end. The C-terminal lysine (residue 447, according to the EU numbering system) in the Fc region can be removed, for example, during the production or purification of an antibody or by recombinant engineering of a nucleic acid encoding the heavy chain of an antibody. Therefore, the composition of a complete antibody can include an antibody population that removes all K447 residues, an antibody population that does not remove the K447 residue, and an antibody population with a mixture of antibodies containing and not containing the K447 residue. Suitable native sequence Fc regions for use in antibodies of the present disclosure include human IgG1, IgG2, IgG3, and IgG4.

[0141] A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence 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.

[0142] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification, in some aspects one or more amino acid substitutions. In some aspects, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or to the Fc region of a parent polypeptide, such as about one to about ten amino acid substitutions in the native sequence Fc region or in the Fc region of a parent polypeptide, and in some aspects about one to about five amino acid substitutions. In some aspects, the variant Fc region has at least 80% homology with the native sequence Fc region and / or with the Fc region of a parent polypeptide, has at least 90% homology therewith, or has at least 95% homology therewith.

[0143] "Fc receptor" or "FcR" describes a receptor that is bound to the Fc region of an antibody. In some aspects, FcR is a native sequence human FcR. In some aspects, FcR is a FcR (gamma receptor) that binds to an IgG antibody and includes receptors of FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors, and FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, with the main difference being its cytoplasmic domain. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif ("ITAM") in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif ("ITIM") in its cytoplasmic domain. In this article, the term "FcR" covers other FcRs, including those to be identified in the future. FcR can also increase the serum half-life of antibodies.

[0144] "Binding affinity" generally refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or antigen-binding fragment thereof and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the equilibrium dissociation constant (K D Affinity can be measured and / or expressed in many ways known in the art, including but not limited to the equilibrium dissociation constant (K D ) and equilibrium association constant (K A ). D According to k off / k on The quotient of K A According to k on / k off Calculate the quotient of k on refers to, for example, the association rate constant of an antibody or antigen-binding fragment thereof with an antigen, and k off k refers to, for example, the dissociation rate constant of an antibody or its antigen-binding fragment from an antigen. on and k off The method can be performed by techniques known to those skilled in the art such as Or KinExA to measure.

[0145] With respect to the binding of an antibody to a target molecule, the term "specific binding" or "binding specifically to" or "specific for" a particular polypeptide or an epitope on a particular polypeptide target means binding that is significantly 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, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, specific binding is indicated. As used herein, the term "specific binding" or "specifically binds to" or "is specific for" a particular polypeptide or an epitope on a particular polypeptide target can be represented, for example, by a molecule having a KD of about 10 for the target: -4 M or lower, 10 -5 M or lower, 10 -6 M or lower, 10 -7 M or lower, 10 -8 M or lower, 10 -9 M or lower, 10 -10 M or lower, 10 -11 M or lower, 10 -12 M or less or 10 -4 M to 10 -6 M or 10 -6 M to 10 -10 M or 10 -7 M to 10 -9 The term "specific binding" refers to a binding in which a molecule binds to a specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0146] The term "joint" or "connected" refers to a covalent bond between two polypeptides or two heterologous molecules. In some aspects, the joint is a chemical joint. In some aspects, the joint comprises a peptide bond, and the two polypeptides or two heterologous molecules are directly or via one or more additional amino acids connected to each other. A glycine joint is a joint comprising one or more glycines but not containing other amino acids, such as GGGG (SEQ ID NO: 285). A glycine-rich joint is a joint comprising one or more glycines and may contain other amino acids, as long as glycine is the main species in the joint, such as GGGNGG, wherein N is any amino acid (SEQ ID NO: 286). A glycine-serine joint is a joint containing glycine and serine in any proportion, such as GGGS (SEQ ID NO: 287). Similarly, a proline joint is a joint comprising one or more prolines but not containing other amino acids. A proline-rich joint is a joint comprising one or more prolines and may contain other amino acids, as long as proline is the main species in the joint.

[0147] As used herein, "percentage (%) of amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences refer to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and any conservative substitutions are not considered part of the sequence identity. Alignment for the purpose of determining percentage of amino acid sequence identity can be performed in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art needed to achieve maximal alignment over the full length of the sequences being compared.

[0148] The term "epitope" includes any determinant that can be bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets that antigen, and when the antigen is a polypeptide, includes specific amino acids that directly contact the antibody. In most cases, the epitope is located on a polypeptide, but in some cases, it may be located on other types of molecules such as nucleic acids. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural features and / or specific charge characteristics. In general, antibodies specific for a particular target antigen will preferentially recognize epitopes on the target antigen in a complex mixture of polypeptides and / or macromolecules.

[0149] 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 approach, a comprehensive mutant library of an antigen or a portion thereof (e.g., an extracellular domain) can be generated by mutating each individual amino acid residue to alanine (or if the amino acid residue is alanine, mutating it to another residue such as serine) and testing each mutant for binding to a target antibody or antigen-binding fragment thereof.

[0150] An antibody is said to "competitively inhibit" binding of a reference antibody to a given epitope if it preferentially binds to the given epitope or an overlapping epitope such that it blocks binding of the reference antibody to the epitope to some extent. Competitive inhibition can be determined by any method known in the art, such as a competition ELISA assay. An antibody is said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0151] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is one that is 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 no longer in the form in which they are found in nature. In some aspects, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0152] As used herein, "substantially pure" refers to a material that is at least 50% pure (ie, free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0153] The term "expression system" refers to one or more nucleic acid molecules comprising operably linked coding and control sequences, and a host cell and / or other in vitro transcription and translation machinery that enables production of one or more proteins encoded by the nucleic acid molecules.

[0154] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA in which an additional DNA segment can be joined. Another type of vector is a bacteriophage vector. Another type of vector is a viral vector, in which an additional DNA segment can be joined to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and free mammalian vectors with bacterial replication origins). Other vectors (e.g., non-free mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and thus replicated with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors". In general, expression vectors with practicality in recombinant DNA technology are usually in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms.

[0155] As used interchangeably herein, "polypeptide" or "nucleic acid" refers to nucleotide polymers of any length, and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.

[0156] "Host cell" includes an individual cell or cell culture that can be or has been a recipient of a vector for incorporating a polynucleotide insert. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with a polynucleotide of the invention.

[0157] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed.

[0158] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology. Desired therapeutic effects include reducing the rate of progression of a particular disease, disorder, or condition, ameliorating or alleviating its pathological state, and alleviating or improving its prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with a particular disease, disorder, or condition are alleviated or eliminated.

[0159] As used herein, the terms "administer," "administering," "administration," and the like refer to methods that can be used to deliver a drug (eg, an anti-human antibody or antigen-binding fragment thereof) to a desired site of biological action.

[0160] "Effective amount" refers to the amount that effectively achieves the desired therapeutic result at least under the necessary dosage and time period. The effective amount can be provided in one or more administrations. The effective amount is also the amount in which any toxic or harmful effects of the treatment are exceeded by the beneficial effects of the treatment. Regarding therapeutic use, the beneficial or desired results include the following clinical results, such as alleviating one or more symptoms caused by the disease, increasing the quality of life of the person suffering from the disease, reducing the dosage of other agents required for treating the disease, enhancing the effect of another agent (such as via targeting), delaying the progression of the disease and / or prolonging survival. The effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to directly or indirectly achieve therapeutic treatment. As should be understood in clinical situations, the effective amount of a drug, compound or pharmaceutical composition may or may not be achieved in the case of combining another drug, compound or pharmaceutical composition. Therefore, "effective amount" can be considered in the case of administering one or more therapeutic agents, and if the desired result can be achieved or achieved in combination with one or more other agents, a single dose of an effective amount can be considered.

[0161] As used herein, the terms "subject" and "patient" are used interchangeably. The 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 aspects, the subject is a cynomolgus monkey. In some aspects, the subject is a human.

[0162] As used herein, administration "in conjunction with" or "in combination with" another compound or composition includes simultaneous administration and / or administration at different times. Administration in conjunction also encompasses administration as a co-formulation or administration as a separate composition, including at different dosing frequencies or intervals and using the same route of administration or different routes of administration. In some aspects, administration in conjunction is administration as part of the same treatment regimen.

[0163] As used herein, "neurological disorder" refers to a disease or disorder that involves the CNS and / or has a cause in the CNS. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, eye diseases or disorders, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, epileptic seizures, behavioral disorders, and lysosomal storage diseases.

[0164] As used herein, "lysosomal storage disease" or (LSD) refers to an inherited metabolic disease characterized by excessive accumulation of substrates (such as undigested or partially digested macromolecules) in various cells of an organ, ultimately leading to cellular dysfunction and clinical abnormalities. LSD has been defined as a defect in lysosomal function, usually classified by the accumulated substrate, and includes sphingolipidosis, oligosaccharidosis, mucolipidosis, mucopolysaccharidosis, lipoprotein storage disease, neuronal ceroid lipofuscinosis, etc. LSD may also include other deficiencies or defects in proteins that lead to the accumulation of macromolecules, such as proteins required for normal post-translational modification of lysosomal enzymes or proteins important for normal lysosomal trafficking. LSD is a disease caused by a single gene defect. Nearly 70% of LSDs are caused by enzyme defects, and the rest are defects in enzyme activators or related proteins.

[0165] "Protein replacement therapy" or "PRT" refers to a medical treatment that supplements or replaces a specific protein in a patient in whom the protein is deficient or absent.

[0166] "Enzyme replacement therapy enzyme" or "ERT enzyme" refers to an enzyme that is deficient in lysosomal storage diseases. "ERT enzyme variant" refers to a functional variant of a wild-type ERT enzyme or a fragment thereof, including allelic variants and splice variants, wherein the ERT enzyme variant, 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 a fragment thereof when measured under the same conditions. A "catalytically active fragment" of an ERT enzyme refers to a portion of a full-length ERT enzyme or a variant thereof, wherein the catalytically active fragment, 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 a variant thereof when measured under the same conditions.

[0167] As used herein, the terms "about" and "approximately" when used to modify a numerical value or numerical range, indicate that deviations of 10% above and 10% below the value or range are still within the intended meaning of the recited value or range. It should be understood that no matter in which aspects are described in the language of "about" or "approximately" numerical values ​​or ranges, similar aspects involving specific numerical values ​​or ranges are also provided.

[0168] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, a reference to an "antibody" is a reference to one to a plurality of antibodies, such as molar amounts, and includes equivalents thereof known to those skilled in the art, etc.

[0169] It should be understood that wherever aspects are described herein using the language "comprising", similar aspects described using "consisting of" and / or "consisting essentially of" are also provided. In the present disclosure, "comprises", "comprising", "containing", and "having" and the like may mean "includes", "including", and the like; "consisting essentially of" or "consists essentially of" are open ended, allowing for more than what is recited, as long as the basic or novel features of the recited are not altered by the presence of more than what is recited, but excluding prior art aspects.

[0170] Anti-TfR antigen binding domain

[0171] Provided herein are antigen binding domains that specifically bind to human TfR.

[0172] Such antigen binding domains are capable of crossing the blood-brain barrier (BBB) ​​and are capable of transporting other agents (e.g., therapeutically active agents) associated with the antigen binding domain across the BBB. Therefore, in some aspects, provided herein is an antigen binding domain that specifically binds to human TfR that is capable of internalization in BBB epithelial cells such as HCMEC / D3 cells.

[0173] In some aspects, the antigen binding domain that specifically binds to human TfR comprises six CDRs of the antibodies listed in Tables 12 and 13 (i.e., three VH CDRs of the antibodies listed in Table 12 and three VL CDRs of the same antibodies listed in Table 13).

[0174] In some aspects, an antigen binding domain that specifically binds to human TfR comprises six CDRs of the antibodies listed in Tables 12 and 13, and 19, 21, 24, and 25. In some aspects, the CDRs of such antigen binding domains can be identified according to the Chothia numbering scheme, which refers to the positions of immunoglobulin structural loops (see, e.g., Chothia C and 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. Pat. No. 7,709,226). Generally, when using the Kabat numbering convention, the Chothia CDR-H1 loop occurs at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop occurs at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop occurs at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop occurs at light chain amino acids 24 to 34, the Chothia CDR-L2 loop occurs at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop occurs at light chain amino acids 89 to 97. When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).

[0175] In some aspects, an antigen binding domain that specifically binds to human TfR comprises six Chothia CDRs of the antibodies listed in Tables 12, 13, 19, 21, 24, and 25. In some aspects, such as 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 aspects, provided herein is an antigen binding domain that specifically binds to human TfR and comprises a combination of Kabat CDRs and Chothia CDRs.

[0176] In some aspects, the CDRs of the 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 Dübel, ed., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In some aspects, provided herein are antigen binding domains that specifically bind to human TfR and comprise the VH CDRs and VL CDRs of the antibodies listed in Tables 12, 13, 19, 21, 24, and 25, as determined by the method of MacCallum RM et al.

[0177] In some aspects, the CDRs of an antigen binding domain that specifically binds to human TfR can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable region, represents a compromise between the Kabat CDRs and the Chothia structural loops, and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In some aspects, provided herein are antigen binding domains that specifically bind to human TfR and comprise the VHCDRs and VL CDRs of the antibodies listed in Tables 12, 13, 19, 21, 24, and 25, as determined by the AbM numbering scheme.

[0178] In some aspects, the antigen binding domain that specifically binds to human TfR comprises six IMGT CDRs of antibodies listed in Tables 12, 13, 19, 21, 24, and 25 according to the IMGT numbering system as 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 at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97.

[0179] In some aspects, the antigen binding domains provided herein that specifically bind to human TfR are described by their individual VL domains, or their individual VH domains, or their three individual VL CDRs, or their three individual VH CDRs. See, for example, Rader C et al., (1998) PNAS 95:8910-8915, which is incorporated herein by reference in its entirety, describing the humanization of mouse anti-αvβ3 antibodies by identifying complementary light chains or heavy chains from human light chain or heavy chain libraries, respectively, and producing humanized antibody variants with an affinity as high as or higher than that of the original antibody. See also Clackson T et al., (1991) Nature 352:624-628, which is incorporated herein by reference in its entirety, describing a method for producing antibodies that bind to specific antigens by using specific VL domains (or VH domains) and screening the complementary variable domains in the library. The screening yielded 14 new partners for specific VH domains and 13 new partners for specific VL domains, all of which were strong binders as determined by ELISA. See also Kim SJ and Hong HJ, (2007) J Microbiol 45:572-577, which is incorporated herein by reference in its entirety, describing methods for generating antibodies that bind to a specific antigen by using a specific VH domain and screening a library (e.g., a human VL library) for complementary VL domains; the selected VL domains can in turn be used to guide the selection of additional complementary (e.g., human) VH domains.

[0180] In some aspects, the antigen binding domain that specifically binds to human TfR comprises the VH of an antibody listed in Tables 19, 21, 24, and 25.

[0181] In some aspects, the antigen binding domain that specifically binds to human TfR comprises the VL of an antibody listed in Tables 19, 21, 24, and 25.

[0182] In some aspects, 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 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).

[0183] In some aspects, the 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 in Tables 11, 19, 21, 24, and 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 in Tables 11, 19, 21, 24, and 25. In some aspects, the antigen binding domain that specifically binds to human TfR also comprises the CDRs of the antibodies in Tables 12, 13, 19, 21, 24, and 25 (e.g., the amino acids that are not identical in VH and / or VL are outside the CDRs).

[0184] In some aspects, the 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 in Tables 11, 19, 21, 24, and 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 in Tables 11, 19, 21, 24, and 25. In some aspects, the antigen binding domain that specifically binds to human TfR also comprises the CDRs of the antibodies in Tables 12, 13, 19, 21, 24, and 25 (e.g., the amino acids that are not identical in VH and / or VL are outside the CDRs).

[0185] In some aspects, the 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 in Tables 11, 19, 21, 24, and 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 in Tables 11, 19, 21, 24, and 25. In some aspects, the antigen binding domain that specifically binds to human TfR also comprises the CDRs of an antibody in Tables 12, 13, 19, 21, 24, and 25 (e.g., the amino acids that are not identical in VH and / or VL are outside the CDRs).

[0186] In some aspects, the 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 in Tables 11, 19, 21, 24, and 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 in Tables 11, 19, 21, 24, and 25. In some aspects, the antigen binding domain that specifically binds to human TfR also comprises the CDRs of an antibody in Tables 12, 13, 19, 21, 24, and 25 (e.g., the amino acids that are not identical in VH and / or VL are outside the CDRs).

[0187] In some aspects, the 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 in Tables 11, 19, 21, 24, and 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 in Tables 11, 19, 21, 24, and 25. In some aspects, the antigen binding domain that specifically binds to human TfR also comprises the CDRs of the antibodies in Tables 12, 13, 19, 21, 24, and 25 (e.g., the amino acids that are not identical in VH and / or VL are outside the CDRs).

[0188] In some aspects, the 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 in Tables 11, 19, 21, 24, and 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 in Tables 11, 19, 21, 24, and 25. In some aspects, the antigen binding domain that specifically binds to human TfR also comprises the CDRs of the antibodies in Tables 12, 13, 19, 21, 24, and 25 (e.g., the amino acids that are not identical in VH and / or VL are outside the CDRs).

[0189] In some aspects, the 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 in Tables 11, 19, 21, 24, and 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 in Tables 11, 19, 21, 24, and 25. In some aspects, the antigen binding domain that specifically binds to human TfR also comprises the CDRs of an antibody in Tables 12, 13, 19, 21, 24, and 25 (e.g., the amino acids that are not identical in VH and / or VL are outside the CDRs).

[0190] In some aspects, the 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 in Tables 11, 19, 21, 24, and 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 in Tables 11, 19, 21, 24, and 25. In some aspects, the antigen binding domain that specifically binds to human TfR also comprises the CDRs of an antibody in Tables 12, 13, 19, 21, 24, and 25 (e.g., the amino acids that are not identical in VH and / or VL are outside the CDRs).

[0191] In some aspects, provided herein is an antigen binding domain that binds to the same TfR epitope as an antibody, wherein the antibody comprises the VH amino acid sequence of an antibody in Tables 11, 19, 21, 24 and 25 and the VL amino acid sequence of the same antibody in Tables 11, 19, 21, 24 and 25.

[0192] In some aspects, provided herein is an antigen binding domain that competitively inhibits the binding of an antibody to TfR, wherein the antibody comprises a VH amino acid sequence of an antibody in Tables 11, 19, 21, 24 and 25 and a VL amino acid sequence of the same antibody in Tables 11, 19, 21, 24 and 25.

[0193] In some aspects, the antigen binding domain that specifically binds to human TfR includes VH and VL (e.g., VH and VL in Tables 11, 19, 21, 24, and 25) on a single polypeptide chain. In some aspects, the antigen binding domain includes scFv. The scFv may include a VH at the N-terminus of the VL or a VL at the N-terminus of the VH. The scFv may include, for example, a joint between VH and VL. Therefore, the scFv may be in the orientation of VH-joint-VL or VL-joint-VH. The length of such a joint may be about 5 to about 25 amino acids. The length of such a joint may be about 5 to about 20 amino acids. The length of such a joint may be about 10 to about 25 amino acids. The length of such a joint may be about 10 to about 20 amino acids. Such a joint may be, for example, a glycine joint, a glycine-rich joint, or a glycine-serine joint. Such a joint may include 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).

[0194] In some aspects, the antigen binding domain that specifically binds to human TfR comprises VH on a first polypeptide and VL on a second polypeptide (eg, Fab).

[0195] In some aspects, 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 a nanobody).

[0196] In some aspects, the antigen binding domain that specifically binds to human TfR is a mouse antigen binding domain. In some aspects, the antigen binding domain that specifically binds to human TfR is a chimeric antigen binding domain. In some aspects, the antigen binding domain that specifically binds to human TfR is a humanized antigen binding domain. In some aspects, the antigen binding domain that specifically binds to human TfR is a human antigen binding domain.

[0197] In some aspects, the antigen binding domain provided herein that specifically binds to human TfR also binds to cynomolgus monkey TfR.

[0198] In some embodiments, the antigen binding domains provided herein bind to human TfR with "low" affinity. In some aspects, the antigen binding domain binds to human TfR with an affinity between 500nM and 10uM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 2μM and 5μM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 1μM and 5μM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 2μM and 8μM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 750nM and 2μM.

[0199] In some embodiments, the antigen binding domains provided herein bind to human TfR with a "medium" affinity. In some embodiments, the antigen binding domains provided herein bind to human TfR with a "medium" affinity. In some aspects, the antigen binding domain binds to human TfR with an affinity between 10nM and 500nM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 50nM and 500nM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 100nM and 250nM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 10nM and 100nM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 250nM and 500nM.

[0200] In some embodiments, the antigen binding domains provided herein bind to human TfR with "high" affinity. In some aspects, the antigen binding domain binds to human TfR with an affinity of less than 10nM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 0.01nM and 10nM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 0.1nM and 10nM. In some aspects, the antigen binding domain binds to human TfR with an affinity between 0.01nM and 1nM.

[0201] In some aspects, the antigen binding domains provided herein specifically bind to human TfR with an affinity of no more than 250 nM (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 no more than 200 nM (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 no more than 150 nM (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 the Carterra LSA platform. In some aspects, such as by ELISA OD 450 The antigen binding domain that specifically binds to human TfR provided herein binds to human TfR as measured.

[0202] In some aspects, the antigen binding domain provided herein that specifically binds to human TfR binds to cynomolgus monkey TfR with an affinity of no greater than 250 nM (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 no greater than 200 nM (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 no greater than 150 nM (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 wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, for example, using a Carterra LSA platform. In some aspects, the antigen binding domains provided herein specifically bind 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.

[0203] In some aspects, an antigen binding domain provided herein that specifically binds to human TfR binds to each of human TfR and cynomolgus monkey TfR with an affinity of no more than 250 nM (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 no more than 200 nM (e.g., 1 μM to 200 nM, 10 μM to 250 nM, 1 nM to 200 nM, or 3 nM to 200 nM), or an affinity of no more than 150 nM (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 aspects, the antigen binding domain provided herein that specifically binds to human TfR binds to each of 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.

