CD98HC antigen-binding domain and methods of use thereof

Abstract

The present disclosure relates generally to antigen-binding domains that specifically bind to human CD98 heavy chain (CD98hc) and their use in transport across the blood-brain barrier (BBB).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application Nos. 63 / 369,885, filed July 29, 2022, 63 / 489,675, filed March 10, 2023, and 63 / 495,514, filed April 11, 2023, each of which is incorporated herein by reference in its entirety.

[0002] Reference to electronically submitted sequence listing The electronically submitted sequence listing (Name: 4503_021PC03_SequenceListing_ST26.xml, Size: 498,296 bytes, Created: July 27, 2023) is incorporated herein by reference in its entirety.

[0003] Field of the Disclosure The present disclosure relates to antigen-binding domains that specifically bind to CD98 heavy chain (CD98hc), which are capable of crossing the blood-brain barrier and transporting other agents (e.g., therapeutically active agents) bound to the antigen-binding domain across the blood-brain barrier. [Background technology]

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

[0005] Summary of the Disclosure Provided herein are antigen-binding domains, fusion proteins, antibodies, and multispecific proteins that specifically bind to CD98hc, and methods of making and using the same.

[0006] In some aspects, provided herein is an antigen-binding domain that specifically binds to a CD98hc heavy chain (CD98hc), comprising complementarity-determining region (CDR) 1, VH CDR2, and VH CDR3 of the heavy chain variable region (VH), and CDR1, CDR2, and CDR3 sequences of the light chain variable region (VL), each comprising the following amino acid sequences: SEQ ID NOs: 413, 414, 112, and 176 to 178, respectively; SEQ ID NOs: 50 to 52 and 116 to 118, respectively; SEQ ID NOs: 53 to 55 and 119 to 121, respectively; SEQ ID NOs: 56 to 58 and 122 to 124, respectively; SEQ ID NOs: 59 to 61 and 125 to 127, respectively; SEQ ID NOs: 62 to 64 and 128 to 130, respectively; and SEQ ID NOs: 65 to 68, respectively. 7 and 131 to 133, SEQ ID NOs: 68 to 70 and 134 to 136, SEQ ID NOs: 71 to 73 and 137 to 139, SEQ ID NOs: 74 to 76 and 140 to 142, SEQ ID NOs: 77 to 79 and 143 to 145, SEQ ID NOs: 80 to 82 and 146 to 148, SEQ ID NOs: 83 to 85 and 149 to 151, SEQ ID NOs: 86 to 88 and 152 to 154, SEQ ID NOs: 89 to 91 and 155 to 157, SEQ ID NOs: 92 to 94 and 1 58 to 160, SEQ ID NOs: 95 to 97 and 161 to 163, SEQ ID NOs: 98 to 100 and 164 to 166, SEQ ID NOs: 101 to 103 and 167 to 169, SEQ ID NOs: 104 to 106 and 170 to 172, SEQ ID NOs: 107 to 109 and 173 to 175, SEQ ID NOs: 110 to 112 and 176 to 178, SEQ ID NOs: 113 to 115 and 179 to 181, SEQ ID NOs: 226 to 228 and 273 to 275, SEQ ID NOs: SEQ ID NOs: 229 to 231 and 276 to 278, respectively; SEQ ID NOs: 232 to 234 and 279 to 281, respectively; SEQ ID NOs: 235 to 237 and 282 to 284, respectively; SEQ ID NOs: 238 to 240 and 285 to 287, respectively; SEQ ID NOs: 241 to 243 and 288 to 290, respectively; SEQ ID NOs: 244 to 246, 273, 274, and 291, respectively; SEQ ID NOs: 247 to 249, 292, 274, and 293, respectively; SEQ ID NOs: 250 to 252, 294, 274, and 291, respectively;SEQ ID NOs: 253 to 255 and 295 to 297, respectively, SEQ ID NOs: 256 to 258, 298, 274, and 299, respectively, SEQ ID NOs: 259 to 261 and 300 to 302, respectively, SEQ ID NOs: 262 to 264, 303, 274, and 304, respectively, SEQ ID NOs: 265 to 267, 305, 306, and 291, respectively, SEQ ID NOs: 268 to 270 and 307 to 309, respectively, SEQ ID NOs: 265, 271, 272, 310, 274, and 299, respectively, SEQ ID NOs: 110, 414, 112, and 176 to 17 8, SEQ ID NOs: 110, 414, 112, and 176 to 178, respectively, SEQ ID NOs: 413, 415, 112, and 176 to 178, respectively, SEQ ID NOs: 110, 415, 112, and 176 to 178, respectively, SEQ ID NOs: 110, 416, 112, and 176 to 178, respectively, SEQ ID NOs: 413, 416, 112, and 176 to 178, respectively, SEQ ID NOs: 413, 415, 112, 417, 418, and 178, respectively, SEQ ID NOs: 419, 422, 112, and 176 to 178, respectively, SEQ ID NOs: SEQ ID NOs: 419, 423, 112, and 176-178, respectively; SEQ ID NOs: 419, 424, 112, and 176-178, respectively; SEQ ID NOs: 419, 425, 112, and 176-178, respectively; SEQ ID NOs: 419, 426, 112, and 176-178, respectively; SEQ ID NOs: 419, 422, 112, 427, 177, and 178, respectively; SEQ ID NOs: 419, 422, 112, 428, 177, and 178, respectively; SEQ ID NOs: 419, 422, 112, 429, 177, and 178, respectively; 419, 422, 112, 430, 177, and 178, respectively, SEQ ID NOs: 419, 422, 112, 431, 177, and 178, respectively, SEQ ID NOs: 419, 422, 112, 432, 177, and 178, respectively, SEQ ID NOs: 419, 422, 112, 433, 177, and 178, respectively, SEQ ID NOs: 420, 422, 112, and 176-178, respectively, SEQ ID NOs: 421, 422, 112, and 176-178, respectively, or SEQ ID NOs: 421, 426, 112, 434, 177, and 178, respectively.