[0204] In some aspects, the antigen binding domains provided herein that specifically bind to human TfR bind to human TfR with an affinity of 6.7nM to 3.5μM and bind to cynomolgus monkey TfR with an affinity of 38nM to 2.3μM. In some aspects, the antigen binding domains provided herein that specifically bind to human TfR bind to human TfR with an affinity of 6.7nM to 340nM and bind to cynomolgus monkey TfR with an affinity of 18nM to 870nM, optionally wherein affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, for example, using a Carterra LSA platform.

[0205] In some aspects, relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, the antigen binding domains specifically binding to human TfR provided herein reduce the cell surface expression of TfR on HCMED / D3 cells by 40-80%. Cell surface expression can be measured, for example, using Western blotting or FACS.

[0206] In some aspects, relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, the antigen binding domains specifically binding to human TfR provided herein do not significantly increase the cell surface expression of TfR on HCMED / D3 cells. Cell surface expression can be measured, for example, using Western blotting or FACS.

[0207] In some aspects, relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, the antigen binding domains specifically binding to human TfR provided herein do not reduce the cell surface expression of TfR on HCMED / D3 cells by more than 40%, 60% or 80%, and relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, the cell surface expression of TfR on HCMED / D3 cells is not significantly increased. Cell surface expression can be measured, for example, using Western blotting or FACS.

[0208] In some aspects, following peripheral injection, the accumulation of an antigen binding domain that specifically binds to human TfR provided herein in the brain of vascular-depleted human TfR knock-in mice is at least 4-fold or 5-fold higher than that of an isotype control.

[0209] In some aspects, after peripheral injection, the accumulation of the antigen binding domain specifically binding to human TfR provided herein in the non-human primate brain with vascular depletion is at least 2 times that of the isotype control. In some aspects, after peripheral injection, the accumulation of the antigen binding domain specifically binding to human TfR provided herein in the non-human primate brain with vascular depletion is at least 4 times or 5 times that of the isotype control. In some aspects, after peripheral injection, the accumulation of the antigen binding domain specifically binding to human TfR provided herein in the non-human primate brain with vascular depletion is at least 8 times that of the isotype control. In some aspects, after peripheral injection, the accumulation of the antigen binding domain specifically binding to human TfR provided herein in the non-human primate brain with vascular depletion is 8 times to 64 times that of the isotype control. In some aspects, after peripheral injection, the accumulation of the antigen binding domain specifically binding to human TfR provided herein in the non-human primate brain with vascular depletion is 8 times to 64 times that of the isotype control. In some aspects, following peripheral injection, the accumulation of an antigen binding domain that specifically binds to human TfR provided herein in the vascular-depleted non-human primate brain is 2- to 11-fold higher than an isotype control.

[0210] In some aspects, the specific binding of the antigen binding domains provided herein to human TfR is at least 5 times greater than the binding to unrelated proteins. In some aspects, the specific binding of the antigen binding domains provided herein to cynomolgus monkey TfR is at least 5 times greater than the binding to unrelated proteins. In some aspects, the specific binding of the antigen binding domains provided herein to human TfR is at least 5 times greater than the binding to unrelated proteins and / or the specific binding to cynomolgus monkey TfR is at least 5 times greater than the binding to unrelated proteins.

[0211] Also provided herein are antigen binding domains that bind to the same TfR epitope 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.

[0212] Agents comprising an anti-TfR antigen binding domain

[0213] Provided herein are agents (e.g., fusion proteins, multispecific (e.g., bispecific) proteins, antibodies, antigen-binding fragments thereof, etc.) comprising an antigen-binding domain that specifically binds to human TfR.

[0214] Fusion Protein

[0215] In some aspects, the fusion protein provided herein comprises: an antigen binding domain that specifically binds to human TfR and a heterologous protein. In some aspects, the fusion protein provided herein comprises: (i) an antigen binding domain that specifically binds to human TfR and (ii) a heterologous protein or polypeptide or a fragment thereof. In some aspects, the heterologous protein is a protein or polypeptide or a fragment thereof that can be used in protein replacement therapy (PRT). In some aspects, the heterologous polypeptide is an enzyme (e.g., an enzyme for enzyme replacement therapy (ERT)) or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide or protein is an ERT enzyme or an ERT enzyme variant or a catalytically active fragment thereof.

[0216] 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 a granulin precursor (PGRN), a sphingolipid activator protein (PSAP) or a survival motor neuron protein (SMN). In some aspects, the heterologous protein is an enzyme selected from the following: ubiquitin protein ligase E3A (UBE3A), α-L iduronidase (IDUA), iduronic acid-2-sulfatase (IDS), N-acetylgalactosamine-6-sulfatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulfatase (arylsulfatase B; ARSB), acidic sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramide β-galactosidase, glucosylceramidase, β-hexosaminidase A, β-hexosaminidase B, arylsulfatase A, β-galactosidase, acid ceramidase, α-glucosidase, lysosomal acid lipase, lysosomal protease, its synthetase surrogate such as larodinase, idursulfatase, elosulphase α or galactosesulfatase (galsuphase), or a variant thereof, or a catalytically active fragment thereof. In some aspects, the heterologous protein is a protein or enzyme selected from the group consisting of clusterin (APOJ), reelin, tripeptidyl peptidase 1 (CLN2 / TPP1), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-α-glucosaminide N-acetyltransferase (HGSNAT), and N-acetyl-α-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 β-galactosidase, glucosylceramidase, β-hexosaminidase A, β-hexosaminidase B, arylsulfatase A, β-galactosidase, acid ceramidase, α-glucosidase, lysosomal acid lipase, lysosomal protease, its synthetic enzyme alternatives such as laronidase, idursulfatase, elosulphase α or galactosylsulfatase, or variants thereof, or catalytically active fragments thereof.

[0217] Exemplary forms of fusion proteins disclosed herein are depicted in Figure 1E middle.

[0218] For example, in some aspects, the heterologous protein or polypeptide in the fusion protein is at the N-terminal of the antigen binding domain that specifically binds to human TfR. In some aspects, the heterologous protein or polypeptide in the fusion protein is at the C-terminal of the antigen binding domain that specifically binds to human TfR. In some aspects, the heterologous fusion protein or polypeptide is directly connected to the antigen binding domain that specifically binds to human TfR via a peptide bond. In some aspects, the heterologous fusion protein or polypeptide is connected to the antigen binding domain that specifically binds to human TfR via a joint such as a peptide joint. In some aspects, the fusion protein comprises an antigen binding domain and a heterologous protein or polypeptide and an Fc part. In some aspects, the antigen binding domain and the heterologous protein or polypeptide are connected to the N-terminal of the Fc part of the fusion protein. In some aspects, the antigen binding domain and the heterologous protein are connected to the C-terminal of the Fc part of the fusion protein. In other aspects, the antigen binding domain is connected to the N-terminal of the Fc part of the fusion protein, and the heterologous protein or polypeptide is connected to the C-terminal of the Fc part of the fusion protein. In other aspects, the antigen binding domain is linked to the C-terminus of the Fc portion of the fusion protein, and the heterologous protein or polypeptide is linked to the N-terminus of the Fc portion of the fusion protein.

[0219] In some aspects, disclosed herein is a fusion protein comprising (i) a single scFv, or VHH, or Fab antigen binding domain that binds to human TfR and (ii) a heterologous protein or polypeptide, wherein the fusion protein comprises two copies of the heterologous protein or polypeptide. In some aspects, the fusion protein comprises an Fc domain. The Fc may be a heterodimeric Fc comprising a first Fc polypeptide having a knob mutation and a second Fc polypeptide having a hole mutation ("knob-hole Fc", described herein). In some aspects, a single scFv, Fab or VHH antigen binding domain that binds to human TfR is connected to the C-terminus of the Fc domain (ie, to one of the two heavy chains of the Fc domain), while two copies of the heterologous protein or polypeptide are connected to the N-terminus of the Fc. In some aspects, a single scFv, Fab or VHH antigen binding domain that binds to human TfR is attached to the N-terminus of the Fc domain (i.e., to one of the two heavy chains of the Fc domain), while two copies of the heterologous protein or polypeptide are attached to the C-terminus of the Fc domain. An example of this 2+1 format is Figure 1E Shown as (v) and (vi) in FIG.

[0220] In some aspects, disclosed herein is a fusion protein comprising (i) an antibody that binds to human TfR, wherein the antibody comprises two heavy chains and two light chains; and (ii) two copies of a heterologous protein or polypeptide linked to the C-termini of the two antibody heavy chains.

[0221] In some aspects, the fusion protein comprises (i) two scFv, Fab or VHH antigen binding domains that bind to human TfR and are 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.

[0222] In some aspects, disclosed herein is a fusion protein comprising (i) a single scFv, VHH or Fab antigen binding domain that binds to human TfR, and (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 connected to the C-terminus of the Fc domain and the heterologous protein or polypeptide is connected to the N-terminus of the Fc domain. In some embodiments, Fc is a single-chain engineered monovalent Fc domain. An example of this single-chain 1+1 format of a fusion protein is Figure 1E It is shown as (i).

[0223] In some aspects, 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 connected to the N-terminus of the Fc domain and the heterologous protein or polypeptide is connected to the C-terminus of the Fc domain. In some embodiments, Fc is a single-chain engineered monovalent Fc. Examples of this 1+1 format of fusion proteins are Figure 1E It is shown as (ii).

[0224] In some aspects, disclosed herein is a fusion protein comprising (i) a single scFv, VHH or Fab antigen binding domain that binds to human TfR, and (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 peptide are both linked to the N-terminus or C-terminus of the Fc domain. Examples of this 1+1 format of fusion proteins are described in Figure 1E It is shown as (iv).

[0225] Bispecific and multispecific proteins

[0226] In some aspects, the antibodies or antigen binding fragments thereof provided herein include an antigen binding domain that specifically binds to human TfR. In some aspects, the antibodies or antigen binding fragments thereof include an antigen binding domain that specifically binds to human TfR and an antigen binding domain that specifically binds to CNS antigens or brain antigens. In some aspects, CNS antigens or brain antigens are not TfR. Also provided herein are antibodies or antigen binding fragments that bind to the same TfR epitope as the TfR antigen binding domains provided herein. Also provided herein are antibodies or antigen binding fragments that competitively inhibit the binding of the TfR antigen binding domains provided herein to TfR.

[0227] In some aspects, the multispecific protein provided herein comprises a first antigen binding domain and a second antigen binding domain that binds to human TfR. The first antigen binding domain that binds to human TfR may be any antigen binding domain that binds to human TfR provided herein. The second antigen binding domain may be an antigen binding domain that specifically binds to a CNS antigen or a brain antigen. In some aspects, the CNS antigen or the brain antigen is not TfR.

[0228] In some aspects, the multispecific proteins provided herein comprise an antigen binding domain that binds to human TfR, the antigen binding domain being 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 aspects, the CNS antigen or brain antigen is not TfR. In some aspects, such multispecific proteins can be in the form of a 2+1 antibody (e.g. Figure 1A ) or a 2+2 antibody format (as shown in Figure 1B and 1C ).

[0229] In some aspects, the multispecific protein provided herein comprises a TfR antigen binding domain, which is a scFv connected to an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains. In some aspects, the scFv is connected to the C-terminus of one of the two antibody heavy chains, for example via a protein linker.

[0230] In some aspects, the multispecific protein comprises 1) an antigen binding domain that binds TfR, 2) a second antigen binding domain that binds different CNS or brain antigens, and 3) an Fc region, wherein the TfR antigen binding domain and the second antigen binding domain are connected or connected to the Fc region of the multispecific protein. In other aspects, the 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 to different CNS or brain antigens, and 3) an Fc region, wherein the TfR antigen binding domain and the second antigen binding domain are connected or connected to the Fc region of the multispecific protein. In some aspects, the multispecific protein comprises an antigen binding domain that binds TfR, a second antigen binding domain that binds different CNS or brain antigens, and an Fc region. In some aspects, the TfR antigen binding domain and the second antigen binding domain are connected or connected to the N-terminus of the Fc portion of the multispecific protein. In other aspects, the TfR antigen binding domain is attached or connected to the N-terminus of the Fc portion of the multispecific protein, and the second antigen binding domain is attached to the C-terminus of the Fc portion of the multispecific protein. In other aspects, the TfR antigen binding domain is attached or connected to the C-terminus of the Fc portion of the multispecific protein, and the second antigen binding domain is attached to the N-terminus of the Fc portion of the multispecific protein.

[0231] In some aspects, the multispecific proteins provided herein comprise two copies of a TfR antigen binding domain as a scFv and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, wherein one of the two copies of the antigen binding domain is connected to the C-terminus of one of the antibody heavy chains, and wherein the other copy of the antigen binding domain is connected to the C-terminus of another antibody heavy chain. In some aspects, the scFv is connected to the heavy chain via a protein linker.

[0232] In some aspects, disclosed herein is a trivalent bispecific protein 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 wherein the single scFv, Fab or VHH antigen binding domain that binds to human TfR is attached to the C-terminus or N-terminus of one of the two antibody heavy chains. The Fc may be a heterodimeric Fc, such as a knob and hole Fc. This example of a 2+1 format with a knob-hole Fc is in Figure 1D It is shown as (i).

[0233] In some aspects, disclosed herein is a tetravalent bispecific protein comprising (i) two scFv, Fab or VHH antigen binding domains bound to human TfR and (ii) an antibody, wherein the antibody comprises two heavy chains and two light chains; and wherein one scFv, Fab or VHH antigen binding domain bound to human TfR is connected to the C-terminus of one of the two antibody heavy chains, and another scFv, Fab or VHH antigen binding domain bound to human TfR is connected to the C-terminus or N-terminus of the other of the two antibody heavy chains. In some aspects, the two scFv, Fab or VHH antigen binding domains bound to human TfR may comprise the same amino acid sequence. In some aspects, Fc is a heterodimeric Fc, such as an Fc domain with a knob mutation and a hole mutation.

[0234] In some aspects, disclosed herein is a bivalent bispecific protein comprising (i) a single scFv, VHH or Fab antigen binding domain that binds to human TfR and is connected to the N-terminus of the Fc domain of the Fc dimer, and (ii) a second antigen binding domain, wherein the second antigen binding domain does not specifically bind to human TfR and is connected to the N-terminus of the second Fc domain of the Fc dimer. In some aspects, the second antigen binding domain binds to a CNS or brain antigen other than TfR. Examples of this 1+1 format are Figure 1D It is shown as (iii).

[0235] In some aspects, disclosed herein is a bivalent bispecific protein comprising (i) a single scFv, VHH or Fab antigen binding domain that binds to human TfR and is connected to the N-terminus or C-terminus of the Fc domain of an Fc dimer, and (ii) a second antigen binding domain, wherein the second antigen binding domain does not specifically bind to human TfR and comprises a single scFv, VHH or Fab connected to the N-terminus of the Fc of the Fc dimer or the C-terminus of the second Fc domain of the Fc dimer. In some aspects, the Fc is a heterodimeric Fc, such as an Fc domain with a knob mutation and a hole mutation. Examples of this 1+1 format are Figure 1D Shown in (ii), wherein 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.

[0236] As provided herein, the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein may be multispecific, such as bispecific. Many different forms and uses of bispecific binding molecules are known in the art (reviewed in, for example, Kontermann; Drug Discov Today, July 2015; 20 (7): 838-47; MAbs, March-April 2012; 4 (2): 182-97). The bispecific protein according to the present invention is not limited to any particular bispecific form or its production method. Therefore, the bispecific protein of the present disclosure may include various configurations having a first antigen binding domain that binds to human TfR and a second antigen binding domain that binds to, for example, a CNS antigen or a brain antigen.

[0237] Bispecific molecules include, for example, κ-λ bodies, dual affinity retargeting molecules (DARTs), knob-hole antibodies, chain exchange engineered domain bodies (SEEDbody) and DuoBody. In some aspects, the bispecific fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein comprise knob mutations and hole mutations. 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 substitution 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 comprising different antigen binding domains; (ii) a single chain antibody specific for two different epitopes, such as via two scFvs linked in series by an additional peptide linker; (iii) a dual variable domain antibody (DVD-Ig), in which each light and heavy chain contains two variable domains linked in series by a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig.TM.) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iv) chemically linked bispecific (Fab')2 fragments; (v) Tandab, which is a fusion of two single-chain diabodies, resulting in a tetravalent bispecific antibody with two binding sites for each target antigen; (vi) flexibody, which is a combination of scFv and diabody, resulting in a multivalent molecule; (vii) so-called "dock and lock" molecules, based on the "dimerization and docking domain" in protein kinase A, which, when applied to Fab, can produce a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments; (viii) so-called Scorpion molecules, which comprise, for example, two scFvs fused to the ends of a human Fab arm; and (ix) diabodies. Other examples of antibody structures are described in WO2019 / 246288, which is incorporated by reference.

[0238] In some aspects, the bispecific protein provided herein is selected from one of the following formats: CrossMab, DAF (two in one), DAF (four in one), DutaMab, DT-IgG, charge pair, Fab-arm exchange, Triomab, LUZ-Y, Fcab, κλ 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 (four in one), nanobody, nanobody-HAS, BiTE, TandAb, scDiabody-CH3, diabody-CH3, triabody, minibody, minibody, TriBi minibody, 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, as described in Spiess, C., et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol. Immunol. (2015), which is incorporated herein by reference.

[0239] In some aspects, the fusion protein, antibody or its antigen binding fragment or multi-specific protein provided herein is bivalent. In some aspects, the multi-specific protein provided herein is multivalent (e.g., bivalent). In some aspects, the fusion protein, antibody or its antigen binding fragment or multi-specific protein provided herein is trivalent (e.g., in the form of 2+1 antibodies). In some aspects, the trivalent form includes a single TfR antigen binding domain provided herein and two antigen binding domains that are bound to CNS antigens or brain antigens. Two antigen binding domains that are bound to CNS antigens or brain antigens may include the same amino acid sequence or may include different amino acid sequences. In some aspects, the TfR antigen binding domain is scFv. In some aspects, the TfR antigen binding domain is VHH.

[0240] In some aspects, the fusion protein, antibody or antigen binding fragment thereof, or multispecific protein provided herein is tetravalent (e.g., in the form of a 2+2 antibody). In some aspects, the tetravalent form comprises two TfR antigen binding domains provided herein and two antigen binding domains that bind to CNS antigens or brain antigens. The two TfR antigen binding domains may comprise the same amino acid sequence or may comprise different amino acid sequences. In some aspects, the two TfR antigen binding domains comprise the same amino acid sequence. In some aspects, one or both of the TfR antigen binding domains are scFv. The two antigen binding domains that bind to CNS antigens or brain antigens may comprise the same amino acid sequence or may comprise different amino acid sequences.

[0241] The fusion proteins, antibodies or antigen binding fragments thereof, or multispecific proteins provided herein may include a linker, for example, to connect the TfR antigen binding domain to a heterologous protein, an antibody or antigen binding fragment thereof, or other antigen binding domains. 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 (GGGGS) x3 (SEQ ID NO: 184). The linker may include the amino acid sequence (GGSGG) x3 (SEQ ID NO: 289).

[0242] The fusion protein, antibody or its antigen-binding fragment or multi-specific protein provided herein may include a constant region. In some aspects, the TfR antigen-binding domain provided herein is connected to a constant region, such as the C-terminus of a constant region. In some aspects, the constant domain is a human constant domain. In some aspects, 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 joint may include an amino acid sequence GGSGG (no repetition) (SEQ ID NO: 304). The length of the joint may be 1 to 20 amino acids.

[0243] In some aspects, the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein comprises a heavy chain and a light chain. Regarding the heavy chain, in some aspects, the heavy chain of the antigen-binding protein described herein may be an alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In some aspects, the heavy chain may include a human alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In some aspects, the heavy chain includes a human gamma (γ) heavy chain constant region. In some aspects, the heavy chain includes an amino acid sequence of an IgG1 heavy chain constant region. In some aspects, the heavy chain includes an amino acid sequence of an IgG2 (eg, IgG2a or IgG2b) heavy chain constant region. In some aspects, the heavy chain includes an amino acid sequence of an IgG4 heavy chain constant region. Regarding the light chain, in some aspects, the light chain is a κ light chain. In some aspects, the light chain is a λ light chain. In some aspects, the light chain is a human κ light chain or a human λ light chain.

[0244] In some aspects, the fusion protein, antibody or its antigen-binding fragment or multispecific protein provided herein comprises a constant region, and the constant region comprises the amino acid sequence of the constant region of IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecules or human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecules. In some aspects, the fusion protein, antibody or its antigen-binding fragment or multispecific protein provided herein comprises a constant region, and the constant region comprises the amino acid sequence of the constant region of IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecules, any category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) of immunoglobulin molecules or any subclass (e.g., IgG2a and IgG2b). In some aspects, the constant region comprises the amino acid sequence of a constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecules.

[0245] Non-limiting examples of human constant region sequences are described in, e.g., U.S. Pat. No. 5,693,780 and in Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

[0246] 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, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein may comprise a constant region comprising a knob mutation and a constant region comprising a hole mutation.

[0247] FC domain

[0248] The fusion protein, antibody or its antigen-binding fragment or multispecific protein provided herein may include an Fc domain or a fragment thereof. In some aspects, the Fc domain is an IgG category, an IgM category or an IgA category. In some aspects, the Fc domain or a fragment thereof is an IgG Fc domain or a fragment thereof. In some aspects, the Fc domain or a fragment thereof is a human IgG Fc domain or a fragment thereof. In some aspects, the Fc domain or a fragment thereof is a human IgG1 Fc domain or a fragment thereof. In some aspects, the Fc domain or a fragment thereof is a human IgG2 Fc domain or a fragment thereof. In some aspects, the Fc domain or a fragment thereof is a human IgG4 Fc domain or a fragment thereof. In some aspects, the Fc domain or a fragment thereof is a monovalent Fc.

[0249] In some aspects provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided include a modified Fc domain or fragment thereof. In some aspects, the modified Fc domain or fragment thereof is a modified IgG1 Fc comprising one or more modifications. For example, in some aspects, the IgG1 modified Fc comprises one or more amino acid substitutions (e.g., relative to a wild-type Fc domain of the same isotype). In some aspects, the one or more amino acid substitutions are selected from 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. (2000) Cell Immunol, 200: 16-26). 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, wherein the amino acid positions are according to the EU numbering convention. In some aspects, Fc comprises amino acid substitutions L234A, L235A and P331S (LALAPS) according to EU numbering. In some aspects, Fc comprises N325S and L328F mutations according to EU numbering. In some aspects, Fc comprises P329G or P329S according to EU numbering. In some aspects, Fc comprises K322A according to EU numbering.