[0007] In some embodiments, the antigen binding domain comprises a VH and a VL, wherein the VH and VL are set forth in SEQ ID NOs: 363 and 364, respectively, SEQ ID NOs: 6 and 7, respectively, SEQ ID NOs: 8 and 9, 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, SEQ ID NOs: 48 and 49, respectively, SEQ ID NOs: 194 and 195, respectively, SEQ ID NOs: 196 and 197, respectively, SEQ ID NOs: 198 and 199, respectively, SEQ ID NOs: 200 and 201, respectively, SEQ ID NOs: 202 and 203, respectively, SEQ ID NOs: 204 and 205, respectively, SEQ ID NOs: 206 and 207, respectively, SEQ ID NOs: 208 and 209, respectively, SEQ ID NOs: 210 and 211, respectively, SEQ ID NOs: 212 and 213, respectively, SEQ ID NOs: 214 and 215, respectively, SEQ ID NOs: 216 and 217, respectively, SEQ ID NOs: 218 and 219, respectively, SEQ ID NOs: 220 and 221, respectively, SEQ ID NOs: 222 and 223, respectively, SEQ ID NOs: 224 and 225, respectively, SEQ ID NOs: 355 and 356, respectively, SEQ ID NOs: 357 and 358, respectively, SEQ ID NOs: 359 and 360, respectively, SEQ ID NOs: 361 and 362, respectively, SEQ ID NOs: 365 and 366, respectively, SEQ ID NOs: 367 and 368, respectively, SEQ ID NOs: 369 and 370, respectively, SEQ ID NOs: 371 and 372, respectively, SEQ ID NOs: 373 and 374, respectively, SEQ ID NOs: 375 and 376, respectively, SEQ ID NOs: 377 and 378, respectively, SEQ ID NOs: 379 and 380, respectively, SEQ ID NOs: 381 and 382, ​​respectively, SEQ ID NOs: 383 and 384, respectively, SEQ ID NOs: 385 and 386, respectively, SEQ ID NOs: 387 and 388, respectively, SEQ ID NOs: 389 and 390, respectively,The amino acid sequences of SEQ ID NOs: 391 and 392, respectively, SEQ ID NOs: 393 and 394, respectively, SEQ ID NOs: 395 and 396, respectively, SEQ ID NOs: 397 and 398, respectively, SEQ ID NOs: 399 and 400, respectively, SEQ ID NOs: 401 and 402, respectively, or SEQ ID NOs: 403 and 404, respectively, are at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequences of SEQ ID NOs: 391 and 392, respectively, SEQ ID NOs: 393 and 394, respectively, SEQ ID NOs: 395 and 396, respectively, SEQ ID NOs: 397 and 398, respectively, SEQ ID NOs: 399 and 400, respectively, SEQ ID NOs: 401 and 402, respectively, or SEQ ID NOs: 403 and 404, respectively.

[0008]

[0023] In some aspects, provided herein is an antigen binding domain that specifically binds to human CD98hc, comprising a VH and a VL, wherein the VH is selected from the group consisting of SEQ ID NOs: 363, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 194, 196, 198, 200, Antigen-binding domains are provided comprising the amino acid sequence of 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 355, 357, 359, 361, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 401, or 403.

[0009]

[0023] In some aspects, provided herein is an antigen binding domain that specifically binds to human CD98hc, comprising a VH and a VL, wherein the VL is selected from the group consisting of SEQ ID NOs: 364, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 195, 197, 199, 201, Antigen-binding domains are provided comprising the amino acid sequence of 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 356, 358, 360, 362, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 400, 402, or 404.

[0010] In some aspects, the antigen binding domains provided herein comprise a VH and a VL, each comprising the following amino acid sequences: SEQ ID NOs: 363 and 364, respectively; SEQ ID NOs: 6 and 7, respectively; SEQ ID NOs: 8 and 9, 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, SEQ ID NOs: 48 and 49, respectively, SEQ ID NOs: 194 and 195, respectively, SEQ ID NOs: 196 and 197, respectively, SEQ ID NOs: 198 and 199, respectively, SEQ ID NOs: 200 and 201, respectively, SEQ ID NOs: 202 and 203, respectively, SEQ ID NOs: 204 and 205, respectively, SEQ ID NOs: 206 and 207, respectively, SEQ ID NOs: 208 and 209, respectively, SEQ ID NOs: 210 and 211, respectively, SEQ ID NOs: 212 and 213, respectively, SEQ ID NOs: 214 and 215, respectively, SEQ ID NOs: 216 and 217, respectively, SEQ ID NOs: 218 and 219, respectively, SEQ ID NOs: 220 and 221, respectively, SEQ ID NOs: 222 and 223, respectively, SEQ ID NOs: 224 and 225, respectively, SEQ ID NOs: 355 and 356, respectively, SEQ ID NOs: 357 and 358, respectively, SEQ ID NOs: 359 and 360, respectively, SEQ ID NOs: 361 and 362, respectively, SEQ ID NOs: 365 and 366, respectively, SEQ ID NOs: 367 and 368, respectively, SEQ ID NOs: 369 and 370, respectively, SEQ ID NOs: 371 and 372, respectively, SEQ ID NOs: 373 and 374, respectively, SEQ ID NOs: 375 and 376, respectively, SEQ ID NOs: 377 and 378, respectively, SEQ ID NOs: 379 and 380, respectively, SEQ ID NOs: 381 and 382, ​​respectively, SEQ ID NOs: 383 and 384, respectively, SEQ ID NOs: 385 and 386, respectively, SEQ ID NOs: 387 and 388,SEQ ID NOs: 389 and 390, respectively, SEQ ID NOs: 391 and 392, respectively, SEQ ID NOs: 393 and 394, respectively, SEQ ID NOs: 395 and 396, respectively, SEQ ID NOs: 397 and 398, respectively, SEQ ID NOs: 399 and 400, respectively, SEQ ID NOs: 401 and 402, respectively, or SEQ ID NOs: 403 and 404, respectively.