[0250] Table 1: Exemplary Fc domains

[0251]

[0252]

[0253]

[0254] In some aspects provided herein, the fusion protein, antibody or its antigen binding fragment, or multispecific protein provided herein is a bispecific fusion protein, antibody or its antigen binding fragment, or multispecific protein. Bispecific molecules include, for example, κ-λ bodies, dual affinity retargeting molecules (DARTs), knob-hole antibodies, chain exchange engineered domain bodies (SEEDbody) and DuoBody. In some aspects, the bispecific fusion protein, antibody or its antigen binding fragment, or multispecific protein provided herein comprises 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 substitution T366S, L368A and Y407V according to EU numbering.

[0255] In some aspects provided herein, the fusion protein, antibody or its antigen-binding fragment or multispecific protein provided herein include one or more mutations to promote heterodimerization of Fc domains. In some aspects, the bispecific dimerization Fc domain provided herein is formed by the Fc domain containing amino acid mutations, substitutions, additions or deletions to promote heterodimerization, wherein different polypeptides including different Fc domains can dimerize to produce heterodimer configurations. In some aspects, the bispecific antibody of the present disclosure includes the first Fc sequence containing the first CH3 district and the second Fc sequence containing the second CH3 district, wherein the first CH3 district is different from the sequence of the second CH3 district and makes the heterodimerization interaction between the first CH3 district and the second CH3 district stronger than each of the homodimerization interactions between the first CH3 district and the second CH3 district.

[0256] Methods for promoting heterodimerization of Fc domains include amino acid deletions, additions or substitutions of the amino acid sequence of the Fc domain, such as by including a set of "knob-into-hole" deletions, additions or substitutions or including amino acid deletions, additions or substitutions to achieve electrostatic steering of the Fc, thereby facilitating attractive interactions between different polypeptide chains. Methods for promoting heterodimerization of complementary Fc polypeptides have been previously described in the literature, for example, by 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, 5:646-654; Gunasekaran et al., 2010, 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; WO2 011 / 143545; WO2014 / 067011; WO2012 / 058768; WO2018 / 027025; US2014 / 0363426; US2015 / 0307628; US2018 / 0016354; US2015 / 0239991; US2017 / 0058054; USPN5731168; USPN7183076; USPN9701759; USPN9605084; USPN9650446; USPN8216805; USPN8765412; and USPN8258268.

[0257] In some aspects, the complementary Fc polypeptides of the Fc heterodimers include mutations that alter the charge polarity across the Fc dimer interface such that co-expression of electrostatically matched Fc domains supports favorable attractive interactions, thereby promoting the desired Fc heterodimer formation; whereas unfavorable repulsive charge interactions inhibit unwanted Fc homodimer formation (Guneskaran et al., 2010, J Biol Chem, 285: 19637-19646). When co-expressed in cells, association between polypeptide chains is possible, but due to charge repulsion, the chains will not substantially self-associate.

[0258] In addition, the complementary Fc polypeptides of the Fc heterodimer include a "knob-and-hole" configuration that promotes the heterodimerization of the two Fc polypeptides. The "knob-and-hole" technology is described in, for example, U.S. Patent Nos. 5,731,168; 7,695,936; 8,216,805; 8,765,412; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). In general, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide, so that the protrusion can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. The protrusion is constructed by replacing the small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine), a compensatory cavity of the same or similar size as the projection is generated in the interface of the second polypeptide. The projection and cavity can be made by changing the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or by peptide synthesis. In some aspects, the knob is modified to include the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole is modified to include the amino acid substitution T366S, L368A and Y407V in the other of the two subunits of the Fc domain. In some aspects, the subunit comprising the knob-modified Fc domain additionally includes the amino acid substitution S354C, and the subunit comprising the hole-modified Fc domain additionally includes the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits in the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). Thus, in such a configuration, the first Fc polypeptide comprises an amino acid modification that forms a "knob" and the second Fc polypeptide comprises an amino acid modification that forms a "hole," thereby forming an Fc heterodimer comprising complementary Fc polypeptides.

[0259] Exemplary paired amino acid modifications of complementary Fc polypeptides in the Fc heterodimer configuration are listed in the table below (EU numbering).

[0260] Table 2: Exemplary paired Fc modifications of heterodimeric Fc domains

[0261] First Fc polypeptide Second Fc polypeptide T366Y Y407T T366W T366S / L368W / Y407V T366W T366S / L368A / Y407V T366W / S354C T366S / L368A / Y407V / Y349C T350V / L351Y / F405A / Y407V T350V / T366L / K392L / T394W K360D / D399M / Y407A E345R / Q347R / T366V / K409V K409D / K392D D399K / E356K K360E / K409W Q347R / D399V / F405T L360E / K409W / Y349C Q347R / D399V / F405T / S354C K370E / K409W E357N / D399V / F405T

[0262] Some agents provided herein include antigen-binding fragments of antibodies. Antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, WO93 / 16185; and U.S. Patent Nos. 5571894 and 5587458. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5869046.

[0263] Diabodies are antibody fragments with two antigen binding sites, which may be bivalent and / or bispecific. See, e.g., EP404097; 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 comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In some aspects, single-domain antibodies are human single-domain antibodies (see, e.g., U.S. Pat. No. 6,248,516).

[0264] Antibody fragments can be made by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein.

[0265] 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 such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has changed relative to the class or subclass of the parent antibody.

[0266] As provided herein, the fusion proteins, antibodies or their antigen-binding fragments, or multispecific proteins provided herein can be humanized. Typically, non-human antibodies are humanized to reduce immunogenicity for people while retaining the specificity and affinity of the parent non-human antibodies. In some aspects, humanized antibodies are substantially non-immunogenic in people. In some aspects, the affinity of humanized antibodies to targets is substantially the same as that of antibodies from another species producing the humanized antibodies. See, for example, U.S. Patent Nos. 5530101; 5693761; 5693762; and 5585089. In some aspects, the amino acids of antibody variable domains that can be modified while reducing their immunogenicity without weakening the natural affinity of the antigen-binding domain are identified. See, for example, U.S. Patent Nos. 5766886 and 5869619. In general, a humanized antibody comprises one or more variable domains, wherein CDR (or a portion thereof) is derived from a non-human antibody, and a framework region (FR) (or a portion thereof) is derived from a human antibody sequence. Humanized antibodies may include at least a portion of a human constant region. In some aspects, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody producing CDR residues), e.g., to restore or improve antibody specificity or affinity.

[0267] Humanized antibodies and methods for making them are reviewed in, e.g., Almagro et al. Front. Biosci. 13: 161 9-1633 (2008), and further described in, e.g., U.S. Pat. Nos. 5821337, 7527791, 6982321, and 7087409. 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 the consensus sequence of human antibodies of a particular subgroup of light chain variable regions 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 (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson et al., J. Immunol. 151: 2623 (1993)); Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al. J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al. J. Biol. Chem. 271: 22611-22618 (1996)).

[0268] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein can be human. Human antibodies can be produced using various techniques 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).

[0269] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic attack. People can engineer mouse strains that lack mouse antibody production with large fragments of human Ig loci, and it is expected that these mice will produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can retain large variable gene diversity and appropriate regulation of antibody production and expression. By utilizing mouse mechanisms for antibody diversification and selection and lack of immune tolerance to human proteins, the human antibody library reproduced in these mouse strains can produce high-affinity fully human antibodies for any antigen of interest (including human antigens). Using hybridoma technology, antigen-specific human MAbs with desired specificity can be produced and selected. Certain exemplary methods are described in U.S. Patent Nos. 5545807, EP546073 and EP 546073. See also, for example, description of XENOMOUSE TM US Patent Nos. 6075181 and 6150584 for the technology; description US Patent No. 5770429 for technology; description KM US Patent No. 7,041,870; and Description Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0270] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, 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, 1 03: 3557-3562 (2006). Additional methods include those described in, for example, U.S. Patent No. 7189826 (describing the production of monoclonal human IgM antibodies by hybridoma cell lines). Human hybridoma technology (Trioma 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 generated by isolating Fv clone variable domain sequences selected from phage display libraries of human origin. Such variable domain sequences can then be combined with desired human constant domains. The technology for selecting human antibodies from antibody libraries is described below.

[0271] In some aspects provided herein, the antibodies are human antibodies isolated by in vitro methods and / or screening combinatorial libraries for antibodies having the desired one or more activities. 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), etc. In certain phage display methods, pools of VH and VL genes are individually cloned by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phages, as described in Winter et al. Ann. Rev. Immunol. 12: 433-455 (1994). For example, various methods for generating phage display libraries and screening the libraries for antibodies having the desired binding characteristics are known in the art. 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. J. Immunol. Methods 284(-2):1 19-132 (2004). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the initial library can be cloned (e.g., from people) to provide a single source of antibodies to a variety of non-self and self antigens without any immunization, such as Griffiths et al. EMBO J. 12: 725-734 (1993). Finally, the initial library can also be prepared synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequences to encode hypervariable CDR3 regions and achieve in vitro rearrangement, such as Hoogenboom et al. J. Mol. Biol., 227: 381-388, 1992. Patents describing human antibody phage libraries include, for example, U.S. Patent No. 5750373 and U.S. Patent Publication No. 2007 / 0292936 and 2009 / 0002360. Antibodies separated from human antibody libraries are considered to be human antibodies or human antibody fragments herein.

[0272] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein can comprise an antigen-binding domain that binds to a CNS antigen or a brain antigen. As provided herein, the antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein can comprise an antigen-binding domain that binds to a CNS antigen or a brain antigen. In some aspects, the CNS antigen or brain antigen can be 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 secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), caspase 6, sortin (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 A 6A (MS4A6A) or transmembrane protein 106B (TMEM106b).In some aspects, the CNS antigen or brain antigen can be β-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), α-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), ubiquitin protein ligase E3A (UBE3A), γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), caspase 6, sorting protein (SORT), myeloid cell line Triggering receptor 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 non-metastatic melanoma 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 expressed on cells.

[0273] In some aspects, the CNS or brain antigen is on a cancer cell within the central nervous system. In some aspects, the CNS or brain antigen is a cell surface target selected from the following on a blood cancer cell: B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b. In some aspects, the CNS or brain antigen is a tumor cell target selected from the following: siglec-3 or CD33, siglec-5, siglec-7, siglec-9, siglec 14, PILRA, IL18-BP, MerTK, ACKR1, ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR(1-4), folate receptor α, GAL-3BP (galectin binding protein), GD2, GD3, GloboH (globohexasylceramide), gp100, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine-rich repeat protein 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon cancer kinase), RON, ROR1, TF (tissue factor) and TROP2.

[0274] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein may include an antigen-binding domain that binds to a CNS antigen or a brain antigen. As provided herein, the multispecific proteins provided herein may include 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 may include VH and VL. Exemplary CNS antigen binding VH and VL sequences are provided 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 herein by reference in its entirety.

[0275] Table 3: Exemplary CNS Antigens

[0276]

[0277] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are able to cross the BBB due to the fact that the anti-TfR antigen-binding domain in the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins are able to cross the BBB.

[0278] In some aspects, the internalization of the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein in the blood-brain barrier epithelial cell is greater than 10 times that of the isotype control. The blood-brain barrier epithelial cell can be, for example, a HCMEC / D3 cell.

[0279] In some aspects, relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, the fusion protein provided herein, antibody or its antigen binding fragment or multi-specific protein will not reduce the cell surface expression of TfR on HCMEC / D3 cells by more than 60%. In some aspects, relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, the fusion protein provided herein, antibody or its antigen binding fragment or multi-specific protein will not reduce the cell surface expression of TfR on HCMEC / D3 cells by more than 40%. Cell surface expression can be measured, for example, using Western blotting or FACS.

[0280] In some aspects, relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, the fusion proteins, antibodies or antigen binding fragments thereof, or multispecific proteins provided herein do not significantly increase the cell surface expression of TfR on HCMEC / D3 cells. Cell surface expression can be measured, for example, using Western blotting or FACS.

[0281] In some aspects, relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, the fusion proteins provided herein, antibodies or their antigen-binding fragments, or multispecific proteins will not reduce the cell surface expression of TfR on HCMEC / D3 cells by more than 60%, and relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, the cell surface expression of TfR on HCMEC / D3 cells will not be significantly increased. Cell surface expression. Cell surface expression can be measured, for example, using Western blot or FACS. In some aspects, relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, the fusion proteins provided herein, antibodies or their antigen-binding fragments, or multispecific proteins will not reduce the cell surface expression of TfR on HCMEC / D3 cells by more than 40%, and relative to the cell surface expression of TfR on HCMEC / D3 cells treated with isotype controls, the cell surface expression of TfR on HCMEC / D3 cells will not be significantly increased. Cell surface expression can be measured, for example, using Western blot or FACS.

[0282] In some aspects, the accumulation of the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein is at least 4 times that of the isotype control in the vascular depleted mouse brain. In some aspects, the accumulation of the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein is at least 5 times that of the isotype control in the vascular depleted mouse brain.

[0283] In some aspects, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein have an equilibrium dissociation constant (K) of about 6.7 nM to about 3.5 μM. D ) binds to human TfR. In some aspects, the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein has an equilibrium dissociation constant (K) of about 6.7 nM to about 340 nM. D ) binds to human TfR. In some aspects, the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein binds to human TfR with a K of about 38 nM to about 2.3 μM. D Binds to cynomolgus monkey TfR. In some aspects, the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein binds to cynomolgus monkey TfR with a K of about 18 nM to about 870 nM. D Binds to cynomolgus monkey TfR. In some aspects, the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein binds to cynomolgus monkey TfR with an equilibrium dissociation constant (K) of about 6.7 nM to about 3.5 μM. D ) binds to human TfR and herein with a K of about 38 nM to about 2.3 μM DBinds to cynomolgus monkey TfR. In some aspects, the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein binds to cynomolgus monkey TfR with an equilibrium dissociation constant (K) of about 6.7 nM to about 340 nM. D ) binds to human TfR and herein with a K of about 18 nM to about 870 nM D Binds to cynomolgus monkey TfR.

[0284] Polynucleotides and methods for preparing anti-TfR antigen binding domains and agents containing the same

[0285] In some aspects, provided herein are polynucleotides comprising nucleotide sequences encoding an antigen binding domain, fusion protein, antibody, antigen binding fragment thereof, or multispecific protein described herein that specifically binds to human TfR, or a domain thereof described herein; and vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells).

[0286] In some aspects, the polynucleotides provided herein include nucleic acid molecules encoding the heavy chain of the antigen binding domain of human TfR provided herein. In some aspects, the polynucleotides provided herein include nucleic acid molecules encoding the light chain of the antigen binding domain of human TfR provided herein. In some aspects, the polynucleotides provided herein include nucleic acid molecules encoding the heavy chain of the antigen binding domain of human TfR provided herein and nucleic acid molecules encoding the light chain of the antigen binding domain of human TfR provided herein.

[0287] In some aspects, provided herein is a combination or composition of polynucleotides. In some aspects, a 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 multi-specific protein provided herein, for example, wherein the first polynucleotide encodes a first heavy chain, the second polynucleotide encodes a second heavy chain and an antigen binding domain specifically bound to human TfR provided herein, and the third polynucleotide encodes a light chain. In some aspects, the antigen binding domain bound to human TfR is a scFv. In some aspects, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation. In some aspects, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0288] In some aspects, 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 second antigen binding domain that specifically binds to human TfR, and the third polynucleotide encodes a light chain. In some aspects, the first antigen binding domain and the second antigen binding domain that bind to human TfR comprise the same amino acid sequence. In some aspects, the first antigen binding domain and the second antigen binding domain that bind to human TfR comprise different amino acid sequences. In some aspects, the first antigen binding domain and / or the second antigen binding domain that bind to human TfR are scFv. In some aspects, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation. In some aspects, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0289] In some aspects, the combination or composition comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide encode a multispecific protein provided herein, wherein the first polynucleotide encodes a heavy chain and an antigen binding domain that binds to human TfR provided herein, and wherein the second polynucleotide encodes a light chain.

[0290] Also provided herein are polynucleotides comprising a nucleotide sequence encoding an antigen binding domain, a fusion protein, an antibody, an antigen binding fragment thereof, or a multispecific protein or a domain thereof as described herein that specifically binds to human TfR, the polynucleotides are optimized, for example, by codon / RNA optimization, replacement with a heterologous signal sequence, and / or elimination of mRNA instability elements. The method of generating an optimized nucleic acid for recombinant expression by introducing codon changes (e.g., codon changes encoding the same amino acid due to the degeneracy of the genetic code) and / or eliminating inhibitory regions in mRNA can be performed by using, for example, the optimization methods described in U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498.

[0291] Polynucleotides comprising nucleotide sequences encoding antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multispecific proteins or domains thereof described herein that specifically bind to human TfR as described herein can be generated from nucleic acids from a suitable source (e.g., hybridomas) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, genomic DNA obtained from hybridoma cells that produce antibodies of interest can be used to perform PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of known sequences. Such PCR amplification methods can be used to obtain nucleic acids comprising sequences encoding, for example, light chains and / or heavy chains of antigen binding domains, antibodies, or antigen binding fragments thereof. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to generate antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multispecific proteins or domains thereof described herein that specifically bind to human TfR as described herein.

[0292] The polynucleotides provided herein may be in the form of, for example, 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 a coding strand or a non-coding (antisense) strand. In some aspects, the polynucleotides are cDNA or DNA lacking one or more endogenous introns. In some aspects, the polynucleotides are non-naturally occurring polynucleotides. In some aspects, the polynucleotides are recombinantly produced. In some aspects, the polynucleotides are separated. In some aspects, the polynucleotides are substantially pure.

[0293] 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 messaging RNA (mRNA). In some embodiments, the synthetic mRNA has at least one nucleoside modification. In some embodiments, at least one nucleoside modification is selected from the group consisting of: pyridin-4-one ribonucleoside, 5-aza-uridine, 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-taurinemethyluridine, 1-taurinemethyl-pseudouridine, 5-taurinemethyl-2-thio-uridine, 1-taurinemethyl-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, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 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, pyrrolocytidine, pyrrolo 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-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthioadenine, 2-methoxyadenine, inosine, 1-methylinosine, wyosine, wyosine, 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-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxoguanosine, 7-methyl-8-oxoguanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine and N2,N2-dimethyl-6-thio-guanosine.

[0294] In some aspects, provided herein are polynucleotides encoding fusion proteins, the fusion proteins comprising antigen binding proteins and heterologous polypeptides provided herein. In some aspects, the heterologous polypeptide comprises an antigen binding domain that binds to the following: β-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), α-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), γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), caspase 6, sorting protein (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 non-metastatic melanoma protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4 domain A4A (MS4A4A), transmembrane 4 domain A 6A (MS4A6A), or transmembrane protein 106B (TMEM106b), or a portion thereof. In some aspects, 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 aspects, the multispecific protein comprises an antigen binding domain that binds to: 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-glucocerebrosidase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), caspase 6, sorting protein (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 non-metastatic melanoma protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4 domain A4A (MS4A4A), transmembrane 4 domain A 6A (MS4A6A), or transmembrane protein 106B (TMEM106b), or a portion thereof. In some aspects, 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).

[0295] In some embodiments, polynucleotides are disclosed herein, which encode fusion proteins disclosed herein that include heterologous polypeptides. 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), prosphingolipid activator protein (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), prosphingolipid activator protein (PSAP) or survival motor neuron protein (SMN). In some aspects, the heterologous protein is an enzyme selected from the group consisting of alpha-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), beta-glucocerebrosidase (GCase or GBA), galactosylceramide 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 surrogate thereof such as laronidase, idurosulfatase, elosulphase alpha or galactosylsulfatase, or a variant thereof, or a catalytically active fragment thereof.In some aspects, the heterologous protein is an enzyme 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-α-glucosaminide N-acetyltransferase (HGSNAT), and N-acetyl-α-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 β-galactosidase, glucosylceramidase, β-hexosaminidase A, β-hexosaminidase B, arylsulfatase A, β-galactosidase, acid ceramidase, α-glucosaminidase glycosidase, lysosomal acid lipase, lysosomal protease, its synthetic enzyme substitute such as laronidase, iduronate sulfatase, elosulphase α or galactose sulfatase, or its variant, or its catalytically active fragment 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 β-galactosidase, glucosylceramidase, β-hexosaminidase A, β-hexosaminidase B, arylsulfatase A, β-galactosidase, acid ceramidase, α-glucosidase, lysosomal acid lipase, lysosomal protease, its synthetic enzyme substitute such as laronidase, idursulfatase, elosulphase α or galactosylsulfatase, or its variant, or its catalytically active fragment. In some embodiments, the polynucleotide is mRNA (e.g., synthetic mRNA).

[0296] In certain aspects, provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multispecific proteins or domains thereof described herein that specifically bind to human TfR as described herein, for recombinant expression in host cells (e.g., mammalian host cells). Vectors for producing antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multispecific proteins or domains thereof described herein that specifically bind to human TfR as described herein can be produced by recombinant DNA technology, for example, using techniques well known in the art. These methods include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo gene recombination. Also provided are replicable vectors comprising nucleotide sequences encoding antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multispecific proteins or domains thereof described herein that specifically bind to human TfR as described herein, and the nucleotide sequences are operably linked to a promoter. Such vectors may, for example, include nucleotide sequences encoding constant regions of antigen binding domains, antibodies, or antigen binding fragments thereof (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Pat. No. 5,122,464), and variable domains of antigen binding domains, antibodies, or antigen binding fragments thereof may be cloned into such vectors for expression of the entire heavy chain, the entire light chain, or both the entire heavy chain and the entire light chain. In some embodiments, the vector is a gene therapy vector (e.g., AAV or lentiviral vector).

[0297] In some aspects, provided herein is an expression system comprising a polynucleotide comprising a nucleotide sequence encoding an antigen binding domain, a fusion protein, an antibody, an antigen binding fragment thereof, or a multispecific protein or a domain thereof described herein that specifically binds to human TfR. The expression system may be contained on a vector. The expression system may also be integrated into a host cell chromosome. 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 a vector provided herein.