[0011] In some embodiments, the antigen-binding domain is capable of crossing the blood-brain barrier (BBB). In some embodiments, the antigen-binding domain binds to CD98hc. In some embodiments, the antigen-binding domain is internalized into blood-brain barrier epithelial cells. In some embodiments, the blood-brain barrier epithelial cells are HCMEC / D3 cells.

[0012] In some embodiments, the antigen-binding domain binds to CD98hc with an affinity of 500 nM to 10 μM. In some embodiments, the antigen-binding domain binds to CD98hc with an affinity of 50 nM to 500 nM. In some embodiments, the antigen-binding domain binds to CD98hc with an affinity of 1 nM to 50 nM. In some embodiments, the antigen-binding domain binds to human CD98hc with an ELISA OD450 of at least 0.45 and / or binds to cynomolgus monkey CD98hc with an ELISA OD450 of at least 0.45. In some embodiments, the antigen-binding domain binds to CD98hc with an affinity of 3.1 nM to 210 nM. In some embodiments, the antigen-binding domain binds to CD98hc with an affinity of 3.2 nM to 1.5 μM. In some embodiments, the affinity is measured by high-throughput surface plasmon resonance (SPR) detection.

[0013] In some embodiments, the antigen binding domain does not decrease cell surface expression of CD98hc on HCMEC / D3 cells by more than 20% compared to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control, hi some embodiments, the antigen binding domain does not increase or decrease cell surface expression of CD98hc on HCMEC / D3 cells by more than 50% compared to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control.

[0014] In some embodiments, the antigen-binding domain accumulates in the brain of vascular-depleted mice at least 1.5-fold or at least 2-fold more than the isotype control, hi some embodiments, the antigen-binding domain exhibits at least a 5-fold increase in brain:serum concentration ratio compared to the isotype control 24 hours after administration to the mice.

[0015] In some embodiments, the antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 46 and 47, respectively, SEQ ID NOs: 367 and 368, respectively, SEQ ID NOs: 369 and 370, respectively, SEQ ID NOs: 371 and 372, respectively, SEQ ID NOs: 373 and 374, respectively, SEQ ID NOs: 375 and 376, respectively, SEQ ID NOs: 377 and 378, respectively, SEQ ID NOs: 379 and 380, respectively, SEQ ID NOs: 381 and 382, ​​respectively, SEQ ID NOs: 383 and 384, respectively, SEQ ID NOs: 385 and 386, SEQ ID NOs: 387 and 388, respectively, SEQ ID NOs: 389 and 390, respectively, SEQ ID NOs: 391 and 392, respectively, SEQ ID NOs: 393 and 394, respectively, SEQ ID NOs: 395 and 396, respectively, SEQ ID NOs: 397 and 398, respectively, SEQ ID NOs: 399 and 400, respectively, SEQ ID NOs: 401 and 402, respectively, or SEQ ID NOs: 403 and 404, respectively. In some embodiments, the antigen binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 14 and 15, respectively.

[0016] In some embodiments, the antigen-binding domain comprises a VH and a VL on a single polypeptide chain. In some embodiments, the antigen-binding domain comprises a single-chain fragment variable (scFv). In some embodiments, the scFv is oriented as VH-linker-VL. In some embodiments, the scFv is oriented as VL-linker-VH. In some embodiments, the linker is about 5 to about 25 amino acids, about 5 to about 20 amino acids, about 10 to about 25 amino acids, or about 10 to about 20 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 182) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 337). In some embodiments, the scFv comprises the amino acid sequence of any one of SEQ ID NOs: 318 to 336. In some embodiments, the antigen-binding domain comprises a VH on a first polypeptide and a VL on a second polypeptide. In some aspects, the antigen binding domain is a murine, chimeric, humanized, or human antigen binding domain. In some aspects, the antigen binding domain is a humanized antigen binding domain.

[0017] In some aspects, provided herein is an antigen-binding domain that specifically binds to human CD98hc, wherein the antigen-binding domain is (i) a VH CDR1, a VH CDR2, and a VH CDR3 of an antigen-binding domain provided herein, or (ii) a VHH comprising a VH of an antigen-binding domain provided herein, optionally wherein the VHH is capable of crossing the blood-brain barrier (BBB).

[0018] In some embodiments, provided herein are fusion proteins comprising an antigen-binding domain provided herein and a heterologous protein or peptide, such as beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, glucocerebrosidase B (GRB), ribosomal protein 1 (RIB), ribosomal protein 2 (RI ... idase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), glycoprotein non-transferase protein B (GPNMB), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 1 (SIG1), and sialic acid-binding Ig-like lectin 2 (SIG2). Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4-domain A4A (MS4A4A), transmembrane 4-domain A6A (MS4A6A), or transmembrane protein 106B (TMEM106b), clusterin (APOJ), reelin, ubiquitin protein ligase E3A (UBE3A), tripeptidyl peptidase Heterologous proteins or peptides are provided that include the amino acid sequences of CLN2 / TPP1, alpha-L-iduronidase (IDUA), iduronate 2-sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosamide N-acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), or N-sulfoglucosamine sulfohydrolase (SGSH), or portions thereof.

[0019] In some aspects, provided herein are antibodies comprising the antigen-binding domains provided herein. In some aspects, provided herein are antibodies or antigen-binding fragments thereof that bind to the same human CD98hc epitope as the antigen-binding domains provided herein. In some aspects, provided herein are antibodies or antigen-binding fragments thereof that competitively inhibit the binding of the antigen-binding domains provided herein to human CD98hc.

[0020] In some embodiments, provided herein are multispecific proteins comprising a first antigen-binding domain provided herein linked to a second antigen-binding domain. In some embodiments, the second antigen-binding domain specifically binds to a CNS antigen. In some embodiments, provided herein are multispecific proteins comprising an antigen-binding domain provided herein linked to an antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a CNS antigen. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region. In some embodiments, the antigen-binding domain provided herein is linked to the C-terminus of the heavy chain constant region, optionally via an amino acid linker. In some embodiments, the multispecific protein is bispecific. In some embodiments, the multispecific protein is bivalent, trivalent, or tetravalent. In some embodiments, the multispecific protein is bivalent.

[0021] In some aspects, provided herein are multispecific proteins that are trivalent, optionally wherein the trivalent protein comprises one antigen-binding domain that binds to human CD98hc and two antigen-binding domains that bind to a CNS antigen.

[0022] In some embodiments, a multispecific protein is provided, wherein the multispecific protein is tetravalent, optionally wherein the tetravalent protein comprises two antigen-binding domains that bind to human CD98hc and two antigen-binding domains that bind to a CNS antigen.

[0023] In some aspects, provided herein are multispecific proteins that are trivalent and bispecific and comprise an antigen-binding domain provided herein linked to an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, and the antigen-binding domain is linked to the C-terminus of one of the two antibody heavy chains in an scFv, optionally via an amino acid linker.

[0024] In some aspects, provided herein is a multispecific protein that is tetravalent and bispecific, comprising two antigen-binding domains provided herein and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, each of the two antigen-binding domains is an scFv, Fab, or VHH, and wherein one of the two antigen-binding domains is linked, optionally via an amino acid linker, to the C-terminus of one antibody heavy chain, and the other of the antigen-binding domains is linked, optionally via an amino acid linker, to the C-terminus of the other antibody heavy chain.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof comprises a constant region comprising a knob mutation and a constant region comprising a hole mutation. In some embodiments, the antigen-binding domain is linked to the constant region comprising the hole mutation, optionally via an amino acid linker. In some embodiments, the antigen-binding domain is linked to the constant region comprising the knob mutation, optionally via an amino acid linker. In some embodiments, the amino acid linker is a glycine-serine linker, optionally comprising the amino acid sequence (GGGGS)x3 (SEQ ID NO: 183). In some embodiments, the amino acid linker is a glycine-serine linker, optionally comprising the amino acid sequence (GGSGG)x3 (SEQ ID NO: 338).

[0026] In some embodiments, the CNS antigen is a brain antigen. In some embodiments, the CNS antigen is not CD98hc.

[0027] In some embodiments, 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. In some embodiments, 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 knob mutations and mutations that reduce effector function, wherein the mutations that reduce effector function include (i) L234A, L235A, and / or P331S and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S.

[0028] In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof. In some embodiments, the IgG antibody or antigen-binding fragment thereof is an IgG1 antibody or antigen-binding fragment thereof or an IgG4 antibody or antigen-binding fragment thereof.

[0029] In some embodiments, the multispecific protein binds to human CD98hc with an equilibrium dissociation constant (KD) of about 3 nM to about 225 nM and / or binds to cynomolgus CD98hc with a KD of about 3 nM to about 225 nM.

[0030] In some embodiments, the multispecific protein is internalized into blood-brain barrier epithelial cells at a greater than 10-fold increase compared to internalization by an isotype control, and optionally the blood-brain barrier epithelial cells are HCMEC / D3 cells. In some embodiments, the multispecific protein does not reduce cell surface expression of CD98hc on HCMEC / D3 cells by more than 20% compared to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. In some embodiments, the multispecific protein does not increase cell surface expression of CD98hc on HCMEC / D3 cells by more than 50% compared to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. In some embodiments, the multispecific protein accumulates in vascular-depleted mouse brains at least 1.5-fold or at least 2-fold more than an isotype control. In some embodiments, the multispecific protein increases the brain:serum concentration ratio by at least 5-fold compared to an isotype control 24 hours after administration to mice.

[0031] In some embodiments, the CNS antigen is beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, beta-glucocerebromide (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), trigger receptor expressed in myeloid cells, or a combination thereof. receptor 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), sialic acid-binding Ig-like lectin 11 (Siglec11), glycoprotein non-transferase protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4-domain A4A (MS4A4A), transmembrane 4-domain A6A (MS4A6A), MS4A4E, transmembrane protein 106B (TMEM106b), ubiquitin protein ligase E3A (UBE3A), CR1, ABCA1, ABCA7, HLA-DR1, HLA-DR5, IL1RAP, TREML2, IL-34, SORL1, or ADAM1. In some embodiments, the CNS antigen is MS4A4A, and optionally, (i) the antigen binding domain, antibody, or antigen binding domain that binds MS4A4A comprises a VH comprising the amino acid sequence of SEQ ID NO: 407 and / or a VL comprising the VL sequence of SEQ ID NO: 405, and / or (ii) the antigen binding domain that binds human CD98hc comprises the amino acid sequence of SEQ ID NO: 316.