[0298] Therefore, cells are also provided herein, such as host cells, which include polynucleotides and / or vectors for recombinantly expressing antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multispecific proteins or domains thereof described herein that specifically bind to human TfR as described herein. In some aspects, in order to express double-chain antigen binding proteins, vectors encoding both heavy and light chains alone can be co-expressed in host cells to express the entire immunoglobulin. In some aspects, the host cell contains two different vectors, the first vector comprising polynucleotides encoding the heavy chain of the antigen-binding proteins described herein, and the second vector comprising polynucleotides encoding the light chain of the antigen-binding proteins. In some aspects, the first host cell comprises a first vector containing polynucleotides encoding heavy chains, and the second host cell comprises a second vector containing polynucleotides encoding light chains. In some aspects, a host cell group is provided herein, which comprises such a first host cell and such a second host cell.

[0299] In some aspects, provided herein is a method for producing an antigen binding domain, fusion protein, antibody, antigen binding fragment thereof, or multi-specific protein or its domain described herein that specifically binds to human TfR as described herein in a host cell. In some aspects, provided herein is a method for producing a single-chain antigen binding domain, Fc domain, and a heterologous protein or polypeptide that specifically binds to human TfR. In some aspects, provided herein is a method for producing a single-chain antigen binding domain, Fc domain, and a second antigen binding domain that specifically binds to human TfR as described herein. The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques, and the resulting cell can be subsequently cultured by conventional techniques to produce an antigen binding domain, fusion protein, antibody, antigen binding fragment thereof, or multi-specific protein or its domain described herein that specifically binds to human TfR as described herein.

[0300] A variety of host expression vector systems can be used to express the antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multispecific proteins or domains thereof described herein that specifically bind to human TfR as described herein (see, e.g., U.S. Pat. No. 5,807,715). Such host expression systems represent not only vehicles by which coding sequences of interest can be produced and subsequently purified, but also cells that, when transformed or transfected with appropriate nucleotide coding sequences, can express in situ the antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multispecific proteins or domains thereof described herein that specifically bind to human TfR as described herein. They include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the antibody coding sequence; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the antibody coding sequence; plant cell systems (e.g., green algae, such as Chlamydomonas reinhardtii) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) containing the coding sequence or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the coding sequence; or mammalian cell systems (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), which have a recombinant expression construct containing a promoter derived from a mammalian cell genome (e.g., a metallothionein promoter) or a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). In some aspects, the cell used to express the antigen binding domain, fusion protein, antibody, antigen binding fragment thereof, or multispecific protein described herein that specifically binds to human TfR or its domain described herein is a CHO cell, such as a CHO GS System TMIn some aspects, the cells used to express the antigen binding domain, fusion protein, antibody, antigen binding fragment thereof, or multispecific protein described herein that specifically binds to human TfR or the domain thereof described herein are human cells, such as human cell lines. In some aspects, the mammalian expression vector is pOptiVEC TM Or pcDNA3.3. In some aspects, bacterial cells such as Escherichia coli or eukaryotic cells (e.g., mammalian cells) are used to express antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multispecific proteins or domains thereof described herein that specifically bind to human TfR. For example, mammalian cells such as Chinese hamster ovary (CHO) cells are combined with vectors such as major intermediate early gene promoter elements from human cytomegalovirus to be effective expression systems for antibodies (Foecking MK and Hofstetter H (1986) Gene 45: 101-105; and Cockett MI et al., (1990) Biotechnology 8: 662-667). In some aspects, antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multispecific proteins or domains thereof described herein that specifically bind to human TfR as described herein are produced by CHO cells or NS0 cells.

[0301] In addition, a host cell strain may be selected that modulates the expression of the inserted sequences, or modifies and processes the gene product in a desired specific manner. Such modification (e.g., glycosylation) and processing (e.g., cleavage) of the protein product may aid in protein function. To this end, eukaryotic host cells may be used that have the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, 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.

[0302] Once the antigen binding domain, fusion protein, antibody, antigen binding fragment thereof, or multispecific protein described herein that specifically binds to human TfR or a domain thereof described herein has been produced by recombinant expression, it can be purified by any method known in the art for purification, such as by chromatography (e.g., ion exchange, affinity, especially affinity for a specific antigen after protein A and fractionated column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. In addition, the antigen binding domain, fusion protein, antibody, antigen binding fragment thereof, or multispecific protein described herein that specifically binds to human TfR or a domain thereof described herein can be fused to a heterologous polypeptide sequence to facilitate purification.

[0303] In some aspects, once the antigen binding domain, fusion protein, antibody, antigen binding fragment thereof, or multi-specific protein described herein that specifically binds to human TfR is separated or purified. In general, the antigen binding domain, fusion protein, antibody, antigen binding fragment thereof, or multi-specific protein described herein that specifically binds to human TfR is substantially free of other proteins. For example, in some aspects, the preparation of the antigen binding domain, fusion protein, antibody, antigen binding fragment thereof, or multi-specific protein described herein that specifically binds to human TfR is substantially free of cellular material and / or chemical precursors.

[0304] Pharmaceutical composition

[0305] Provided herein is a composition comprising an antigen binding domain, fusion protein, antibody, antigen binding fragment thereof, or multi-specific protein specifically bound to human TfR as described herein. In some aspects, the antigen binding domain, fusion protein, antibody, antigen binding fragment thereof, or multi-specific protein specifically bound to human TfR with the desired purity is present in a preparation comprising, 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 nontoxic to the recipient at the dose and concentration used. Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may include antioxidants, buffers, antibacterial agents, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives.

[0306] In some aspects, the pharmaceutical composition comprises an antigen binding domain, fusion protein, antibody, antigen binding fragment thereof, or multispecific protein described herein that specifically binds to human TfR and a pharmaceutically acceptable carrier (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th Edition (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd Edition, Pharmaceutical Press (2000)). In some aspects, the pharmaceutical composition described herein is used as a medicament. The composition to be used for in vivo administration may be sterile. This is easily achieved by filtration through, for example, a sterile filtration membrane.

[0307] Also provided herein are pharmaceutical compositions comprising polynucleotides encoding antigen binding domains, fusion proteins, antibodies or antigen binding fragments thereof, or multi-specific proteins specifically binding to human TfR as described herein. In some embodiments, the polynucleotides are RNA. In some embodiments, the polynucleotides are synthetic mRNA. In some embodiments, the polynucleotides are modified mRNA. In some embodiments, the pharmaceutical composition comprising polynucleotides further comprises a lipid-based transfection reagent.

[0308] The pharmaceutical compositions described herein can be used to exert a biological effect in vivo or in vitro. For example, the pharmaceutical compositions described herein can be used to cross the blood-brain barrier, for example, in a subject.

[0309] In some aspects, provided herein is a pharmaceutical composition for treating a disease or illness, such as neuropathy, neurodegenerative disease, cancer, eye disease, epileptic seizure, lysosomal storage disease, amyloidosis, viral or microbial disease, ischemia, behavioral disorders and CNS inflammation. In some aspects, provided herein is a pharmaceutical composition for treating a disease or illness, 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 aspects, provided herein is a pharmaceutical composition for treating frontotemporal dementia.

[0310] 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.

[0311] Methods of using anti-TfR antigen binding domains and agents comprising the same

[0312] As provided herein, antigen binding domains that specifically bind to human TfR, fusion proteins comprising such antigen binding domains, antibodies, antigen binding fragments thereof, and multispecific proteins are preferably transported across the blood-brain barrier. Therefore, provided herein is a method for administering or transporting an antigen binding protein that specifically binds to human TfR or a fusion protein, antibody, antigen binding fragment thereof, or multispecific protein that specifically binds to an antigen binding protein that specifically binds to human TfR across the blood-brain barrier of a subject, comprising 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 that specifically binds to an antigen binding protein that specifically binds to human TfR.

[0313] In view of the ability of the antigen binding domains specifically bound to human TfR and fusion proteins, antibodies, antigen binding fragments thereof, and multi-specific proteins comprising such antigen binding domains to transport across the blood-brain barrier as provided herein, they can be used to treat nervous system diseases or disorders. In some aspects, the method for treating a nervous system 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 multi-specific protein comprising an antigen binding protein that specifically binds to human TfR. Nervous system diseases or disorders may be, for example, neuropathy disorders, neurodegenerative diseases, cancer, eye disease disorders, epileptic seizures, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, or CNS inflammation.The nervous system disease or disorder can be, for example, a neurodegenerative disease (such as Lewy body disease, post-polio syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, Gaucher disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), a tauopathies (such as Alzheimer's disease and supranuclear palsy), a prion disease (such as bovine spongiform encephalopathy, scrapie, Creutz-feldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), a bulbar palsy, a motor neuron disease, a degenerative disease of the nervous system (such as Canavan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander's disease, Tourette's syndrome, syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, Laforadisease, Rett syndrome, Wilson's disease, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome. syndrome), dementia (such as Pick's disease and spinocerebellar ataxia), cancer of the CNS and / or brain (such as glioblastoma or brain metastases caused by 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's 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 aspects, the neurological disease or disorder is Alzheimer's disease. In some aspects, 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.

[0314] In some aspects, the present invention provides a method of treating a lysosomal storage disease with a fusion protein disclosed herein. In some aspects, the lysosomal storage disease is selected from Gaucher disease, ceroid lipofuscinosis (Patton disease), mucopolysaccharidosis (MPS) type I, MPS type II, and MPS type III.

[0315] Antigen binding domains that specifically bind to human TfR as provided herein, as well as fusion proteins, antibodies, antigen binding fragments thereof, and multispecific proteins comprising such antigen binding domains, can be used to detect antigens (e.g., CNS antigens or brain antigens). Antigen binding domains that specifically bind to human TfR and fusion proteins, antibodies, antigen binding fragments thereof, and multispecific proteins comprising such antigen binding domains can be labeled for the purpose. Exemplary labels include, for example, radioisotopes (e.g. 64 CU) and fluorescent labeling. Therefore, there is provided a method for detecting antigens using an antigen-binding protein specifically bound to human TfR as described herein or a fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein comprising an antigen-binding protein specifically bound to human TfR. In some aspects, the method for detecting an antigen in the CNS (e.g., brain) of a subject includes administering an antigen-binding protein specifically bound to human TfR or a fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein comprising an antigen-binding protein specifically bound to human TfR to the antigen in the CNS (e.g., brain). Such methods may further include, for example, positron emission tomography (PET) imaging of the subject. In some aspects, disclosed herein is a method for detecting CNS antigens in vitro, comprising contacting an in vitro sample with a fusion protein, antibody, or antigen-binding fragment thereof, or multi-specific protein provided herein and positioning an imaging agent in the sample.

[0316] As provided herein, antigen binding domains that specifically bind to human TfR and fusion proteins, antibodies, antigen binding fragments thereof, and multi-specific proteins comprising such antigen binding domains can be used for prognosis, diagnosis, monitoring and / or screening applications, including in vivo applications that are well known and standard to those skilled in the art and based on this specification. In some aspects, there is provided herein a fusion protein, antibody, antigen binding fragment thereof, or multi-specific protein that specifically binds to an antigen binding protein of human TfR or comprises an antigen binding protein that specifically binds to human TfR, which is used as a diagnostic agent. In some aspects, an antigen binding protein that specifically binds to human TfR or a fusion protein, antibody, antigen binding fragment thereof, or multi-specific protein that comprises an antigen binding protein that specifically binds to an antigen binding protein of human TfR comprises a detectable label.

[0317] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0318] The present disclosure will be more fully understood by reference to the following examples. However, they should not be considered to limit the scope of the present disclosure. All references throughout the present disclosure are hereby expressly incorporated by reference.

[0319] Example

[0320] Example 1: Generation of Avi-His tagged variants of transferrin receptor (TfR)

[0321] By cloning the synthetic gene based on cDNA into mammalian expression vector, transient transfection and expression in Expi293 cells subsequently, mammalian expression of transferrin receptor antigen (SEQ ID NO:1-7) people, cynomolgus macaque (cynomolgusmacaque, cyno) and mouse variants is carried out.Construct comprises heterologous signal peptide and C-terminal Avi-His tag, so that purification and biotinylation are carried out.In brief, according to manufacturer's specification sheets, the plasmid of encoding antigen is transfected using Expifectamine 293 transfection kit (ThermoFisher A14524).Five days after transfection, harvest culture supernatant, by centrifugal clarification, and use HisPur Ni-NTA resin (Thermo Scientific 88223) to purify in drip column (drip-column) form.Use 0.2um filter device to filter 200mL culture supernatant, and 3mL resin slurry in PBS is added in the supernatant filtered. The sample was incubated overnight at 4 ° C under shaking, and the next day, the beads were loaded onto a 20 mL drip column, washed with 10 mL His-Select wash buffer (Millipore Sigma H5288), and eluted with 5 mL His-Pur elution buffer (Millipore Sigma H5413). The eluent buffer was replaced with PBS using an Amicon Ultra-15 centrifugal filter device (MilliporeUFC9010). The antibody concentration was quantified by measuring the absorbance at 280 nm using Nanodrop 8000 (ThermoFisher). Antigen purity was determined by SDS-PAGE. Size exclusion chromatography (SEC) was used to analyze the aggregation of antigens. According to the manufacturer's instructions, some antigens were biotinylated using a BirA biotin-protein ligase kit (AVIDITY).

[0322] The apical domain of TfR provides a pathway 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 interfering with the native function of the receptor. Tagged variants of the apical domain of Tfr were generated. These constructs contained amino acids 69-263 of the full-length human TfR sequence, with 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 by HisPur Ni-NTA resin as described above.

[0323] DNA fragments encoding the apical domains of human TfR (huTfR) and cynomolgus monkey TfR (cynoTfR) and the arrayed forms of the N-terminal His / Avi tags were ordered from GeneArt and cloned into the pcDNA 3.4 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.

[0324] Table 4. Avi-His tagged TfR variants

[0325]

[0326]

[0327] Example 2: Generation of a CHO cell line overexpressing TfR

[0328] Several cell lines were generated for screening the binding of anti-TfR antibodies obtained as described herein. Briefly, CHO cells stably expressing human TfR and mouse TfR were generated using pLenti-EF1a constructs expressing full-length human TfR and mouse TfR (SEQ ID NOs: 8 and 9, respectively). Human TfR with hygromycin selection and mouse TfR with hygromycin selection were expressed using lentiviral constructs (Genecopoeia). ViraSafe TMThe lentivirus packaging system (CellBiolabs VPK-206) generates lentivirus by transfection of 293T cells. Subsequently, the supernatant containing the lentivirus was used to transduce Chinese hamster ovary (CHO) cells. Two days after transduction, puromycin or hygromycin was added to the culture medium as a selection pressure for human TfR and mouse TfR. The cell surface expression of the obtained CHO cells stably expressing human TfR and mouse TfR was analyzed by flow cytometry.

[0329] Table 5. Full-length TfR sequences

[0330]

[0331]

[0332] Example 3: Generation of TfR humanized mice

[0333] Mouse strains were generated at Taconic Biosciences GmbH (Germany) for humanization of the extracellular domain of TfR. Mouse ECD was replaced with a human version using CRISPR, while retaining the mouse intracellular and transmembrane parts under the control of a mouse promoter. Brain sections from these mice were evaluated by IHC and / or Western blotting to confirm the expression and location of human ECD in vivo (data not shown).

[0334] Example 4: Generation of anti-TfR hybridoma antibodies

[0335] To obtain antibodies against TfR, the following procedure was used to generate hybridomas. BALB / c mice or Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) were immunized twice a week by subcutaneous or intraperitoneal injection of purified extracellular domain polypeptides of human, cynomolgus monkey and / or mouse TfR with or without adjuvant (obtained as described in Example 1 above). Lymph nodes were harvested from mice or rats for hybridoma cell line generation after a total of 7-8 injections and three days after the last boost.

[0336] The reactivity of serum from animals to TfR was analyzed by FACS on CHO cells overexpressing human or mouse TfR and by ELISA against human, cynomolgus monkey and mouse TfR Avi-His protein (Example 1). Lymphocytes of animals whose serum expressed strong binding to CHO cells overexpressing human or mouse TfR were isolated and fused with SP2 / mIL-6 (CRL-2016, American Type Culture Collection, Rockville, MD) or Sp2ab (ENZ-70008, Enzo Life Sciences, Farmingdale, NY) mouse myeloma cells via electrofusion (Hybrimune, BTX, Holliston, MA) and incubated overnight at 37°C, 5% CO2 in Clonacell-HY medium C (Stemcell Technologies, Vancouver, BC, Canada, catalog number 03803).

[0337] The next day, the fused cells were centrifuged and resuspended in Clonacell-HY medium E (Stemcell Technologies, catalog number 03805) containing HAT (Sigma Aldrich, catalog number H0262). The cells were inoculated into a T225 flask and grown for 7 days at 37°C, 5% CO2, and then the hybridoma library was cryopreserved. The library was then thawed and recovered overnight in Clonacell-HY medium E. The next day, IgG positive hybridoma single cells were sorted into a Falcon 96-well U-bottom plate using a FACS Aria II cell sorter (BD Biosciences, San Jose, CA). 768 hybridomas were sorted from mouse fusions, and 768 hybridomas were sorted from rat fusions. After growing the cells in 200 ul Clonacell-HY medium E at 37°C, 5% CO2 for 11 days, tissue culture supernatant from each well was screened by FACS on CHO cells overexpressing human or mouse TfR (as described below).

[0338] Example 5: Screening of anti-TfR antibody hybridoma supernatants by FACS

[0339] A total of 1536 anti-TfR hybridoma supernatants were initially screened by FACS to determine their ability to differentially bind to CHO cells overexpressing human or mouse TfR cells compared to CHO parental cells. Overexpressing cells were harvested, washed, and labeled with Pacific Blue, FITC, and Pacific Orange dyes (ThermoFisher) to create unique barcoded cell populations. Barcoded cells (5×10 per cell population) were then added to the 4% 4% 2 ... 4 ) were aliquoted into 96-well U-bottom plates and incubated on ice for 30 minutes with 50 μl hybridoma cell culture supernatant or 1 μg / ml commercially purified mouse anti-human TfR monoclonal antibody (Sigma Aldrich, catalog number SAB4700515) and rat anti-mouse TfR antibody (Invitrogen, Waltham, MA, catalog number R17217). After this primary incubation, the supernatant was removed by centrifugation, the cells were washed twice with 175 μl ice-cold FACS buffer (PBS + 1% FBS + 2mM EDTA), and then the cells were further incubated on ice with anti-mouse IgG Fc-allophycocyanin (APC) or anti-rat IgG Fc-APC (Jackson Labs, catalog numbers 115-136-071 and 112-136-071, respectively) (1:1000 dilution) for 20 minutes. After this secondary antibody incubation, cells were washed twice again with ice-cold FACS buffer and resuspended in a final volume of 50 μl of FACS buffer. Binding intensity on cells was analyzed using a FACS Canto system (BD Biosciences), and the APC mean fluorescence intensity (MFI) ratio on each barcoded cell population was determined for each anti-TfR hybridoma supernatant tested.

[0340] A total of 189 mouse hybridomas and 115 rat hybridoma clones showed greater than 3-fold difference in binding to CHO cells stably overexpressing human TfR (as determined by MFI) compared to isotype controls. A total of 15 rat hybridomas showed greater than 2-fold difference in binding to CHO cells stably overexpressing mouse TfR compared to isotype controls (data not shown). None of the hybridoma clones cross-reacted with human and mouse TfR by FACS.

[0341] Table 6. FACS results of selected mouse hybridoma supernatants (shown as fold relative to CHO parent)

[0342]

[0343] Table 7. FACS results of selected rat hybridoma supernatants (shown as fold relative to CHO parent)

[0344]

[0345]

[0346] Example 6: Screening of anti-TfR antibody hybridoma supernatants by recombinant TfR protein binding assay

[0347] 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 (as described in Example 1) compared to binding to an unrelated Avi-His-tagged control protein. 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 with ELISA diluent (PBS + 0.5% BSA + 0.05% Tween 20) for one hour. Blocking buffer was removed, and Avi-His-tagged human TfR, human TfR apical domain, mouse TfR, and unrelated Avi-His tagged proteins were added to ELISA diluent at 1 μg / ml and captured at room temperature for 1 hour. After washing three times with 300 μl of PBST (PBS+0.05% Tween20, Thermo 28352), hybridoma cell culture supernatant (50 μl / well) was added to each well. After incubation at room temperature for 30 min, the plate was washed three times with 300 μl 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 in ELISA diluent at 1:5000, added to each well at 50 μl / well, and incubated for 30 minutes at room temperature under shaking. After the last set of washes (3×300 μl in PBST), 50 μl / well of BioFx TMB substrate (Surmodics, catalog number TMBW-1000-01) was added to the wells. After 5-10 minutes, the reaction was quenched with 50 μl / well of 2N sulfuric acid. The absorbance of the plate at 450 nm was read on a SpectraMax M5 (Molecular Devices, Sunnyvale, CA) using SoftMax Pro software.

[0348] From this hybridoma supernatant screening, a total of 112 anti-TfR hybridoma clones were identified that exhibited greater than a 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 huTfR apical domain. The binding data for selected mouse anti-TfR hybridoma clones and rat anti-TfR hybridoma clones (shown as a ratio of binding to recombinant huTfR Avi-His relative to an unrelated protein) are shown in Tables 8 and 9, respectively.

[0349] Table 8: ELISA (OD450) results of selected mouse hybridoma supernatants (shown as fold relative to irrelevant proteins)

[0350]

[0351]

[0352] Table 9: ELISA (OD450) results of selected rat hybridoma supernatants (shown as fold relative to irrelevant proteins)

[0353]

[0354] Example 7: Internalization of anti-TfR antibodies into blood-brain barrier endothelial cell lines in vitro

[0355] Anti-TfR hybridoma antibodies were purified and screened for their ability to internalize into the hCMEC / D3 cell line.

[0356] The supernatant from anti-TfR hybridoma clones was purified on Hamilton STAR platform (Hamilton Company, Reno, NV) using ProPlus Phytip columns (Biotage, Uppsala, Sweden, catalog number PTH 91-20-07). In brief, the antibody from the supernatant was captured by coupled protein A on the resin-filled pipette 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 1M Tris-HCl pH 8. The concentration of purified antibodies was determined by measuring the absorbance at 280nm using Nanodrop 8000 (ThermoFisher). The internalization ability of hybridoma purified antibodies was then tested.