[0032] In some embodiments, the multispecific protein comprises the amino acid sequence of SEQ ID NOs: 405-410.

[0033] In some embodiments, the fusion proteins provided herein, the antibodies or antigen-binding fragments thereof, or the multispecific proteins provided herein are capable of crossing the BBB. In some embodiments, the fusion proteins provided herein, the antibodies or antigen-binding fragments thereof, or the multispecific proteins provided herein are linked to an imaging agent.

[0034] In some aspects, provided herein are compositions comprising a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode a multispecific protein provided herein, wherein the first polynucleotide encodes a first heavy chain, the second polynucleotide encodes a second heavy chain and an antigen-binding domain that specifically binds to human CD98hc, and the third polynucleotide encodes a light chain.

[0035] In some aspects, provided herein are compositions comprising a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides 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 CD98hc, the second polynucleotide encodes a second heavy chain and a second antigen-binding domain that specifically binds to human CD98, and the third polynucleotide encodes a light chain, and optionally, the first and second antigen-binding domains that bind to human CD98hc comprise the same amino acid sequence.

[0036] In some embodiments, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation.

[0037] In some embodiments, the ratio of the first, second, and third polynucleotides is about 1:3:6.

[0038] In some embodiments, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0039] In some embodiments, provided herein are compositions comprising a first polynucleotide and a second polynucleotide, wherein the first and second polynucleotides encode a multispecific protein provided herein, wherein the first polynucleotide encodes a heavy chain and an antigen-binding domain that binds to human CD98hc, and the second polynucleotide encodes a light chain.

[0040] In some aspects, provided herein are host cells comprising the compositions provided herein.

[0041] In some aspects, provided herein is an isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain of an antigen-binding domain provided herein. In some aspects, provided herein is an isolated polynucleotide comprising a nucleic acid molecule encoding the light chain variable region of an antigen-binding domain provided herein.

[0042] In some aspects, provided herein is an isolated vector comprising a polynucleotide provided herein. 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 provided herein.

[0043] In some aspects, provided herein is a host cell comprising a polynucleotide provided herein or a vector provided herein. In some aspects, the host cell is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS1, COS7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture.

[0044] In some aspects, provided herein are methods of producing an antigen-binding domain or a multispecific protein, comprising producing the antigen-binding domain or multispecific protein by culturing a host cell provided herein, and optionally further comprising isolating the antigen-binding domain or multispecific protein from the culture.

[0045] In some aspects, provided herein is an isolated antigen-binding domain or multispecific protein thereof produced by the methods provided herein.

[0046] In some aspects, provided herein are pharmaceutical compositions comprising (i) an antigen-binding domain, fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein, and (ii) a pharmaceutically acceptable carrier. In some aspects, the concentration of the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein is increased in the brain after administration to a subject compared to an isotype control. In some aspects, the administration increases delivery of the fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition into the brain by at least 50%, at least 100%, at least 200%, at least 500%, or at least 1000% compared to an isotype control.

[0047] In some aspects, provided herein are methods of treating a neurological disease or disorder in a subject, comprising administering to the subject a fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition provided herein. In some aspects, the administration increases delivery of the fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition into the brain by at least 50%, at least 100%, at least 200%, at least 500%, or at least 1000% compared to an isotype control. In some aspects, the 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. In some aspects, the neurological disease or disorder is selected from a neuropathic disorder, a neurodegenerative disease, a cancer, an ocular disorder, a seizure disorder, a lysosomal storage disorder, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and inflammation of the CNS. In some aspects, the neurological disease or disorder is selected from Alzheimer's disease (AD), Huntington's disease, dystonia, ataxia, Bell's palsy, stroke, dementia, dementia with Lewy bodies, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, encephalitis, traumatic brain injury, and limbic-predominant age-related TDP-43 encephalopathy (LATE). In some aspects, the dementia is frontotemporal dementia (FTD). In some embodiments, the neurological disease or disorder is Alzheimer's disease. In some aspects, the Alzheimer's disease is early-onset Alzheimer's disease, prodromal Alzheimer's disease, mild Alzheimer's disease, or late-onset Alzheimer's disease. In some embodiments, the neurological disease or disorder is Parkinson's disease. In some embodiments, the neurological disease or disorder is frontotemporal epilepsy. In some embodiments, the neurological disease or disorder is autism. In some embodiments, the neurological disease or disorder is lissencephaly.

[0048] In some aspects, provided herein are methods of treating a lysosomal storage disease in a subject, comprising administering a fusion protein 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.

[0049] In some embodiments, provided herein are methods for transporting a fusion protein, an antibody or antigen-binding fragment thereof, or a multispecific protein across the BBB 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 provided herein. In some embodiments, the concentration of the fusion protein, the antibody or antigen-binding fragment thereof, or the multispecific protein is increased in the brain after administration compared to an isotype control. In some embodiments, the concentration of the fusion protein, the antibody or antigen-binding fragment thereof, the multispecific protein, or the 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. In some embodiments, administration of the fusion protein, the antibody or antigen-binding fragment thereof, or the multispecific protein does not result in more than a 10% decrease in the subject's reticulocyte count compared to administration of an isotype control. In some embodiments, administration of the fusion protein, the antibody or antigen-binding fragment thereof, or the multispecific protein does not result in a decrease in the subject's reticulocyte count compared to an isotype control.

[0050] In some aspects, provided herein are methods of increasing the concentration of a CNS-binding antigen in the CSF of a subject, comprising administering to the subject a multispecific protein provided herein, wherein the concentration of the CNS-binding antigen is increased compared to administering the CNS-binding antigen alone to the subject.