[0357] Internalization into endothelial cells at the blood-brain barrier is the first stage of transcytosis across the BBB into the brain. In order to identify anti-TfR antibodies with internalization ability, HCMEC / D3 cells were seeded in black-walled transparent bottom 96-well plates (#3904, Corning) at 2.5*10^4 cells / well. The next day, cells were treated with anti-TfR antibodies pre-conjugated with 6 μg / ml and an equal concentration of pHrodo-Red labeling reagent (Z25612, Invitrogen) in 100 μl culture medium (EBM2, Lonza). Human IgG isotypes and anti-TfR antibodies with known internalization ability were included in the assay as negative and positive controls, respectively.

[0358] The plate was then placed in an IncuCyte machine (Live Cell Analysis System) and images were captured every 2 hours over a 24 hour time course. Images were then processed and analyzed using the IncuCyte software. Internalization data (pHrodo Red positive area μm) at the 24 hour time point 2 / image) as relative fold change of isotype hIgG1, summarized in Table 10 below.

[0359] Data are reported as total red area per well and are shown as fold change relative to an irrelevant mouse IgG antibody used as a negative control in Table 10. An anti-TfR antibody was also included in this assay as a positive control.

[0360] Table 10: Internalization of purified anti-TfR hybridoma antibodies

[0361]

[0362]

[0363] Example 8: Molecular cloning of anti-TfR antibodies

[0364] Anti-TfR antibodies from the above hybridomas were cloned as follows. 1-2x10 5 Hybridoma cells were washed with PBS and resuspended in 200 μl RNAlater (Invitrogen, catalog number AM7021). The samples were stored at -80°C and sent to Abterra Biosciences (San Diego, CA) for sequencing. In brief, RNA was extracted and cDNA synthesis was performed. The variable regions of IgG / IgM, IgK and IgL were amplified using a 5'RACE strategy using proprietary primers. The hybridoma variable region amplicons were sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA). Reads from hybridomas were processed by Abterra's Reptor analysis process.

[0365] A total of 32 unique sequences were identified among the 72 apical domain-positive anti-TfR hybridoma clones. The amino acid sequences of the variable heavy and variable light chains of the unique apical domain-binding anti-TfR antibodies of mouse origin are provided in Table 11 below. An additional 21 unique sequences were identified among the 24 apical domain-positive anti-TfR hybridoma clones from rats.

[0366] Table 11: Anti-huTfR apical domain antibody sequences from mouse hybridoma

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374] Table 12: Anti-huTfR apical domain antibody sequences from mouse hybridoma (Kabat)

[0375]

[0376]

[0377]

[0378]

[0379] *HVR = Hypervariable Region; also called CDR

[0380] Table 13: Anti-huTfR apical domain antibody sequences from mouse hybridoma (Kabat)

[0381]

[0382]

[0383]

[0384]

[0385] Example 9: Hybridoma antibody reformatting into 2+1 bispecific antibody

[0386] Antibodies for formatting into a 2+1 bispecific format were selected based on various criteria: 1) the antibodies covered a broad range of affinities based on ELISA and FACS binding assays, 2) the antibodies covered a broad range of internalization activities, 3) no prominent high-risk modification sites were identified in the CDRs, and 4) the antibodies were phylogenetically distinct from each other in the hybridoma sequences obtained.

[0387] A non-target specific IgG isotype antibody with a knob-mutation in the heavy chain constant domain ("inert isotype control antibody") 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 a "hole" mutation (the "hole side" of the IgG isotype antibody). The IgG isotype antibody attached to the scFv is called a 2+1 bispecific antibody, such as Figure 1A and Figure 1D As shown in (i). The 20 amino acid linker connecting the VH domain and the VL domain in the scFv is GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 183) (Bird et al., Science October 21, 1988; 242(4877): 423-6), and the linker connecting the C-terminus of the Fc "hole" domain to the scFv is (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).

[0388] The scFv (VH and VL) sequences were formatted into expression constructs of IgG heavy chains with "hole" mutations ("heavy chain-hole" constructs) using the framework in SEQ ID NO: 185. In some cases, the "heavy chain-hole" constructs comprised a set of mutations (H435R, Y436F) to minimize binding to protein A (Tustian et al., MAbs. 2016 May-Jun; 8(4):828-38) (SEQ ID NO: 186). The 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 comprised mutations that reduced effector function (SEQ ID NOs: 189-194).

[0389] The exemplary structure of a 2+1 bispecific antibody comprises the following components: 1) an isotype control hIgG1 wild-type antibody having a knob ((T366W) mutation and a hole mutation (T366S_L368A_Y407V) in the constant region, 2) a (G4S)x3 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.

[0390] Table 14. 2+1 bispecific antibody sequences

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398] To generate 2+1 bispecific antibodies, transient transfection of Expi293 cells (Invitrogen) was performed according to the manufacturer's instructions. For 20 mL culture, 20 μg of total DNA consisting of 3 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 achieve high purity of 2+1 bispecific antibodies. Cell culture supernatant was harvested 5 days after transfection. The clarified supernatant was purified using a drip column with MabSelectSure resin (Cytiva), washed with PBS, eluted with pH 3.5 elution buffer, and neutralized to a final pH of 5.5-6.0 with Tris-HCl. The neutralized eluate containing the antibody was dialyzed into PBS. Antibody concentration was quantified by measuring the absorbance at 280 nm using Nanodrop 8000 (ThermoFisher) or Lunatic (Unchained Labs). The purity of the 2+1 bispecific antibody was determined by SDS-PAGE. Aggregation of the 2+1 bispecific antibody was analyzed using size exclusion chromatography (SEC). Antibodies were then produced and purified as 2+1 bispecific antibodies and analyzed via ELISA, FACS, and internalization to confirm binding retention in the 2+1 format.

[0399] Example 10: Generation and characterization of 2+1 anti-TfR bispecific antibodies

[0400] Anti-TfR hybridoma antibodies were converted to 2+1 format and expressed and purified as described in Example 9. Binding retention was confirmed via ELISA and FACS.

[0401] To determine the binding retention of the reformatted scFv 2+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. The 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, room temperature). The plates were washed three times again with PBST and incubated with biotinylated human, cynomolgus monkey and mouse TfR and an irrelevant antigen, human transferrin receptor (1 μg / ml, 1 h, room temperature). The plates were washed three times again with PBST and incubated with anti-TfR antibody (1 μg / ml, 1 h, room temperature). The plates were washed three times again with PBST and incubated with the secondary detection antibody anti-human horseradish peroxidase-conjugated antibody (1:5000 dilution, 30 min, room temperature). The plates were washed three times for the final time and incubated with TMB-ELISA substrate solution (Thermo Fisher) and then quenched with H2SO4. The OD values ​​at 450 nm (OD 450 ) was used to analyze anti-TfR binding. The binding characteristics of scFv 2+1 anti-TfR antibodies to TfR are summarized in Table 15 below as fold changes relative to isotype hIgG.

[0402] In order to determine the binding retention of the reformulated 2+1 scFv anti-TfR antibody by FACS, the binding of the antibody to HCMEC / D3 cells (a cell line derived from human brain endothelial cells), a CHO cell line overexpressing human and mouse TfR, and CHO cells was evaluated for nonspecific binding. The cells were seeded in tissue culture plates (50,000 cells / well), washed and resuspended in FACS buffer (PBS+2% BSA+1mM 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 an anti-mouse secondary antibody conjugated to allophycocyanin (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 on an iQue flow cytometer (IntelliCyt) by measuring mean fluorescence intensity (MFI). FACS data are summarized in Table 15 below as fold change in MFI relative to isotype hIgG1.

[0403] In summary, 28 of the 32 reformatted antibodies retained binding affinity for 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, so only those antibodies were considered for further maturation.

[0404] Table 15: Binding characteristics of anti-TfR antibodies reformatted into a 2+1 bispecific format.

[0405]

[0406]

[0407] Example 11: In vitro internalization of 2+1 anti-TfR bispecific antibodies in BBB endothelial cell lines

[0408] Internalization of 2+1 anti-TfR bispecific antibodies into hCMEC / D3 cell lines was performed as previously described (Example 7). The 2+1 anti-TfR bispecific antibodies displayed a wide range of internalization capabilities, many of which displayed significantly higher internalization capabilities than the anti-TfR control antibody.

[0409] Table 16: Internalization characteristics of anti-TfR antibodies reformatted into 2+1 bispecific antibody format (fold change of cumulative intensity / phase area per well)

[0410]

[0411]

[0412] Example 12: Humanization of anti-TfR mouse antibodies

[0413] The scFv portions of TfR9, TfR12, and TfR15 were selected for humanization. These antibodies were selected for cynomolgus monkey cross-reactivity, but also for their function (internalization), functional stability (ability to internalize after heat stress), and sequence diversity.

[0414] Humanization is carried out using the CDR transplantation method. One of the most commonly used methods for humanizing non-human antibodies is to transplant the CDR from non-human antibodies onto the human antibody receptor framework. Typically, such CDR transplants can cause its affinity to weaken or completely lose due to the disturbance of the humanized antibody framework. As a result, it may be necessary to retain certain residues in the mouse framework to replace the human residues (back mutation) at the corresponding position to restore the weakened or lost affinity. Therefore, it is crucial to accurately predict the residues retained in the selected human antibody germline receptor framework, and the framework can retain the function and paratope of the humanized antibody. In addition, for good manufacturability and downstream development, it is necessary to retain or improve thermal stability and solubility.

[0415] Structure-based antibody modeling was applied in the method of humanizing anti-TfR mouse monoclonal antibodies (mAbs) using the BioMOE module of MOE (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada). In brief, the VH and VL sequences of the mouse mAb sequences to be humanized were compared with the functional germline sequences of people VL, VH, LJ and HJ taken from IMGT (http: / / www.imgt.org / ). Pseudogenes and ORFs were excluded. For each mouse mAb (query), five most similar VL and five most similar VH germline sequences were selected, and combined with the most similar VJ and HJ genes to produce 25 humanized sequences. The CDRs to be transplanted to the human framework are defined according to AbM definitions (http: / / www.bioinf.org.uk / abs / #cdrdef).

[0416] Two humanized sequences are selected according to the frequency of VH framework and VL framework in the human library. The query and humanized sequences are used to create Fv homology models. The Fv homology model is created using the BioMOE module or Antibody Modeler module of MOE (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada). Through the complete antibody homology modeling process, AMBER10: EHT force field is used for energy minimization. Based on the Fv homology model, the scoring metric provided by MOE is used to calculate, analyze and sort molecular descriptors, such as the interaction energy between VL and VH, isoelectric point (3D pI) based on coordinates, hydrophobic patches and charged surface areas. These molecular descriptors are used to prioritize humanized mAbs for downstream experimental procedures, including protein expression, purification, binding affinity testing and functional assays.

[0417] The BioMOE module of MOE provides a tool, i.e., mutation site characterization, to visualize and classify potential residues for back mutation. Back mutation is defined as amino acid substitutions that return to the original query sequence to replace the humanized sequence. Using this tool, the original query (reference) is individually compared with the humanized variants selected for the primary amino acid sequence and the 3D structure of the 3D Fv homology model.

[0418] Changes between the reference and humanized variants were categorized based on differences in amino acid type, interaction potential with CDR residues, impact potential of VL / VH pairing, and potential changes in hydrophobic and charged surface areas in and near the CDRs.

[0419] Mutations near the CDRs or VL / VH interface, with significant charge differences or containing strong H-bond interactions are evaluated individually, and significantly disruptive mutations are reverted to the original query residues. As a result, the humanized sequence may contain up to five back mutations.

[0420] The humanized sequences of TfR9, TfR12 and TfR15 are described in Table 17 below.

[0421] Table 17: Heavy and light chain sequences in humanized variant scFvs of TfR9, TfR12 and TfR15 are underlined.

[0422]

[0423]

[0424]

[0425]

[0426]

[0427] Table 18 - Variable heavy chain domains and variable light chain domains of humanized variants of TfR9, TfR12 and TfR15

[0428]

[0429]

[0430]

[0431]

[0432]

[0433] Example 13: Effects of 2+1 anti-TfR bispecific antibodies on TfR recycling and degradation in hCEMC / D3 cells

[0434] An ideal BBB transport molecule would be efficiently transported into the brain without affecting the expression level or localization of the target receptor (in this case TfR).To determine the effect of the 2+1 anti-TfR bispecific antibody on the recycling of transferrin receptor, both FACS and Western blotting were employed.

[0435] FACS method: HCMEC / D3 cells were seeded in 100 μL culture medium (EBM2, Lonza) in a 24-well plate at 1.5*10^5 cells / well. The cells were then treated with 20 mg / ml of 2+1 anti-TfR bispecific antibody for 24 hours and then processed by the FACSCanto system (BD Biosciences). The human IgG1 isotype was included in the assay as a negative control. Since the 2+1 anti-TfR bispecific antibody is specific for the TfR apical domain, the cells were stained with b3 / 25Ab that does not bind to this domain (Luria-Pérez R., et al.; Bol Med Hosp Infant Mex. 2016; 73(6): 372-379), thereby avoiding antibody competition for receptor binding. To define the effect of 2+1 anti-TfR bispecific antibodies on TfR recycling, the mean fluorescence intensity (MFI) of cells treated with each individual 2+1 anti-TfR bispecific antibody was normalized to the mean fluorescence intensity obtained for the human IgG isotype. Treatment of HCMEC / D3 cells with 2+1 anti-TfR bispecific antibodies resulted in a 40-80% reduction in cell surface TfR expression ( Figure 2 ).

[0436] Two of these clones, TfR9 and TfR12, were selected for further engineering and a panel of humanized antibodies was generated (Examples 8 and 12). Interestingly, some antibody clones derived from TfR9 (i.e., TfR9.1B and TfR9.5B) showed no adverse effects on receptor recycling. In contrast, all clones generated from TfR12 severely impaired receptor recycling ( Figure 3 Using the same strategy, a panel of humanized antibodies was generated based on TfR15 (Examples 8 and 12), and another antibody clone, TfR15.WH8, was identified that had no adverse effects on TfR recycling (data not shown).

[0437] 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 blotting.

[0438] Western blotting: HCMEC / D3 cells were seeded in 1 mL of culture medium (EBM2, Lonza) in a 12-well plate at 3*10^5 cells per well. The cells were then exposed to 20 mg / ml of 2+1 anti-TfR bispecific antibody for 24 hours and then processed by Western blotting (WB). As a control, other wells were treated with human IgG1 isotype control (20 mg / ml) or PBS. After washing twice with ice-cold PBS, cells were collected in 1.5 ml microcentrifuge tubes using a scraper. After centrifugation (2000g, 5 min), the supernatant was removed from the tube and lysed in a mixture containing protease inhibitors (cOmplete TM, mini protease inhibitor cocktail, Roche, #11836153001) in RIPA buffer (R0278, Sigma-Aldrich). The cells were then incubated on a nutator at 4°C and then centrifuged at maximum speed in a tabletop centrifuge for 20 minutes at 4°C. Next, the supernatant was transferred to a new tube, mixed with LDS sample buffer (Invitrogen, #NP0007), and then incubated on a heating block (95°C) for 5 minutes. Subsequently, the samples were subjected to protein gel electrophoresis and the proteins were then transferred to a nitrocellulose membrane. The membrane was 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, #MAB2474) at 4°C for 24 hours. Thereafter, the membrane was washed 4 times with PBST and then incubated with a 1:3000 dilution of HRP-conjugated goat anti-mouse IgG1 antibody (Jackson Immunoresearch, #115-035-003) at room temperature for 1 hour. After washing 4 times with PBST, the membrane was developed using Supersignal West Pico reagent (ThermoFisher, #34580) and imaged and analyzed using an iBright instrument. Next, the membrane was incubated with stripping buffer (ThermoFisher, #21059) to remove bound antibodies and then probed with anti-human b-actin (Abcam, #ab8227) for quantification purposes. The TfR band density of each antibody was normalized to its corresponding b-actin band density. These values ​​were then normalized to the values ​​obtained from cells treated with the human IgG1 isotype.

[0439] Similar to the FACS data, treatment of HCMEC / D3 cells with 2+1 anti-TfR bispecific antibodies had little or no effect on the total amount of TfR protein, especially TfR9.1B had no effect on total protein levels. In contrast, total TfR protein in HCMEC / D3 was greatly reduced after exposure to the humanized antibody generated from TfR12 ( Figure 4 ).

[0440] Example 14: Engineering of humanized variants of TfR9.1B

[0441] Humanized TfR9.1B was selected for further affinity optimization. The anti-TfR scFv portion of TR9.1B was cloned into pComb3X phagemid vector (Addgene, Watertown MA), and overlap extension polymerase chain reaction (PCR) mutagenesis was used to generate randomized VH and VL libraries, which consisted of a theoretical library size of 9E+07 different sequences. They were packaged into phages, each of which carried and expressed a unique VH / VL combination. Soluble biotinylated TfR Avi-his protein was used to carry out three rounds of panning of phage libraries with gradually increasing stringency. After the third round, a single colony was picked, and the scFv in the periplasmic extract (PPE) was screened for binding by ELISA. After initial ELISA screening, 46 of the 9-1B scFv variants were selected for cloning into a 2+1 'hole' vector between the BamHI and NotI sites (described in Example 9 above), which is 3' to the 3x (G4S) linker connecting the Fc portion to the scFv portion. 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 listed in Table 19 below.

[0442] Table 19: Affinity optimized TfR9.1B variant sequences Heavy and light chain sequences are underlined.

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452] Table 20 - Variable heavy and light chain domains of affinity optimized TfR9.1B variants

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460] Clone TfR9.1B.39 was selected as a template for another round of random mutagenesis to remove two potential isomerization sites (D34 in VL and D62 in VH). Biotinylated human and cynomolgus monkey TfR Avi-His proteins were used as bait for panning and ELISA screening, and seven mutants with two potential fixed isomerization sites were selected and cloned into the same vector in a 2+1 bispecific format and used for further characterization (see Table 21).

[0461] Table 21: TfR9.1B.39 variant sequences (VH and VL sequences are underlined)

[0462]

[0463]

[0464] Table 22 - Variable heavy chain domain and variable light chain domain of TfR9.1B.39 variants

[0465]

[0466]

[0467] Table 23: CDR sequences of affinity-optimized TfR9.1B.39 variants (Kabat definition)

[0468]

[0469]

[0470] Example 15: Engineering of humanized TfR15 antibody

[0471] Humanized TfR15 is engineered to optimize affinity to target and remove potential oxidation sites in VH (Trp 38). DNA fragments of WH7, WH8 and WH12 (wherein Trp 38 is replaced by Tyr) were ordered from GeneArt and cloned into the same vector as above. Proteins were expressed and purified from the Expi293 expression system and further characterized. The sequences of these engineered variants are provided in Table 24 below. TfR15.WH8.1.1 is optimized (via alanine scanning of CDR) to produce variants with a range of binding affinities (see Examples 16 and 17 below).

[0472] Table 24: TfR15 variants with removed authenticity sites or reduced affinity

[0473]

[0474]

[0475] Example 16: Engineering of humanized TfR.15.WH8.1 variants

[0476] Humanized TfR.15.WH8.1 variants are engineered to optimize target affinity. The first step is to carry out alanine scanning to CDR, and then carry out amino acid substitution at the selected position in CDR. In order to carry out alanine scanning to CDR, DNA fragments containing variants are ordered from Integrated DNA Technologies (IDT), and cloned into expression vectors. Proteins are expressed and purified from Expi293 cells, and further characterized. According to the alanine scanning results (data not shown), key amino acid positions are selected to carry out further following 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). There are 3 additional variants, wherein in addition to amino acid substitutions, VL and VH orientations are also exchanged or joints are changed. For WH8.1.42A.LH variants, VH and VL orientations are exchanged to VL-joint-VH. For the WH8.1.42A.3x(G4S) variant, the linker was changed to (GGGGS)x3 (SEQ ID NO: 184). For 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 the form of 2+2 antibodies. As described above, 2+2 antibodies were expressed and purified from the Expi293 expression system and further characterized. The sequences are shown in Tables 25-27 below.

[0477] Table 25: Affinity optimized TfR.15.WH8.1 variant sequences Heavy and light chain sequences are underlined.

[0478]

[0479]

[0480]

[0481] Table 26 - Variable heavy and light chain domains of affinity optimized TfR.15.WH8.1 variants

[0482]

[0483]

[0484]

[0485]

[0486] Table 27: CDR sequences of affinity-optimized TfR.15.WH8.1 variants (Kabat definition)

[0487]

[0488]

[0489]

[0490] Example 17: Binding kinetics of TfR.15.WH8.1 variants

[0491] The binding kinetics of 2+2TfR.15.WH8.1 variants (isotype IgG with 2 anti-TfR scFv) to the Avi-His-tagged human and cynomolgus monkey TfR apical domains were evaluated using a Carterra LSA instrument (Carterra, Salt Lake City, UT). Capture pads were prepared on HC30M sensor chips (Carterra) using goat anti-human Fcγ polyclonal antibodies (Jackson ImmunoResearch) according to the instrument manufacturer's instructions. Two independent experiments were performed as follows, so that the N of each antibody was between one and two determinations. Points that produced less than 20RU of analyte binding were excluded from further analysis.

[0492] 2+2 anti-TfR bispecific antibodies were prepared by diluting with running buffer (HBS-EP+; Teknova) and adding 0.5mg / mL BSA (MP Biomedicals) to obtain a final concentration of 15μg / mL and captured on a goat anti-human Fcγ capture surface in array format. The ability of the captured antibodies to bind to recombinant human and cynomolgus monkey heterologs of Avi-His at the top of TfR was tested. An estimate of affinity was generated by injecting each analyte across the antibody array using a single-channel flow cell. The TfR top Avi-His analyte was diluted with running buffer, starting with 2500nM human and cynomolgus monkey TfR top Avi-His, eight three-fold serial dilutions were performed, and then two 10-fold dilutions were performed. The analyte was injected for 5 minutes, followed by 15 minutes of dissociation. After each analyte injection series, the antibody was regenerated with 10mM glycine pH 2.0 buffer (Carterra). NextGenKIT high-throughput kinetic analysis software (Carterra) was used to process and analyze the data.