[0051] In some aspects, provided herein are methods of imaging a CNS antigen in a subject, the method comprising administering to the subject a fusion protein, an antibody or antigen-binding fragment thereof, or a multispecific protein provided herein, and locating an imaging agent within the subject.

[0052] In some aspects, provided herein are methods of detecting a CNS antigen in vitro, comprising contacting an in vitro sample with a fusion protein, antibody, or multispecific protein provided herein, and localizing an imaging agent in the sample.

[0053] In some aspects, provided herein is the use of a fusion protein, an antibody or antigen-binding fragment thereof, or a multispecific protein, or a composition provided herein in a method provided herein.

[0054] In some aspects, provided herein are fusion proteins, antibodies or antigen-binding fragments thereof, multispecific proteins, or compositions for use in the methods provided herein.

[0055] It will be understood that one, some, or all of the features of the various aspects described herein may be combined to form other aspects of the present disclosure. These and other aspects of the present disclosure will be readily apparent to those skilled in the art. These and other embodiments of the present disclosure are further described in the detailed description that follows. [Brief explanation of the drawings]

[0056] [Figure 1A] 2+1 bispecific antibodies are shown. [Figure 1B] 2+2 bispecific antibodies having two scFvs with the same amino acid sequence. [Figure 1C] 2 shows a 2+2 bispecific antibody having two scFvs with different amino acid sequences. [Figure 2]The fold change in CD98hc surface levels on hCMEC / D3 cells after treatment with 2+1 anti-CD98hc bispecific antibodies compared to untreated cells is shown, as assessed by FACS (see Example 16). [Figure 3] 1 shows total CD98hc protein levels in hCMEC / D3 cells after treatment with 2+1 anti-CD98hc bispecific antibody as assessed by Western blot (see Example 16). [Figure 4] Figure 1 shows brain penetration of 2+1 anti-CD98hc bispecific antibodies in hCD98hc+ / - mice, as measured by antibody levels in vascular-depleted brains 24 hours after peripheral injection. Antibody CD98hc.04.064 is increased approximately 2-fold compared to controls. Antibody CD98hc.04.063 is increased approximately 1.5-fold compared to controls (see Example 17). [Figure 5] 1 shows serum PK of 2+1 anti-CD98hc bispecific antibodies in hCD98hc+ / - mice. Brain-penetrating antibodies have the highest serum clearance rate (see Example 17). [Figure 6] FIG. 1 shows the brain / serum ratio of 2+1 anti-CD98hc bispecific antibody in hCD98hc+ / − mice 24 hours after peripheral injection (see Example 17). [Figure 7] FIG. 1 shows the moderate antibody-dependent cellular cytotoxicity (ADCC) response of 2+1 anti-CD98hc bispecific antibodies against BBB cell lines (see Example 20). [Figure 8]

[0033] Figure 2 shows antibody levels in the blood vessel-depleted brain fraction of huCD98hc knock-in mice after administration of a 2+1 anti-CD98hc bispecific antibody and a matched control (having the same Fab and Fc domains but lacking the scFv that specifically binds to huCD98hc). Antibody levels are shown as fold change relative to the matched control (see Example 22). [Figure 9]FIG. 1 shows antibody levels in the serum of huCD98hc knock-in mice after administration of 2+1 anti-CD98hc bispecific antibody and a matched control (having the same Fab and Fc domains but no scFv that specifically binds to huCD98hc) (see Example 22). [Figure 10] FIG. 1 shows the activity of 2+1 anti-CD98hc bispecific antibodies in an in vitro sTREM2 assay (see Example 24). [Figure 11] Absolute reticulocyte counts in non-human primates (NHPs) following administration of 2+1 anti-CD98hc bispecific antibody (see Example 26). [Figure 12] Serum and CSF levels of 2+1 anti-CD98hc bispecific antibody after the first and second doses (see Example 27). [Figure 13] 1 shows antibody concentrations in NHP brain fractions following administration of 2+1 anti-CD98hc bispecific antibody (see Example 28). [Figure 14] 1 shows the levels of soluble TREM2 in serum and CSF of NHPs after administration of 2+1 anti-CD98hc bispecific antibody (see Example 29). [Figure 15] 1 shows the levels of CSF-1 in the CSF of NHPs following administration of 2+1 anti-CD98hc bispecific antibody (see Example 29). DETAILED DESCRIPTION OF THE INVENTION

[0057] Detailed Description of the Disclosure The present disclosure relates to antigen-binding domains that specifically bind to human CD98 heavy chain (CD98hc), as well as antibodies and antigen-binding fragments thereof comprising such antigen-binding domains, methods of making and using such antigen-binding domains, antibodies, and antigen-binding fragments thereof, pharmaceutical compositions comprising such antigen-binding domains, antibodies, and antigen-binding fragments thereof, nucleic acids encoding such antigen-binding domains, antibodies, and antigen-binding fragments thereof, and host cells comprising nucleic acids encoding such antigen-binding domains, antibodies, and antigen-binding fragments thereof.

[0058] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are commonly employed by those skilled in the art using conventional methods, for example, widely used methods such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M.A.usubel, et al. eds., (2003); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000).

[0059] definition The terms "central nervous system" and "CNS" refer to the complex of nervous tissue that controls bodily functions and includes the brain and spinal cord.

[0060] The terms "blood-brain barrier" and "BBB" refer to a network of brain capillary endothelial cells that are tightly sealed by tight junctions and characterized by low levels of nonspecific paracellular and transcellular transport.