[0493] The equilibrium dissociation constant (K) was calculated from the fitted association and dissociation rate constants (k-on and k-off) of the 2+2 anti-TfR bispecific antibodies of the present disclosure binding to the apical Avi-His of human and cynomolgus monkey TfR. DFor measurements with N>1, the mean and standard deviation were calculated, and the measurements of all samples were combined and plotted using GraphPad Prism. D The values ​​are summarized below in Table 28. As shown in the table, some antibodies displayed heterogeneous binding profiles that did not fit to a 1 :1 binding model, so their rate constants could not be determined in this series of experiments.

[0494] Table 28: Equilibrium dissociation constants (KD) of 2+2 anti-TfR bispecific antibodies

[0495]

[0496] N = number of assays; NT = not tested; NA = not applicable; LB = low binding (below the limit of measurement); NB = no binding detected; HET = heterogeneous binding.

[0497] These results illustrate that the 2+2 anti-TfR bispecific antibody exhibits an affinity range of about 3nM to >2.5μM for TfR apical domain binding. Specifically, the 2+2 anti-TfR bispecific antibody binds to human TfR Avi-His with an affinity range of 6nM to >2.5μM; and the anti-TfR antibodies of the present disclosure bind to cynomolgus monkey TfR Avi-His with an affinity range of 3nM to >2.5μM.

[0498] Example 18: Binding kinetics of 2+1 anti-TfR bispecific antibodies

[0499] The binding kinetics of humanized 2+1 anti-TfR bispecific antibodies to human and cynomolgus monkey TfR Avi-His were evaluated using a Carterra LSA instrument (Carterra, Salt Lake City, UT). Briefly, 2+1 anti-TfR bispecific antibodies were prepared in duplicate by 20 to 180-fold dilution into 10mM acetate pH 4.5 (Carterra) to obtain a final concentration of 30 μg / mL. A single-channel flow cell was used to activate the HC30M sensor chip (Carterra) with a 7-minute injection of a 1:1:1 mixture of 100mM MES pH 5.5, 100mM sulfo-NHS, 400mM EDC (all reconstituted in MES pH 5.5; 100 μl of each substance was mixed in a vial just before the assay was run). After switching to a multichannel array flow cell, the antibodies were injected onto the activation chip in four 96-point arrays for 15 minutes. The remaining unconjugated active groups on the chip were then blocked by injecting 1 M ethanolamine pH 8.5 (Carterra) for 7 minutes 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, such that N for each antibody was between one and eight determinations. Spots that produced less than 20 RU of analyte binding were excluded from further analysis.

[0500] The running buffer HBS-EP containing 0.5 mg / ml BSA (MP Biomedicals) was used. + After initiation with Teknova, the immobilized anti-TfR 2+1 antibody was tested for its ability to bind to two forms of TfR Avi-His, which are human and cynomolgus monkey heterologs as described above. Affinity estimates were generated by injecting each analyte across the antibody array using a single-channel flow cell. The TfR Avi-His analyte was diluted in running buffer and eight three-fold serial dilutions were performed starting with 1 μM human and cynomolgus monkey TfRAvi-His. The analytes were injected for 5 minutes, followed by a 10-minute dissociation. After each injection of the analyte, the analyte was diluted with Pierce TM IgG elution buffer (ThermoScientific) was used to regenerate the antibodies. NextGenKIT high-throughput kinetic analysis software (Carterra) was used to process and analyze the data.

[0501] The equilibrium dissociation constant (K) was calculated from the fitted association and dissociation rate constants (k-on and k-off) of the anti-TfR Avi-His antibodies of the present disclosure. D ). The values ​​were combined, the mean and standard deviation were calculated, and graphs were drawn using GraphPad Prism. DThe values ​​are summarized below in Table 29. As shown in the table, some antibodies displayed heterogeneous binding profiles that did not fit to a 1 :1 binding model, therefore their rate constants could not be determined.

[0502] Table 29. Equilibrium dissociation constants (K) of anti-TfR 2+1 bispecific antibodies D )

[0503]

[0504]

[0505] N = number of assays; NT = not tested; NA = not applicable; LB = low binding (below the limit of measurement); NB = no binding detected

[0506] These results illustrate that the 2+1 anti-TfR bispecific antibody exhibits an affinity range of about 6nM to 900nM for TfR Avi-His. Specifically, the 2+1 anti-TfR bispecific antibody of the present disclosure binds to huTfR Avi-His with an affinity ranging from 6.7nM to 340nM. The anti-TfR antibody of the present disclosure binds to cynomolgus monkey TfR Avi-His with an affinity ranging from 18nM to 870nM.

[0507] Example 19: PK of 2+1 anti-TfR bispecific antibody in huTfR ECD KI mice

[0508] 2+1 anti-TfR bispecific antibody production: The 2+1 anti-TfR bispecific antibody for in vivo PK studies was expressed and purified on a 2+1 backbone with isotype control (non-binding) Fab arms.

[0509] The antibody for in vivo PK study was generated by transient transfection of ExpiCHO cells (Invitrogen) according to the manufacturer's instructions. For 400 mL culture, 320 μg total DNA consisting of 3 expression plasmids (heavy chain-hole-scFv, heavy chain-knob and light chain) was used. The molar ratio of 3 plasmids was optimized to obtain a highly purified 2+1 anti-Tfr bispecific antibody. Cell culture supernatant was harvested 10 days after transfection. Antibodies were purified using protein A affinity chromatography, and then ion exchange chromatography was performed on AKTA Avant 25 (Cytiva) to remove product-related impurities. Purified antibodies were dialyzed into PBS. Characterization was analyzed by absorbance at 280 nm, CE-SDS, size exclusion chromatography and endotoxin measurement.

[0510] Study design: To establish an in vivo proof-of-concept for brain penetration of 2+1 anti-TfR bispecific antibodies, a hybrid knock-in (KI) mouse was generated in which the extracellular domain region of mouse TfR was replaced by the extracellular domain region of human TfR in one of the two alleles. This is because the 2+1 anti-Tfr bispecific antibody specifically binds to primates but does not bind to mouse TfR. It is worth noting that the total amount of TfR protein in the resulting TfRmu / hu KI mice was no different from the total amount observed in wild-type (WT) animals (data not shown). A two-dose strategy was used to determine the brain uptake and peripheral clearance of the injected Ab over time. A group of WT and hu-TfRmu / hu mice were given 10 mg / kg isotype IgG or 2+1 anti-TfR bispecific antibodies on days 1 and 14. Blood samples were collected from antibody-treated animals at different time points in serum separation tubes and then allowed to coagulate at room temperature (RT) before centrifugation. The resulting supernatant (representing serum) was then transferred to a fresh tube and stored at -80°C until analysis.

[0511] Sample preparation: Brain tissue was collected from antibody-treated mice on day 15 (i.e., 24 hours after injection of secondary antibody). Prior to this, mice were anesthetized and perfused by heart with 15 ml PBS to clear blood vessels. The brain tissue was then minced and homogenized using a manual grinder in 1 ml HBSS buffer (MilliporeSigma #55037C) containing 10 mM HEPES (#15630130, Gibco). Subsequently, the sample was centrifuged at 1000 g for 5 minutes to precipitate the vascular portion. The supernatant representing the substance was then transferred to a new tube and mixed with a mixture of protease and phosphatase inhibitors (cOmplete TM , mini protease inhibitor cocktail, Roche, #11836153001) in 10x RIPA buffer (final concentration 1x). After incubation on a nutator at 4°C for 20 minutes, the samples were frozen on dry ice and stored at -80°C until analysis. The vascular fraction was cleared from the myelin debris by centrifugation through % ​​18 dextran (70kDa, #31390, Sigma-Aldrich) followed by 2 washes in HBSS buffer. Thereafter, the samples were lysed in RIPA buffer (R0278, Sigma-Aldrich) and stored at -80°C until analysis. The effectiveness of this method was confirmed using WB analysis, where markers of brain endothelial cells (e.g., CD31 and tight junction protein-5) were absent in the parenchymal fraction (data not shown).

[0512] Pharmacokinetic (PK) analysis: The antibody concentration in serum and brain samples was measured using the MSD (Meso Scale Discovery) method. Briefly, 50 μl / well of goat anti-human IgG1 Ab (Jackson ImmunoResearch, #109-005-097) was added to the MSD plate with a PBS solution of 1 μg / ml and incubated overnight at 4 ° C. The plate was then washed 3 times with a wash buffer (0.05% Tween-20 in 1xPBS) and then incubated with a blocking buffer (wash buffer containing 3% BSA) at room temperature for 1 hour. Subsequently, serum (1: 10000 dilution in PBS) and brain (1: 2 dilution in PBS) samples were added to the plate in duplicate and incubated on a shaker at 500RPM for 1 hour. Human IgG1 isotype Ab of known concentration was included in the assay to create a standard curve. Next, the plate was washed 3 times with wash buffer, and then 30 μl / well of sulfo-Tag goat anti-human antibody (R32AJ-5, Meso Scale Discovery) was added in PBS solution at 0.2 μg / ml and incubated on a shaker at 500 RPM for 1 hour. Finally, after washing 3 times with wash buffer, 150 μl / well of 1x READ buffer (R92PC-2, Meso Scale Discovery) was added to the plate, and then read using a Sector Imager S600 instrument.

[0513] By measuring antibody concentrations in vascular-depleted brains, the levels of 2+1 anti-TfR bispecific antibodies in hu-TfRmu / hu mice were 4-5 times higher than isotype IgG ( Figure 5 ). In contrast, brain uptake of the 2+1 anti-TfR bispecific antibody in WT animals that do not express the target receptor was similar to that observed for isotype IgG. Taken together, these results suggest that the 2+1 anti-TfR bispecific antibody has enhanced transferrin receptor-mediated entry into the brain. 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 isotype antibodies ( Figure 6 ). In contrast, the two 2+1 anti-TfR bispecific antibodies showed a significant difference in hu-TfR expression compared to isotype IgG. mu / hu Mice showed higher plasma clearance, which is consistent with TfR-mediated peripheral disposal.

[0514] Example 20: Effect of 2+1 anti-TfR bispecific antibody on TfR expression on brain endothelial cells in vivo

[0515] To determine whether the 2+1 anti-Tfr bispecific antibody would abnormally regulate receptor recycling in target cells in vivo, the total amount of TfR protein in the vascular fraction of brain tissue collected from antibody-treated animals was quantified. To this end, the 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 the vascular fraction of brain tissue collected from antibody-treated animals was quantified. + / - No significant differences in TfR band density were observed between samples obtained from KI mice compared with those from isotype-treated animals ( Figure 7 ). It is noteworthy that in this assay, the membrane was probed with an anti-human TfR antibody (R&D Systems Inc, #MAB2474) that does not have cross-reactivity with mouse TfR. As a result, no TfR band was detected in blood vessels obtained from WT animals. In summary, these observations suggest that the 2+1 anti-TfR bispecific antibody does not alter TfR recycling at the blood-brain barrier (BBB) ​​in vivo.

[0516] Example 21: In vivo reticulocyte depletion after 2+1 anti-TfR bispecific antibody treatment in mice

[0517] It is known that anti-TfR antibodies can induce cell death of reticulocytes in vivo. To test this, a panel of WT and hu-TfR + / -KI mice were injected with 2+1 anti-TfR bispecific antibodies (TfR-9.1B.39 and TfR-15.WH8), human IgG1 isotype, or the previously published anti-ms TfR antibody 8D3 (Boado et al., Biotechnol Bioeng. 2009 Mar 1; 102(4): 1251–1258), which is known to drive strong depletion of mouse reticulocytes. The latter two antibodies were included in the experiment as negative and positive controls, respectively. All tested antibodies were on the WT human IgG1 backbone, which can drive ADCC, ADCP, and CDC reactions 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 the cells with anti-CD71 (Invitrogen, #Catalog No. 17-0719-42), thiazole orange (MilliporeSigma, #390062), and anti-mouse CD45 to exclude circulating leukocytes (Invitrogen, #12-0451-83). Thiazole orange binds to residual ribosomal RNA molecules that are present in reticulocytes but not in mature RBCs. Reticulocytes express high levels of transferrin receptor (CD71) compared to RBCs. As expected, injection of 8D3 antibody resulted in a complete loss of reticulocytes in WT animals (data not shown). In contrast, this cell population was unchanged in isotype-treated animals, regardless of genotype. Similarly, no changes in the number of reticulocytes were observed in WT mice receiving anti-TfR 2+1 bispecific antibodies, which makes sense because 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 a decrease in reticulocyte numbers of approximately 80% to 90% ( Figure 8 ). Based on published results (Yamamoto et al., Molecular Genetics and Metabolism Reports, Vol. 27, June 2021, 100758), the reticulocyte depletion seen here may be mediated by the induction of ADCC (antibody-dependent cellular cytotoxicity) response, which is further evaluated in subsequent examples.

[0518] Example 22: Evaluation of in vitro effector function (CDC and ADCC)

[0519] To investigate the mechanism of reticulocyte depletion seen in 2+1 anti-TfR bispecific antibodies, the ability of these same antibodies to elicit in vitro effector responses, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), was evaluated. In addition, given that anti-TfR antibodies on the wt huIgG1 backbone have been shown to elicit ADCC responses, alternative Fc formats such as N325S / L328F (NSLF) and L234A / L235A / P331S (LALAPS) were tested, which strongly reduce binding to activating FcgRs (particularly FcgRIIIa, a key driver of ADCC).

[0520] CDC Methods and Results: The ability of the 2+1 anti-TfR bispecific antibody to drive complement deposition was measured on the hCMEC / D3 cell line, which expresses high levels of both receptors. Target cells were detached, washed 1x in PBS, and diluted to 2x10 in RPMI1640 medium. 6 Cells / mL. In a round-bottom 96-well plate (Falcon#351177), 50uL target cells (1x105 cells per well) were aliquoted per well. 25uL 4x antibodies diluted in the same culture medium were added to these cells. The cells were incubated with antibodies at 37°C for 15 minutes, and then 25uL of pooled complement human serum (Innovative Research, IPLA-CSER) was added to each well as a complement source, and the plate was incubated for another 2 hours at 37°C. Thereafter, the cells were washed twice with FACS buffer (PBS+2% FBS+1mM EDTA) and resuspended in 50μL FACS buffer+0.25μL / well of propidium iodide (Fischer Scientific, BD 556463), and then analyzed on an iQue flow cytometer (IntelliCyt). CDC was analyzed as the ratio of PI+ single cells in each well. In this assay, neither 2+1 anti-TfR bispecific antibody induced a significant CDC response (data not shown).

[0521] ADCC Methods and Results: The ability of 2+1 bispecific antibodies to induce antibody-dependent cellular cytotoxicity (ADCC) was evaluated using Promega's ADCC Reporter Bioassay System (#G7010). This system relies on an engineered Jurkat T cell line that stably expresses the FcgRIIIa receptor (V158 variant) and a NFAT response element driving the expression of firefly luciferase. Target cells were plated at 1.2x10 6Target cells were diluted in assay buffer (RPMI + 4% low IgG serum) at a concentration of 10 / mL and 25 μL of cells (30,000 per well) were aliquoted into appropriate wells of a 96-well white assay plate. To the wells containing cells, 25 μL of 3x antibody, also diluted in assay buffer, was added. After the antibody was added to the target cells, the effector cells (at 2x10 7 Thaw 630uL of 75,000 cells / mL frozen) at 37°C and add 630uL to 3.6mL warm (37°C) assay buffer, mix gently, and immediately add 25μL of effector cells (75,000 per well, E:T ratio of 2.5) to the wells containing target cells and antibody. The plates were then incubated at 37°C and 5% CO2 for 6 hours to allow receptor cell activation and luciferase expression. After 6 hours, the plates were equilibrated to room temperature (15 minutes) while the Bio-Glo luciferase assay reagent was prepared. 75μL of luciferase assay reagent was added to each well, the plates were incubated for 10 minutes, and luminescence was measured on a BioTek plate reader. In wild-type human IgG1 format, some 2+1 bispecific antibodies against TfR were able to drive significant ADCC signals ( Fig. 9 Further experiments were performed to test whether these responses could be improved by abrogating binding to various FcRs.

[0522] Fc Engineering and ADCC Results: Two 2+1 anti-TfR bispecific antibodies (TfR9.1B.39 and TfR15.WH8) that elicited ADCC signals were re-expressed in a variety of Fc formats (including wild-type hIgG1, hIgG1 NSLF, and hIgG1 LALAPS) and retested in the same ADCC assay format described previously. Both NSLF and LALAPS have been previously described to strongly reduce binding to FcgRIIIA (Leoh et al., Molecular Immunology, July 29, 2015, 67(2Pt B):407-415, Shang et al., J Biol Chem. 2014; 289:15309–15318), which is the main driving force for ADCC activity. In this assay, both NSLF and LALAPS were able to significantly reduce ADCC signals, with NSLF showing a >90% signal reduction at the highest concentration tested for the tested 2+1 anti-Tfr bispecific antibodies ( Fig.10 ).

[0523] Example 23: Binding kinetics of 2+1 anti-TfR bispecific antibody affinity and reliability optimized clones

[0524] The binding kinetics of the 2+1 anti-TfR bispecific antibody to human TfR Avi-His were evaluated using a GatorBio BLI instrument (GatorBio, Palo Alto, CA). Briefly, the 2+1 anti-TfR bispecific antibody was diluted in running buffer (HBS-EP+, Teknova) containing 0.5 mg / ml BSA (MP Biomedicals) for a series of seven-fold, five-fold, three-fold serial dilutions, and then two 10-fold dilutions, starting from 4 μM concentrations of Tf9.1B.39.36, TfR9.1B.39.37, TfR9.1B.39.38 and WH8 parents and starting from 2 μM concentrations of 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. The streptavidin probe (Gator) is immersed in the biotinylated TfR Avi-His solution for five seconds to load the protein onto the tip. The probe is then immersed in the corresponding antibody concentration for three minutes of association, and then dissociated in the running buffer for three minutes. The washing step in the running buffer is carried out between the loading step and the association step. The probe with biotinylated TfR Avi-His is regenerated with 10mM glycine-HCl pH 2.5 buffer. Biotinylated TfRAvi-His is supplemented for five seconds at the beginning of each binding cycle. The data are double referenced with reference probe and buffer (blank) hole, and processed and analyzed using Gator analysis software (GatorBio).

[0525] The equilibrium dissociation constant (KD) was calculated from the fitted association rate constant and dissociation rate constant (k-on and k-off) of the 2+1 anti-Tfr bispecific antibody. The values ​​were combined, the mean and standard deviation were calculated, and the graph was plotted using GraphPad Prism. As shown in the table, some antibodies exhibited heterogeneous binding profiles that did not fit to a 1:1 binding model, so their rate constants could not be determined. K D The values ​​are summarized in Table 30 below.

[0526] Table 30. Equilibrium dissociation constants (K) of anti-TfR 2+1 antibodies D )

[0527]

[0528] N = number of assays; NT = not tested; NA = not applicable; LB = low binding (below the limit of measurement); NB = no binding detected.

[0529] These results illustrate that the 2+1 anti-TfR bispecific antibody exhibits an affinity range of about 30 nM to 4 μM for TfR Avi-His. The 2+1 anti-TfR bispecific antibody binds to human TfR Avi-His with an affinity range of 65 nM to 3.5 μM; the 2+1 anti-TfR bispecific antibody binds to cynomolgus monkey TfR Avi-His with an affinity range of 38 nM to 2.3 μM.

[0530] Example 24: TD1-TfR bispecific antibody in vitro sTREM2 assay

[0531] To evaluate the pharmacokinetics and pharmacodynamics of 2+1 anti-TfR antibodies, 2+1 anti-TfR bispecific antibodies were generated using the following substances: (i) TfR9.1B.39.38scFv; and (ii) a human MS4A4A (referred to as "TD1") specific monoclonal antibody (referred to as "TD1-TfR") in a 2+1 (hole) format (described in Example 25). To confirm that TD1-TfR retains the function of the parent TD1 antibody, a soluble TREM2 (sTREM2) assay was performed using human monocyte-derived macrophages. These cells are expected to show an increase in sTREM2 production after antibody treatment with TD1.

[0532] Human monocytes were isolated from whole blood using RosetteSep human monocyte enrichment mixture (Stemcell technologies) and Ficoll centrifugation according to the manufacturer's protocol. After lysing red blood cells with ACK lysis buffer, monocytes were resuspended in complete medium (RPMI, 10% FBS, Pen / Strep, 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% volume / volume human serum were added to the cells for 5-7 days.

[0533] Each well was stimulated with 10 μg / mL of test antibody (huIgG1, TD1, iso-TfR (isotype control IgG antibody linked to TfR9.1B.39.38) or TD1-TfR) 5 Macrophages were cultured for 48 hours. Duplicate wells were treated as technical replicates. sTREM2 in the supernatant was tested by the MSD platform.

[0534] like Fig.11As shown, TD1-TfR showed activity in in vitro assays by increasing sTREM2 levels compared to hIgG1 and Iso-TfR. TD-1 and TD1-TfR showed similar increases in sTREM2 levels in in vitro assays. This data suggests that adding anti-TfR scFv to the TD1 antibody does not impair the function of TD1.

[0535] Example 25: TD1-TfR bispecific antibodies and methods for intravenous administration to non-human primates

[0536] To evaluate the pharmacokinetics and pharmacodynamics of the 2+1 anti-TfR antibodies disclosed herein, 2+1 anti-TfR bispecific antibodies were generated using the following substances: (i) TfR9.1B.39.38scFV; and (ii) a human MS4A4A (TD1) specific antibody in a 2+1 (hole) format (referred to as "TD1-TfR"). 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:

[0537] TD1 heavy chain hIgG1 NSLF(N325S, L328F) knob(T366W)

[0538] QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGVPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT

[0539] HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE

[0540] DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

[0541] WLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDE

[0542] LTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

[0543] GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0544] GK (SEQ ID NO:405).

[0545] TD1 heavy chain hIgG1 NSLF(N325S, L328F) hole scFv(T366S, L368A, Y407V)-(G4S)3 linker-VH-20AA linker-VL with mutations (H435R, Y436F)

[0546] QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGVPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTEYAMHWVRQAPGQGLEWMGGIAPNTGGTSYNQKFKGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCASQGWLLRGGDYWGQGTLVTVSSGGSEGKSSGSGSESKSTGGSDVVMTQSPLSLPVTLGQPASISCKASQSLLDEGGKTYLNWLQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPQTFGGGTKVEIK(SEQ ID NO:406).

[0547] TD1 light chain

[0548] DVVMTQSPLSLPVTLGQPASISCKSSRSLLYSAGKTYLSWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGIDFHQTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:407).

[0549] In addition, for control purposes, a bispecific antibody (referred to as "Iso-TfR") was generated with (i) TfR-specific scFv and (ii) IgG1 NSLF in a 2+1 (hole) format. The complete amino acid sequence of Iso-TfR is as follows:

[0550] Isotype Control Heavy Chain hIgG1 NSLF(N325S, L328F) Knob(T366W)

[0551] EVRLLESGGGLVQPGGSLRLSCAASGFTFSNYAMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTTSRDDSKNALYLQMNSLRAEDTAVYYCARGGPGWYAADVWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:408).

[0552] Isotype control heavy chain hIgG1 NSLF (N325S, L328F) -scFv (T366S, L368A, Y407V)-(G4S)3 linker-VH-20AA linker-VL, with mutations (H435R, Y436F)

[0553] EVRLLESGGGLVQPGGSLRLSCAASGFTFSNYAMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTTSRDDSKNALYLQMNSLRAEDTAVYYCARGGPGWYAADVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTEYAMHWVRQAPGQGLEWMGGIAPNTGGTSYNQKFKGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCASQGWLLRGGDYWGQGTLVTVSSGGSEGKSSGSGSESKSTGGSDVVMTQSPLSLPVTLGQPASISCKASQSLLDEGGKTYLNWLQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPQTFGGGTKVEIK (SEQ ID NO:409).

[0554] Isotype control light chain

[0555] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQADLPAFAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:410).

[0556] The complete amino acid sequence of the antibody specific for the individual human MSA4A (TD1) is as follows:

[0557] TD1 heavy chain hIgG1 NSLF (N325S, L328F)

[0558] QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGVPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:411).

[0559] TD1 light chain

[0560] DVVMTQSPLSLPVTLGQPASISCKSSRSLLYSAGKTYLSWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGIDFHQTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:407).

[0561] A PK / PD study was performed in naive male cynomolgus monkeys (n=12) after intravenous administration (2 administrations total) of 20 mg / kg (n=3) of each of the 4 antibodies in Table 31 on days 1 and 29. Due to the poor brain penetration of antibodies, TD1 at this dose level has been shown to produce an incomplete PD response in NHPs. The goal of the study was to look for increased antibody levels in the brain, as well as a concurrent increase in relevant PD biomarkers.

[0562] Table 31. In vivo administration of anti-TfR bispecific antibodies

[0563] Group Number of animals (males only) Test Items Route of administration 1 3 Iso-TfR IV 2 3 TD1-TfR IV 3 3 TD1 IV 4 3 huIgG1 (isotype control) IV

[0564] The intravenous dose was administered as a slow bolus via the saphenous vein. The target dose level (mg / kg), target dose concentration (mg / mL), and target dose volume (mL / kg) were consistent between Groups 1-4.

[0565] Example 26: Hematological parameters after intravenous administration of TD1-TfR bispecific antibodies to non-human primates

[0566] Blood samples (~2 mL) for hematology were collected from the femoral vein into tubes containing potassium EDTA prior to dosing and on days 3, 8, 15, 29, and 31 post-dose.

[0567] Table 32. Hematological parameters tested after intravenous administration of anti-TfR bispecific antibodies

[0568]

[0569] The normal range of the reticulocyte count at the testing facility is 25.7-122.3 10 3 / μl.

[0570] Fig.12The absolute reticulocyte counts over the duration of the study are illustrated. The normal range is represented by the dotted line. In summary, no significant changes in any hematological parameters were observed during the study, and no sustained decrease in reticulocytes was observed.

[0571] Example 27: CSF and serum levels of TD1-TfR bispecific antibodies after IV administration in non-human primates

[0572] Cerebrospinal fluid (CSF) and serum were collected from NHPs at various times after the first and second administrations as described in Example 25. Serum and CSF were tested for antibody levels as described below.

[0573] Sandwich ELISA method: 100 μL / well of goat anti-human IgG, monkey adsorbed BIOTIN (Southern Biotech; catalog number 2049-08) working solution (0.5 μg / mL) was added to the SuperBlock TM 96-well Pierce TMStreptavidin-coated high binding plates were added and incubated at room temperature (RT) on a plate shaker at 350 revolutions per minute (RPM) for 1 hour ± 10 minutes. Serum or CSF quality control (QC) and test samples were diluted to the minimum required dilution (MRD) in assay buffer (1x TBS, 0.05% Tween-20, and 0.1% BSA) and then loaded onto the assay plate, while the standard calibration curve and assay buffer QC (AB-QC) were added directly to the assay plate. After incubation, the plate was washed 3 times with wash buffer (1X PBS and 0.05% Tween-20) using a plate washer, and then 100 μL / well of calibration standards, QC, and MRD diluted serum or CSF QC and test samples were added to the plate and incubated at room temperature on a plate shaker at 350RPM for 2 hours ± 15 minutes. 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 at room temperature on a plate shaker at 350RPM for 1 hour ± 10 minutes. After 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 at room temperature (RT) for 15 ± 10 minutes in the dark by covering with aluminum foil or placing in a drawer. After the incubation is complete, 100 μL / well of stop solution (Surmodics Product No. NSTP-1000-01) is added to the plate to terminate the reaction. The plate is then placed on a plate shaker at 350RPM for at least 2 minutes at room temperature. The plate was then read on a Molecular Devices SpectraMax M5 instrument and raw optical density (OD450-540) signals were generated from each well. The concentration of the analyte in each unknown sample was determined by concentration interpolation using the calibration standard curve. The analyte concentration in each unknown sample was determined by SoftMax Pro (Molecular Devices / Version 7.1) using a weighting factor of 1 / Y 2 The 4-parameter logistic (4-PL) model performs standard regression.

[0574] Fig.13 For example, an increase in CSF Cmax was seen with TD1-TfR compared to TD1. An increase in CSF Cmax was also seen with Iso-TfR compared to an isotype control. Fig.13 As exemplified, TD1-TfR exhibited faster serum clearance in NHPs compared to Iso-TfR or TD1 alone.

[0575] Example 28: Effects of the TfR Binding Arm on Brain Uptake and Brain Pharmacodynamics in Non-Human Primates

[0576] Vascular-depleted brain lysates were also used to directly assess antibody levels in multiple NHP brain regions (frontal cortex, entorhinal cortex, and hippocampus).

[0577] Brain sample preparation: Brain tissue was collected from antibody-treated cynomolgus monkeys at day 30 (i.e., 48 hours after administration of the secondary antibody). Prior to this, the animals were anesthetized and perfused cardiacally with PBS to clear blood vessels. For details of administration, see Example 25. The brain tissue was then minced and homogenized in HBSS buffer (MilliporeSigma #55037C) containing 10 mM HEPES (#15630130, Gibco) using a manual grinder. Subsequently, the sample was centrifuged at 1000 g for 5 minutes to precipitate the vascular portion. The supernatant representing the substance was then transferred to a new tube and mixed with a mixture of protease and phosphatase inhibitors (cOmplete TM , mini protease inhibitor cocktail, Roche, #11836153001) in 10x RIPA buffer (final concentration 1x). 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. The vascular fraction was cleared from the myelin debris by centrifugation through 18% dextran (70kDa, #31390, Sigma-Aldrich) followed by 2 washes in HBSS buffer. Thereafter, the samples were lysed in RIPA buffer (R0278, Sigma-Aldrich) and stored at -80°C until analysis. The effectiveness of this method has been previously demonstrated using Western blot analysis, where markers of brain endothelial cells (e.g., CD31 and tight junction protein-5) were absent in the parenchymal fraction (data not shown).

[0578] Pharmacokinetic (PK) analysis: The antibody concentration in brain samples was measured using the MSD (Meso Scale Discovery) method. In brief, 50 μl / well of goat anti-human IgG (monkey absorption) antibody was added to the MSD plate with a PBS solution of 1 μg / ml and incubated overnight at 4 ° C on a shaker at 500RPM. The plate was then washed 3 times with wash buffer (0.05% Tween-20 in 1xPBS) and then incubated with blocking buffer (wash buffer containing 3% BSA) at room temperature for 1 hour. Subsequently, brain (diluted in PBS) samples were added to the plate in duplicate and incubated at room temperature on a shaker at 500RPM for 2 hours. The plate was then washed 3 times with wash buffer, and then 40 μl / well of sulfo-Tag goat anti-human antibody (R32AJ-5, Meso Scale Discovery) was added with a PBS solution of 0.5 μg / ml, and incubated on a shaker at 500RPM for 1 hour. Finally, after washing 3 times with washing buffer, 150 μl / well of 1x READ buffer (R92PC-2, MesoScale Discovery) was added to the plate and then read using a Sector Imager S600 instrument. Eleven 2-fold serial dilutions (100-0.019 ng / ml) of the corresponding antibody were used to generate a standard curve for each treatment. Four-parameter logistic regression fitting curves were used to calculate the antibody concentration in the sample.

[0579] Fig.14 As an example, the TfR BBB targeting arm was able to increase the brain uptake of (i) TD1-TfR compared to TD1 in all 3 brain regions tested (increase ranged from 2- to 11-fold) and (ii) Iso-TfR compared to the isotype parental antibody in all 3 brain regions tested (increase ranged from 8- to 64-fold).

[0580] Example 29: Pharmacodynamic Analysis of TD1-TfR Bispecific Antibody in NHP Samples

[0581] CSF and serum from NHP test subjects as described in Example 25 and NHP brain lysates as described in Example 28 were further tested for downstream biomarkers of TD1 function, including sTREM2 and CSF1R, as described below.

[0582] CSF1R Method Summary (Brain Lysate)

[0583] The concentration of colony stimulating factor 1 receptor (CSF1R) in cynomolgus monkey brain lysates was measured using an enzyme-linked immunosorbent assay (ELISA) kit from R&D Systems (Cat. No. DY329). 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; REF 21-040-CM) and coated on 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 plates were 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 shaking. Prepare human M-CSF R standards and QCs (R&D Systems, part number 841248) in reagent diluent. Dilute test samples and QCs at the minimum required dilution (MRD) in assay buffer (1x PBS, 1% BSA, and 0.05% Tween-20). Then wash the plate and add 100 μL / well of human M-CSF1R standards, QCs diluted in assay buffer, and diluted samples to the appropriate wells and incubate for 2 hours at room temperature. Wash the plate and add 100 μL / well of a 100 ng / mL working solution of biotinyl...

Claims

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

2. An antigen binding domain that specifically binds to human transferrin receptor (TfR), wherein the antigen binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, CDR2 and CDR3 sequence, and the sequence comprises the following amino acid sequence: (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: 94-96 and 149-151, respectively; (xi) SEQ ID NOs: 97, 86, 98 and 135-137, respectively; (xii) SEQ ID NOs: 99, 100, 101 and 152-154, respectively; (xiii) are SEQ ID NOs: 99, 102, 103 and 155-157, respectively; (xiv) are SEQ ID NOs: 104-106, 138, 158 and 159, respectively; (xv) SEQ ID NOs: 107-109 and 160-162, respectively; (xvi) are SEQ ID NOs: 99, 110, 103 and 155-157, respectively; (xvii) are SEQ ID NOs: 99, 111, 112, 152, 163 and 164, respectively; (xviii) are SEQ ID NOs: 90, 113, 114, 165, 166 and 157, respectively; (xix) are SEQ ID NOs: 83, 115, 85, 141, 142 and 167, respectively; (xx) are SEQ ID NOs: 116, 110, 103 and 155-157, respectively; (xxi) are SEQ ID NOs: 117-119 and 168-170, respectively; (xxii) SEQ ID NOs: 116, 110, 103 and 168-170, respectively; (xxiii) are SEQ ID NOs: 90, 120, 114, 165, 166 and 157, respectively; (xxiv) are SEQ ID NOs: 90, 113, 114, 171, 166 and 157, respectively; (xxv) are SEQ ID NOs: 97, 121, 122, 135, 136 and 172, respectively; (xxvi) are SEQ ID NOs: 74, 123, 124, 173, 136 and 137, respectively; (xxvii) are SEQ ID NOs: 74, 125, 126, 174, 136 and 137, respectively; (xxviii) are SEQ ID NOs: 99, 127, 128, 155, 175 and 176, respectively; (xxix) are SEQ ID NOs: 90, 129, 130, 165, 166 and 157, respectively; (xxx) are SEQ ID NOs: NO: 131, 132, 133, 177, 166 and 178; (xxxi) are SEQ ID NO: 131, 132, 133 and 179-181, respectively; (xxxii) are SEQ ID NOs: 90, 134, 114, 165, 166 and 157, respectively; (xxxiii) are SEQ ID NOs: 83, 302, 303, 141, 142 and 182, respectively; (xxxiv) are SEQ ID NOs: 99, 305, 103, 307, 308 and 157, respectively; (xxxv) are SEQ ID NOs: 99, 306, 103, 307, 308 and 157, respectively; (xxxvi) are SEQ ID NOs: 99, 306, 103, 307, 309 and 157, respectively; (xxxvii) are SEQ ID NOs: 99, 306, 103, 310, 311 and 157, respectively; (xxxviii) are SEQ ID NOs: 99, 305, 103, 310, 311 and 157, respectively; (xxxix) are SEQ ID NOs: 99, 305, 103, 310, 311 and 157, respectively NO:99, 312, 103, 307, 308 and 157; (xl) are SEQ ID NO:99, 312, 103, 307, 309 and 157, respectively; (xli) are SEQ ID NOs: 99, 312, 103, 310, 311 and 157, respectively (xlii) are SEQ ID NOs: 94, 95, 418, 421, 150 and 151, respectively; (xliii) are SEQ ID NOs: 94, 412, 419, 422, 150 and 151, respectively; (xliv) are SEQ ID NOs: 94, 412, 420, 422, 150 and 151, respectively; (xlv) are SEQ ID NOs: 94, 413, 418, 422, 150 and 151, respectively; (xlvi) are SEQ ID NOs: 94, 414, 418, 422, 150 and 151, respectively; (xlvii) are SEQ ID NOs: 94, 416, 418, 422, 150 and 151, respectively; (xlviii) are SEQ ID NOs: 94, 417, 418, 424, 150 and 151, respectively; (xlix) are SEQ ID NOs: 460, 312, 103, 307, 309 and 157, respectively; (l) are 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) are SEQ ID NOs: 460, 312, 465, 307, 309 and 157, respectively; (lv) are SEQ ID NOs: 460, 312, 466, 307, 309 and 157, respectively; (lvi) are SEQ ID NOs: 460, 312, 467, 307, 309 and 157, respectively; (lvii) are SEQ ID NOs: 460, 312, 468, 307, 309 and 157, respectively; (lviii) SEQ ID NOs: 460, 312, 469, 307, 309 and 157, respectively; (lix) are SEQ ID NOs: 460, 312, 470, 307, 309 and 157, respectively; (lx) are SEQ ID NOs: 460, 312, 103, 471, 309 and 157, respectively; (lxi) are SEQ ID NOs: 460, 312, 103, 472, 309 and 157, respectively; (lxii) are SEQ ID NOs: 460, 312, 103, 473, 309 and 157, respectively; (lxiii) are SEQ ID NOs: 460, 312, 103, 474, 309 and 157, respectively; (lxiv) are SEQ ID NOs: 460, 312, 103, 475, 309 and 157, respectively; (lxv) are SEQ ID NOs: 460, 312, 103, 476, 309 and 157, respectively; or (lxvi) are SEQ ID NOs: 460, 312, 103, 471, 309 and 157, respectively.

3. The antigen binding domain of claim 1 or 2, wherein the antigen binding domain comprises a VH and a VL, wherein the VH and the VL comprise 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 following amino acid sequence: (i) SEQ ID NO: 399 and 400, respectively; (ii) SEQ ID NOs: 10 and 11, respectively; (iii) are SEQ ID NOs: 12 and 13, respectively; (iv) are SEQ ID NOs: 14 and 15, respectively; (v) are SEQ ID NOs: 16 and 17, respectively; (vi) are SEQ ID NOs: 18 and 19, respectively; (vii) are SEQ ID NOs: 20 and 21, respectively; (viii) are SEQ ID NOs: 22 and 23, respectively; (ix) are SEQ ID NOs: 24 and 25, respectively; (x) are SEQ ID NOs: 26 and 27, respectively; (xi) are SEQ ID NOs: 28 and 29, respectively; (xii) are SEQ ID NOs: 30 and 31, respectively; (xiii) are SEQ ID NOs: 32 and 33, respectively; (xiv) are SEQ ID NOs: 34 and 35, respectively; (xv) are SEQ ID NOs: 36 and 37, respectively; (xvi) are SEQ ID NOs: 38 and 39, respectively; (xvii) are SEQ ID NOs: 40 and 41, respectively; (xviii) are SEQ ID NOs: 42 and 43, respectively; (xix) are SEQ ID NOs: NO:44 and 45; (xx) are SEQ ID NO:46 and 47, respectively; (xxi) are SEQ ID NO:48 and 49, respectively; (xxii) SEQ ID NOs: 50 and 51, respectively; (xxiii) are SEQ ID NOs: 52 and 53, respectively; (xxiv) SEQ ID NOs: 54 and 55, respectively; (xxv) are SEQ ID NOs: 56 and 57, respectively; (xxvi) are SEQ ID NOs: 58 and 59, respectively; (xxvii) are SEQ ID NOs: 60 and 61, respectively; (xxviii) are SEQ ID NOs: 62 and 63, respectively; (xxix) are SEQ ID NOs: 64 and 65, respectively; (xxx) are SEQ ID NOs: 66 and 67, respectively; (xxxi) are SEQ ID NOs: 68 and 69, respectively; (xxxii) SEQ ID NOs: 70 and 71, respectively; (xxxiii) are SEQ ID NOs: 72 and 73, respectively; (xxxiv) are SEQ ID NOs: 313 and 314, respectively; (xxxv) are SEQ ID NOs: 315 and 316, respectively; (xxxvi) are SEQ ID NOs: 317 and 314, respectively; (xxxvii) are SEQ ID NOs: 318 and 316, respectively; (xxxviii) are SEQ ID NOs: 319 and 314, respectively; (xxxix) are SEQ ID NOs: 320 and 316, respectively; (xl) are SEQ ID NOs: 321 and 314, respectively; (xli) are SEQ ID NOs: 322 and 316, respectively; (xlii) are SEQ ID NOs: 323 and 314, respectively; (xliii) are SEQ ID NOs: 324 and 316, respectively; (xliv) are SEQ ID NOs: 325 and 326, respectively; (xlv) are SEQ ID NOs: 327 and 328, respectively; (xlvi) are SEQ ID NOs: 329 and 330, respectively. NO:325 and 329; (xlvii) are SEQ ID NO:330 and 331, respectively; (xlviii) are SEQ ID NO:325 and 332, respectively; (xlix) are SEQ ID NO:330 and 333, respectively; (l) are SEQ ID NO:334 and 332, respectively; (li) are SEQ ID NOs: 335 and 336, respectively; (lii) are SEQ ID NOs: 337 and 338, respectively; (liii) are SEQ ID NOs: 38 and 33, respectively; (liv) are SEQ ID NOs: 339 and 340, respectively; (lv) are SEQ ID NOs: 339 and 341, respectively; (lvi) are SEQ ID NOs: 339 and 342, respectively; (lvii) are SEQ ID NOs: 343 and 340, respectively; (lviii) are SEQ ID NOs: 343 and 342, respectively; (lix) are SEQ ID NOs: 344 and 340, respectively; (lx) are SEQ ID NOs: 344 and 341, respectively; (lxi) are SEQ ID NOs: 344 and 342, respectively; (lxii) are SEQ ID NOs: 345 and 340, respectively; (lxiii) are SEQ ID NOs: 345 and 341, respectively; (lxiv) are SEQ ID NOs: 345 and 342, respectively; (lxv) are SEQ ID NOs: 346 and 347, respectively; (lxvi) are SEQ ID NOs: 348 and 316, respectively; (lxvii) are SEQ ID NOs: NO:349 and 347; (lxviii) are SEQ ID NO:349 and 350, respectively; (lxix) are SEQ ID NO:351 and 352, respectively; (lxx) are SEQ ID NO:349 and 353, respectively; (lxxi) are SEQ ID NO:346 and 354, respectively; (lxxii) are SEQ ID NO:349 and 355, respectively; (lxxiii) are SEQ ID NO:349 and 356, respectively; (lxxiv) are SEQ ID NO:357 and 316, respectively; (lxxv) are SEQ ID NO:349 and 358, respectively; (lxxvi) are SEQ ID NO:349 and 316, respectively; (lxxvii) are SEQ ID NO:359 and 360, respectively; (lxxviii) are SEQ ID NO:361 and 362, respectively; (lxxix) are SEQ ID NO:363 and 316, respectively; (lxxx) are SEQ ID NO: NO:364 and 365; (lxxxi) are SEQ ID NO:349 and 366, respectively; (lxxxii) are SEQ ID NO:346 and 316, respectively; (lxxxiii) are SEQ ID NO:346 and 367, respectively; (lxxxiv) are SEQ ID NO:349 and 368, respectively; (lxxxv) are SEQ ID NO:369 and 316, respectively; (lxxxvi) are SEQ ID NO:346 and 370, respectively; (lxxxvii) are SEQ ID NO:371 and 316, respectively; (lxxxviii) are SEQ ID NO:372 and 356, respectively; (lxxxix) are SEQ ID NO:357 and 358, respectively;(xc) are SEQ ID NOs: 349 and 373, respectively; (xci) are SEQ ID NOs: 346 and 374, respectively; (xcii) are SEQ ID NOs: 375 and 316, respectively; (xciii) are SEQ ID NOs: 376 and 316, respectively; (xciv) are SEQ ID NOs: 346 and 377, respectively; (xcv) are SEQ ID NOs: 378 and 379, respectively; (xcvi) are SEQ ID NOs: 380 and 381, respectively; (xcvii) are SEQ ID NOs: 349 and 382, ​​respectively; (xcviii) are SEQ ID NOs: 357 and 383, respectively; (xcix) are SEQ ID NOs: 349 and 358, respectively; (c) are SEQ ID NOs: 384 and 316, respectively; (ci) are SEQ ID NOs: 385 and 316, respectively; (cii) are SEQ ID NOs: 357 and 386, respectively; (ciii) are SEQ ID NOs: 387 and 388, respectively; (civ) are SEQ ID NOs: 359 and 316, respectively; (cv) are SEQ ID NOs: 389 and 316, respectively; (cvi) are SEQ ID NOs: 390 and 316, respectively; (cvii) are SEQ ID NOs: 391 and 392, respectively; (cviii) are SEQ ID NOs: 393 and 356, respectively; (cix) are SEQ ID NOs: 390 and 392, respectively; (cx) are SEQ ID NOs: 357 and 386, respectively; (cxi) are SEQ ID NOs: 394 and 395, respectively; (cxii) are SEQ ID NOs: 396 and 395, respectively; (cxiii) are SEQ ID NOs: 397 and 395, respectively; (cxiv) are SEQ ID NOs: 390 and 392, respectively; NO:398 and 395; (cxv) are SEQ ID NO:401 and 395, respectively; (cxvi) are SEQ ID NO:402 and 403, respectively; (cxvii) are SEQ ID NOs: 443 and 341, respectively; (cxviii) are SEQ ID NOs: 444 and 341, respectively; (cxix) are SEQ ID NOs: 445 and 341, respectively; (cxx) are SEQ ID NOs: 446 and 341, respectively; (cxxi) are SEQ ID NOs: 447 and 341, respectively; (cxxii) are SEQ ID NOs: 448 and 341, respectively; (cxxiii) are SEQ ID NOs: 449 and 341, respectively; (cxxiv) are SEQ ID NOs: 450 and 341, respectively; (cxxv) are SEQ ID NOs: 451 and 341, respectively; (cxxvi) are SEQ ID NOs: 452 and 341, respectively; (cxxvii) are SEQ ID NOs: 453 and 341, respectively; (cxxviii) are SEQ ID NOs: 443 and 454, respectively; (cxxix) are SEQ ID NOs: 443 and 455, respectively; (cxxx) are SEQ ID NOs: 443 and 456, respectively; (cxxxi) are SEQ ID NOs: 443 and 457, respectively; (cxxxii) are SEQ ID NOs: 443 and 458, respectively; or (cxxxiii) are SEQ ID NOs: 443 and 459, respectively.