[0061] A "central nervous system antigen" or "CNS antigen" is an antigen expressed in the CNS, including the brain, that can be targeted by an antibody or small molecule. Examples of such antigens include, but are not limited to, beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebroidase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interleukin 6 receptor (IL6), and the like. R), TNF receptor 1 (TNFR1), interleukin 1β (IL1(3)), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), glycoprotein nonmetastatic melanoma protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4-domain A4A (MS4A4A), transmembrane 4-domain A6A (MS4A6A), ubiquitin protein ligase E3A (UBE3A), or transmembrane protein 106B (TMEM106b).

[0062] A "brain antigen" is a CNS antigen that is expressed in the brain.

[0063] The terms "CD98hc," "CD98hc polypeptide," and "CD98hc protein" are used interchangeably herein and refer to any native CD98hc from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (cyno)) and rodents (e.g., mice and rats), unless otherwise specified. CD98hc is also referred to as 4F2 cell surface antigen heavy chain, 4F2hc, 4F2 heavy chain antigen, lymphocyte activation antigen 4F2 large subunit, solute carrier family 3 member 2, and CD98. The CD98hc protein is encoded by the SLC3A2 gene and is part of the large amino acid transporter (LAT) complex. In some embodiments, the terms encompass both wild-type and naturally occurring variant sequences, such as splice or allelic variants. In some embodiments, the term encompasses "full-length," unprocessed CD98hc, as well as any form of CD98hc that results from processing within the cell. In some embodiments, the CD98hc is human CD98hc. As used herein, the term "human CD98hc" refers to a polypeptide having the amino acid sequence of SEQ ID NO:435. (SEQ ID NO: 435)

[0064] The term "antibody" refers to an immunoglobulin molecule that recognizes and binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, via at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term "antibody" encompasses monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific (e.g., bispecific) antibodies, and any other immunoglobulin molecule so long as it exhibits the desired biological activity. Antibodies can be of any of five major immunoglobulin classes—IgA, IgD, IgE, IgG, and IgM—or their subclasses (isotypes) (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2)—based on the type of heavy chain constant region, designated alpha, delta, epsilon, gamma, and mu, respectively. Different classes of antibodies have distinct and well-known subunit structures and three-dimensional structures. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0065] The terms "anti-CD98hc antibody," "antibody that binds to CD98hc," and "antibody that specifically binds to CD98hc" refer to an antibody that can bind to CD98hc with sufficient affinity such that the antibody is useful in targeting CD98hc as a diagnostic and / or therapeutic agent. In one embodiment, the extent of binding of an anti-CD98hc antibody to an unrelated, non-CD98hc polypeptide is less than about 10% of the binding of the antibody to CD98hc, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to CD98hc has an affinity of less than 10 μM, less than 1 μM, less than 100 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 0.01 nM, or less than 0.001 nM (e.g., less than 10 -8 M or less, e.g., 10 -8 M~10-13 M, e.g., 10 -9 M~10 -13 M) In certain embodiments, the anti-CD98hc antibody binds to an epitope of CD98hc that is conserved among CD98hc from different species.

[0066] The term "antibody fragment" refers to a portion of an antibody. An "antigen-binding fragment" refers to a portion of an antibody that binds to an antigen. An antigen-binding fragment of an antibody may include an antigenic determining region of the antibody (e.g., a complementarity-determining region (CDR)). Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. An antigen-binding fragment of an antibody may be monovalent or multivalent (e.g., bivalent). An antigen-binding fragment of an antibody may be monospecific or multispecific (e.g., bispecific). An antigen-binding fragment of an antibody may be derived from any animal species, such as, for example, rodents (e.g., mice, rats, or hamsters) and humans, or may be artificially generated.

[0067] "Antigen-binding fragment" or "antigen-binding region" refers to a monovalent portion of an antibody that binds to an antigen. An "antigen-binding domain" may comprise an antigenic determining region of an antibody (e.g., a complementarity determining region (CDR)). Antibodies or antigen-binding fragments thereof (including monospecific and multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof) may comprise an antigen-binding domain.

[0068] The terms "anti-CD98hc antigen-binding domain," "antigen-binding domain that binds to CD98hc," "anti-CD98hc antigen-binding region," "antigen-binding region that binds to CD98hc," and "CD98hc binding domain" refer to an antigen-binding domain that binds to CD98hc with sufficient affinity such that the antigen-binding domain is useful in targeting CD98hc and / or is useful as a diagnostic and / or therapeutic agent or in transporting molecules or compounds across the BBB. In one aspect, the extent of binding of an anti-CD98hc antigen-binding domain to an unrelated, non-CD98hc polypeptide is less than about 10% of the binding of the antigen-binding domain to CD98hc, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, antibodies that bind to CD98hc have a binding affinity of less than 0.1 μM, less than 1 μM, less than 10 μM, less than 100 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 0.01 nM, or less than 0.001 nM (e.g., less than 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 In certain embodiments, the anti-CD98hc antigen-binding domain binds to an epitope of CD98hc that is conserved among CD98hc from different species.

[0069] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, whole antibodies include those having heavy and light chains, including the Fc region. The constant regions may be native-sequence constant regions (e.g., human native-sequence constant regions) or amino acid sequence variants thereof. In some cases, intact antibodies may have one or more effector functions.

[0070] "Native IgG antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains at one end a variable domain (V H ), followed by several constant domains. Each light chain has at one end a variable domain (V L ) and a constant domain at the other end, with the light-chain constant domain aligned with the first constant domain of the heavy chain, and the light-chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain and heavy-chain variable domains.

[0071] Papain digestion of antibodies yields two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, a designation reflecting its tendency to crystallize easily. The Fab fragment contains the entire light chain plus the variable region domain (V) of one heavy chain. H ) and the first constant domain of the heavy chain (C H 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of antigen cross-linking. The Fab' fragment contains one or more cysteines from the antibody hinge region, C H F(ab')2 antibody fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of one domain. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) in the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical bonds for antibody fragments are also known.