4. An antigen binding domain that specifically binds to human TfR, wherein the antigen binding domain comprises VH and VL, wherein the VH comprises the following amino acid sequence: SEQ ID NO: 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, 334, 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.

5. An antigen binding domain that specifically binds to human TfR, wherein the antigen binding domain comprises VH and VL, wherein the VL comprises the following amino acid sequence: SEQ ID NO: 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, 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.

6. The antigen binding domain of claim 1, 4 or 5, wherein the antigen binding domain comprises VH and VL, and the VH and the VL comprise the following amino acid sequences: (i) SEQ ID NO: 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) are SEQ ID NOs: 18 and 19, respectively; (vii) are SEQ ID NOs: 20 and 21, respectively; (viii) are SEQ ID NOs: 22 and 23, respectively; (ix) are SEQ ID NOs: 24 and 25, respectively; (x) are SEQ ID NOs: 26 and 27, respectively; (xi) are SEQ ID NOs: 28 and 29, respectively; (xii) are SEQ ID NOs: 30 and 31, respectively; (xiii) are SEQ ID NOs: 32 and 33, respectively; (xiv) are SEQ ID NOs: 34 and 35, respectively; (xv) are SEQ ID NOs: 36 and 37, respectively; (xvi) are SEQ ID NOs: 38 and 39, respectively; (xvii) are SEQ ID NOs: 40 and 41, respectively; (xviii) are SEQ ID NOs: 42 and 43, respectively; (xix) are SEQ ID NOs: 44 and 45, respectively; (xx) are SEQ ID NOs: 46 and 47, respectively; (xxi) are SEQ ID NOs: 48 and 49, respectively; (xxii) are SEQ ID NOs: NO:50 and 51; (xxiii) are SEQ ID NO:52 and 53, respectively; (xxiv) are SEQ ID NO:54 and 55, respectively; (xxv) are SEQ ID NO:56 and 57, respectively; (xxvi) are SEQ ID NOs: 58 and 59, respectively; (xxvii) are SEQ ID NOs: 60 and 61, respectively; (xxviii) are SEQ ID NOs: 62 and 63, respectively; (xxix) are SEQ ID NOs: 64 and 65, respectively; (xxx) are SEQ ID NOs: 66 and 67, respectively; (xxxi) are SEQ ID NOs: 68 and 69, respectively; (xxxii) SEQ ID NOs: 70 and 71, respectively; (xxxiii) are SEQ ID NOs: 72 and 73, respectively; (xxxiv) are SEQ ID NOs: 313 and 314, respectively; (xxxv) are SEQ ID NOs: 315 and 316, respectively; (xxxvi) are SEQ ID NOs: 317 and 314, respectively; (xxxvii) are SEQ ID NOs: 318 and 316, respectively; (xxxviii) are SEQ ID NOs: 319 and 314, respectively; (xxxix) are SEQ ID NOs: 320 and 316, respectively; (xl) are SEQ ID NOs: 321 and 314, respectively; (xli) are SEQ ID NOs: 322 and 316, respectively; (xlii) are SEQ ID NOs: 323 and 314, respectively; (xliii) are SEQ ID NOs: 324 and 316, respectively; (xliv) are SEQ ID NOs: 325 and 326, respectively; (xlv) are SEQ ID NOs: 327 and 328, respectively; (xlvi) are SEQ ID NOs: 325 and 329, respectively; (xlvii) are SEQ ID NOs: 330 and 331, respectively; (xlviii) are SEQ ID NOs: 325 and 332, respectively; (xlix) are SEQ ID NOs: 330 and 333, respectively; (l) are SEQ ID NOs: 334 and 332, respectively; (li) are SEQ ID NOs: 335 and 336, respectively; (lii) are SEQ ID NOs: 337 and 338, respectively; (liii) are SEQ ID NOs: 38 and 33, respectively; (liv) are SEQ ID NOs: 339 and 340, respectively; (lv) are SEQ ID NOs: 339 and 341, respectively; (lvi) are SEQ ID NOs: 339 and 342, respectively; (lvii) are SEQ ID NOs: 343 and 340, respectively; (lviii) are SEQ ID NOs: 343 and 342, respectively; (lix) are SEQ ID NOs: 344 and 340, respectively; (lx) are SEQ ID NOs: 344 and 341, respectively; (lxi) are SEQ ID NOs: 344 and 342, respectively; (lxii) are SEQ ID NOs: 345 and 340, respectively; (lxiii) are SEQ ID NOs: 345 and 341, respectively; (lxiv) are SEQ ID NOs: 345 and 342, respectively; (lxv) are SEQ ID NOs: 346 and 347, respectively; (lxvi) are SEQ ID NOs: 348 and 316, respectively; (lxvii) are SEQ ID NOs: NO:349 and 347; (lxviii) are SEQ ID NO:349 and 350, respectively; (lxix) are SEQ ID NO:351 and 352, respectively; (lxx) are SEQ ID NO:349 and 353, respectively; (lxxi) are SEQ ID NO:346 and 354, respectively; (lxxii) are SEQ ID NO:349 and 355, respectively; (lxxiii) are SEQ ID NO:349 and 356, respectively; (lxxiv) are SEQ ID NO:357 and 316, respectively; (lxxv) are SEQ ID NO:349 and 358, respectively; (lxxvi) are SEQ ID NO:349 and 316, respectively; (lxxvii) are SEQ ID NO:359 and 360, respectively; (lxxviii) are SEQ ID NO:361 and 362, respectively; (lxxix) are SEQ ID NO:363 and 316, respectively; (lxxx) are SEQ ID NO: NO:364 and 365; (lxxxi) are SEQ ID NO:349 and 366, respectively; (lxxxii) are SEQ ID NO:346 and 316, respectively; (lxxxiii) are SEQ ID NO:346 and 367, respectively; (lxxxiv) are SEQ ID NO:349 and 368, respectively; (lxxxv) are SEQ ID NO:369 and 316, respectively; (lxxxvi) are SEQ ID NO:346 and 370, respectively; (lxxxvii) are SEQ ID NO:371 and 316, respectively; (lxxxviii) are SEQ ID NO:372 and 356, respectively; (lxxxix) are SEQ ID NO:357 and 358, respectively;(xc) are SEQ ID NOs: 349 and 373, respectively; (xci) are SEQ ID NOs: 346 and 374, respectively; (xcii) are SEQ ID NOs: 375 and 316, respectively; (xciii) are SEQ ID NOs: 376 and 316, respectively; (xciv) are SEQ ID NOs: 346 and 377, respectively; (xcv) are SEQ ID NOs: 378 and 379, respectively; (xcvi) are SEQ ID NOs: 380 and 381, respectively; (xcvii) are SEQ ID NOs: 349 and 382, ​​respectively; (xcviii) are SEQ ID NOs: 357 and 383, respectively; (xcix) are SEQ ID NOs: 349 and 358, respectively; (c) are SEQ ID NOs: 384 and 316, respectively; (ci) are SEQ ID NOs: 385 and 316, respectively; (cii) SEQ ID NOs: 357 and 386, respectively; (ciii) are SEQ ID NOs: 387 and 388, respectively; (civ) are SEQ ID NOs: 359 and 316, respectively; (cv) are SEQ ID NOs: 389 and 316, respectively; (cvi) are SEQ ID NOs: 390 and 316, respectively; (cvii) are SEQ ID NOs: 391 and 392, respectively; (cviii) are SEQ ID NOs: 393 and 356, respectively; (cix) are SEQ ID NOs: 390 and 392, respectively; (cx) are SEQ ID NOs: 357 and 386, respectively; (cxi) are SEQ ID NOs: 394 and 395, respectively; (cxii) are SEQ ID NOs: 396 and 395, respectively; (cxiii) are SEQ ID NOs: 397 and 395, respectively; (cxiv) are SEQ ID NOs: 398 and 395, respectively; (cxv) are SEQ ID NOs: 401 and 395, respectively; (cxvi) are SEQ ID NOs: 402 and 403, respectively; (cxxxiv) are SEQ ID NOs: 443 and 341, respectively; (cxxxv) are SEQ ID NOs: 444 and 341, respectively; (cxxxvi) are SEQ ID NOs: 445 and 341, respectively; (cxxxvii) are SEQ ID NOs: 446 and 341, respectively; (cxxxviii) are SEQ ID NOs: 447 and 341, respectively; (cxxxix) are SEQ ID NOs: 448 and 341, respectively; (cxl) are SEQ ID NOs: 449 and 341, respectively; (cxli) are SEQ ID NOs: 450 and 341, respectively; (cxlii) are SEQ ID NOs: 451 and 341, respectively; (cxliii) are SEQ ID NOs: 452 and 341, respectively; (cxliv) are SEQ ID NOs: 453 and 341, respectively; (cxlv) are SEQ ID NOs: 443 and 454, respectively; (cxlvi) are SEQ ID NOs: 443 and 455, respectively; (cxlvii) are SEQ ID NOs: 443 and 456, respectively; (cxlviii) are SEQ ID NOs: 443 and 457, respectively; (cxlix) are SEQ ID NOs: 443 and 458, respectively; or (cxvii) are SEQ ID NOs: 443 and 459, respectively.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

24. An antigen binding domain as described in any one of claims 1-23, wherein the antigen binding domain does not significantly increase the cell surface expression of TfR on HCMEC / D3 cells relative to the cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control.

25. The antigen binding domain of any one of claims 1-24, wherein the accumulation of the antigen binding domain in the vascular depleted mouse brain is at least 4-fold or at least 5-fold higher than an isotype control.

26. An antigen binding domain as described in any one of claims 1-25, wherein the specific binding of the antigen binding domain to human TfR is at least 5 times that of the binding to an unrelated protein.

27. The antigen binding domain of any one of claims 1-26, wherein the specific binding of the antigen binding domain to cynomolgus monkey TfR is at least 5 times greater than the binding to an unrelated protein.

28. An antigen binding domain as described in claim 26 or 27, wherein the binding of the antigen binding domain to human TfR is at least 5 times that of binding to an unrelated protein and / or the specific binding to cynomolgus monkey TfR is at least 5 times that of binding to an unrelated protein.

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

30. The antigen binding domain of any one of claims 1-29, wherein the antigen binding domain comprises VH and VL on a single polypeptide chain.

31. The antigen binding domain of any one of claims 1-30, wherein the antigen binding domain comprises a single chain variable 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. The antigen binding domain of claim 32 or 33, wherein the linker (i) 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, and / or (ii) comprises the amino acid sequence of GGSEGKS SGSGSESKSTGGS (SEQ ID NO: 183) or GGGGSGGGGSGGGGSGG GGS (SEQ ID NO: 288).

35. An antigen binding domain as described in any one of claims 2-34, wherein the antigen binding domain binds to the apical domain of human transferrin receptor (TfR), wherein the antigen binding domain is localized to the brain parenchyma of a subject after peripheral injection, and wherein the antigen binding domain is not within a modified CH3 domain.

36. The antigen binding domain of any one of claims 1-33, wherein 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.

37. The antigen binding domain of any one of claims 1-29, wherein the antigen binding domain comprises a VH on a first polypeptide and a VL on a second polypeptide.

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

39. An antigen binding domain that specifically binds to human TfR, wherein the antigen binding domain is a VHH, which comprises (i) VH CDR1, VH CDR2 and VHCDR3 of an antigen binding domain as described in any one of claims 1-36 and 38, or (ii) VH of an antigen binding domain as described in any one of claims 1-36 and 38, optionally wherein the VHH is capable of crossing the blood-brain barrier (BBB).

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

41. The fusion protein of claim 40, wherein the heterologous protein or peptide comprises the following amino acid sequence: 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-glucocerebrosidase (GCase or GBA), progranulin (PGRN), prosphingolipid activator protein (PSAP), glycoprotein non-transfer protein B (GPNMB), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), caspase 6, sortin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or salivaricin. 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), nebulin Protein, ubiquitin protein ligase E3A (UBE3A), tripeptidyl peptidase 1 (CLN2 / TPP1), α-L-iduronidase (IDUA), iduronate 2-sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-α-glucosaminide N-acetyltransferase (HGSNAT), and N-acetyl-α-glucosaminidase (NAGLU), N-sulfoglucosamine sulfohydrolase (SGSH), or a portion thereof.

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

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

44. A fusion protein as described in 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. A fusion protein as described in claim 44, wherein the single scFv, Fab or VHH antigen binding domain that binds to human TfR is connected to the C-terminus of one of the two copies of the heterologous protein or peptide.

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

47. A fusion protein as described in claim 44, wherein the single scFv, Fab or VHH antigen binding domain that binds to human TfR is connected to the N-terminus of the Fc domain.

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

49. A fusion protein as described in any one of claims 40-43, comprising (i) an antibody that binds to human TfR, wherein the antibody comprises two heavy chains and two light chains; and (ii) two copies of the heterologous protein or peptide, wherein each copy of the heterologous protein or peptide is connected to the C-terminus of one of the two antibody heavy chains.

50. A fusion protein as described in any one of claims 40-43, 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 connected to the C-terminus of the Fc domain, and the two copies of the heterologous protein or peptide are connected to the N-terminus of the Fc domain.

51. A fusion protein as described in any one of claims 40-43, which comprises (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 connected to the C-terminus of the Fc domain, and the heterologous protein or polypeptide is connected to the N-terminus of the Fc domain.

52. A fusion protein as described in any one of claims 40-43, 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 connected to the N-terminus of the Fc domain, and the heterologous protein or polypeptide is connected to the C-terminus of the Fc domain.

53. A fusion protein as described in any one of claims 40-43, 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 polypeptide are both connected to the N-terminus of the Fc domain.

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

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

56. The fusion protein of any one of claims 42-55, wherein the Fc is a modified Fc having modifications listed in Table 1 or 2.

57. A fusion protein as described in any one of claims 40-56, wherein the Fc comprises 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.

58. An antibody comprising the antigen binding domain of any one of claims 1-39.

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 1-39.

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

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

62. A multispecific protein comprising an antigen binding domain as described in any one of claims 1-39 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. The multispecific protein of claim 62, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region.

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

65. The multispecific protein of any one of claims 61-64, wherein the multispecific protein is bispecific.

66. The multispecific protein of any one of claims 61-65, wherein the multispecific protein is bivalent, trivalent, or tetravalent.

67. The multispecific protein of claim 66, wherein the multispecific protein is bivalent.

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

70. A multispecific protein that is trivalent and bispecific and comprises an antigen binding domain as described in any one of claims 1-36 and 38 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 a scFv optionally linked to the C-terminus of one of the two antibody heavy chains via an amino acid linker.

71. A multispecific protein that is trivalent and bispecific and comprises an antigen binding domain as described in any one of claims 1-36 and 38 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 a scFv optionally linked to the N-terminus of one of the two antibody heavy chains via an amino acid linker.

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

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

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

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

76. A multispecific protein that is bivalent and bispecific, comprising: (i) an Fc domain, (ii) a single antigen binding domain as described in any one of claims 1-39, wherein the antigen binding domain is a single scFv, VHH or Fab antigen binding domain that binds to human TfR and is connected to the N-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 connected to the C-terminus of the Fc domain.

77. A multispecific protein that is bivalent and bispecific, comprising: (i) an Fc domain, (ii) a single antigen binding domain as described in any one of claims 1-39, wherein the antigen binding domain is a single scFv, VHH or Fab antigen binding domain that binds to human TfR and is connected 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 connected to the N-terminus of the Fc domain.

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

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

80. The multispecific protein of any one of claims 76-79, wherein the Fc comprises 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.

81. The multispecific protein of any one of claims 62-75, 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. The multispecific protein of claim 81, wherein the antigen binding domain is linked to the constant region containing the hole mutation, optionally via an amino acid linker.

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

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

85. The multispecific protein of claim 84, wherein the 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. The multispecific protein of any one of claims 61-85, wherein the CNS antigen is a brain antigen.

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

88. The multispecific protein of any one of claims 62-75 and 81-87, wherein the antibody or antigen-binding fragment thereof comprises 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.

89. The multispecific protein of any one of claims 62-75 and 81-88, 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. The multispecific protein of any one of claims 62-75 and 81-88, wherein the antibody or antigen-binding fragment thereof comprises a constant region comprising a hole 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.

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

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. The multispecific protein of any one of claims 61-92, wherein the multispecific protein has an equilibrium dissociation constant (K) of about 65 nM to about 4 μM. D ) binds to human TfR and / or with a K of about 37 nM to about 1.3 μM D Binds to cynomolgus monkey TfR.

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

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

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

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

98. The multispecific protein of any one of claims 61-97, wherein accumulation of the multispecific protein in the vascular depleted mouse brain is at least 4-fold or 5-fold higher than an isotype control.

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

100. The multispecific protein of any one of claims 61, 65-69, 76-80, and 93-98, wherein the second antigen binding domain specifically binds to cynomolgus monkey TfR at least 5 times more than to an unrelated protein.

101. A multispecific protein as described in claim 99 or 100, wherein the second antigen binding domain binds to human TfR at least 5 times more than to an unrelated protein and / or specifically binds to cynomolgus monkey TfR at least 5 times more than to an unrelated protein.

102. The multispecific protein of any one of claims 61-101, wherein the CNS antigen is β-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebrosidase (Gcase or GBA), progranulin (PGRN), prosphingolipid activator protein (PSAP), γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), caspase 6, sorting protein (SORT), expressed on myeloid cells triggering 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.

103. A multispecific protein as described in claim 102, wherein the CNS antigen is MS4A4A, optionally wherein (i) the antigen binding domain, antibody or antigen binding domain that binds to MS4A4A comprises VH and / or VL, the VH comprises the MS4A4A binding VH sequence of SEQ ID NO:406, and the VL comprises 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. The multispecific protein of claim 103, wherein the multispecific protein comprises the amino acid sequence of SEQ ID NOs: 405-407.

105. The multispecific protein of any one of claims 61-104, wherein the multispecific protein is capable of binding FcRn.

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

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

108. 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 a multi-specific protein as described in any one of claims 61-105, wherein the first polynucleotide encodes a first heavy chain, the second polynucleotide encodes a second heavy chain and the antigen binding domain that specifically binds to human TfR, and the third polynucleotide encodes a light chain.

109. 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 a multi-specific protein as described in any one of claims 61-105, 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, optionally wherein the first antigen binding domain and the second antigen binding domain that bind to human TfR comprise the same amino acid sequence.

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

6.

112. 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. A composition comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide encode a multi-specific protein as described in any one of claims 61-105, wherein the first polynucleotide encodes a heavy chain and the antigen binding domain that binds to human TfR, and wherein the second polynucleotide encodes a light chain.

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

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

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

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 the heavy chain variable region of an antigen binding domain as described in any one of claims 1-39 and a nucleic acid molecule encoding the light chain variable region of the antigen binding domain.

119. A host cell comprising the polynucleotide of claim 115 or 116 or the vector of claim 117 or 118.

120. The host cell of claim 114 or 119, wherein the host cell is selected from the group consisting of: Escherichia coli, Pseudomonas, Bacillus, Streptomyces, Saccharomyces, CHO, YB / 20, NSO, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture.

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

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

123. A pharmaceutical composition comprising (i) an antigen binding domain, fusion protein, antibody or antigen binding fragment thereof, or multispecific protein as described in any one of claims 1-107 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. 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 to 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. A method of treating a nervous system 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 as described in any one of claims 40-107 and 123-125.

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 to 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. 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. The method of claims 126-128, wherein the nervous system disease or disorder is selected from a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, epileptic seizures, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation.

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's syndrome, Liddle's 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. The method of claim 130, wherein the dementia is frontotemporal dementia (FTD).

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

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

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

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

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

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

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

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

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 following administration compared to an isotype control.

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

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

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

147. An in vitro method for detecting CNS antigens, comprising contacting an in vitro sample with the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein of claim 106 and locating the imaging agent within the sample.

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

149. The fusion protein, antibody or antigen-binding fragment thereof, multispecific protein or pharmaceutical composition of any one of claims 40-107 and 123-125, for use in the method of any one of claims 126-147.

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