[0072] The Fc fragment contains the carboxy-terminal portions of both heavy chains linked by disulfides. The effector functions of the antibody are determined by sequences within the Fc region, which is also recognized by Fc receptors (FcRs) found on certain types of cells.

[0073] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. The folding of these two domains generates six hypervariable loops (three loops each from the H and L chains) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to antigen, albeit with lower affinity than the entire binding site.

[0074] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising a VH and VL antibody domain linked as a single polypeptide chain. In some embodiments, the scFv polypeptide comprises a VH and VL antibody domain linked as a single polypeptide chain. H Domains and V L It further comprises a polypeptide linker between the domains which enables the sFv to form the desired structure for antigen binding.

[0075] The term "diabody" refers to a V diabody in which inter-chain (rather than intra-chain) pairing of variable domains is achieved. H Domain and V L These refer to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the domains, thereby obtaining bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific diabodies are small antibody fragments prepared by combining the V of two antibodies. H and V L It is a heterodimer of two "crossover" sFv fragments in which the domains are present on different polypeptide chains.

[0076] As used herein, a "2+1 antibody format" refers to a trivalent, bispecific antibody format comprising (i) a single antigen-binding domain that binds to human CD98hc, and (ii) an antibody comprising two heavy chains and two light chains, wherein the single antigen-binding domain that binds to human CD98hc is linked to the C-terminus of one of the two antibody heavy chains. This format is illustrated in Figure 1A.

[0077] As used herein, a "2+2 antibody format" refers to a tetravalent, bispecific antibody format comprising (i) two antigen-binding domains that bind to human CD98hc and (ii) an antibody, wherein the antibody comprises two heavy chains and two light chains, and wherein one of the antigen-binding domains that bind to human CD98hc is linked to the C-terminus of one of the two antibody heavy chains and the other of the antigen-binding domains that bind to human CD98hc is linked to the C-terminus of the other of the two antibody heavy chains. The two antigen-binding domains that bind to human CD98hc may comprise the same amino acid sequence. This format is shown in Figure 1B. In some embodiments, the two scFv antigen-binding domains that bind to human CD98hc may comprise different amino acid sequences. This format is shown in Figure 1C.

[0078] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a light or heavy chain, typically consisting of approximately 110-120 or 110-125 amino acids at the amino terminus of a mature heavy chain and approximately 90-115 amino acids in a mature light chain, which differs significantly in sequence among antibodies and is responsible for the binding and specificity of a particular antibody to a particular antigen. While sequence variability is concentrated in regions called complementarity-determining regions (CDRs), the more highly conserved regions of the variable domain are called framework regions (FRs). While not wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of an antibody with an antigen. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In some embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs). "Kabat numbering" and like terms are art-recognized and refer to a system for numbering amino acid residues within the heavy and light chain variable regions of an antibody or antigen-binding fragment thereof. In certain embodiments, CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).Using the Kabat numbering system, the CDRs in an antibody heavy chain molecule are typically located at amino acids 31-35 (optionally including one or two additional amino acids after 35, designated 35A and 35B in the Kabat numbering scheme) (CDRH1), 50-65 (CDRH2), and 95-102 (CDRH3). Using the Kabat numbering system, the CDRs in an antibody light chain molecule are typically located at amino acids 24-34 (CDRL1), 50-56 (CDRL2), and 89-97 (CDRL3). In some embodiments, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme. Chothia instead refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDRH1 loop, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the loop (this is due to the insertions at H35A and H35B in the Kabat numbering scheme: if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions are a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. In some embodiments, the CDRs may also be "Contact" CDRs. The "Contact" CDRs are based on analysis of available complex crystal structures. Residues from each of these CDRs are listed below. TIFF2025528751000001.tif54148

[0079] Each CDR may comprise the following "extended CDRs": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) for VL, and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) for VH. The variable domain residues are numbered according to Kabat et al. (supra) for each of these extended CDR definitions.

[0080] The terms "VH" and "VH domain" are used interchangeably and refer to the heavy chain variable region of an antibody.

[0081] As used herein, the term "heavy chain," when used in reference to an antibody, can refer to any of the distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant region that gives rise to the IgA, IgD, IgE, IgG, and IgM antibody classes (including IgG subclasses, e.g., IgG1, IgG2, IgG3, and IgG4), respectively. Heavy chain amino acid sequences are well known in the art. In some embodiments, the heavy chain is a human heavy chain.

[0082] The terms "VL" and "VL domain" are used interchangeably and refer to the light chain variable region of an antibody.

[0083] As used herein, the term "light chain," when used in reference to an antibody, can refer to either distinct type based on the amino acid sequence of the constant region, e.g., kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In some embodiments, the heavy chain is a human light chain.

[0084] As used herein, the term "constant region" refers to a region of an antibody that is not the variable region of the antibody, e.g., the carboxyl-terminal portions of the light and / or heavy chains that are not directly involved in binding the antibody to an antigen but can exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a highly conserved amino acid sequence compared to the immunoglobulin variable domain. In certain embodiments, the antibody or antigen-binding fragment comprises a constant region or portion thereof sufficient for antibody-dependent cellular cytotoxicity (ADCC).

[0085] By "constant domain" is meant a domain within the constant region that is capable of forming an immunoglobulin fold. Constant domains include CH1, CH2, CH3, and CL domains.

[0086] The term "monoclonal," when referring to antibodies or antigen-binding fragments thereof, refers to a population of homogeneous antibodies or antigen-binding fragments involved in highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies directed against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof encompasses not only intact, full-length monoclonal antibodies, but also antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) variants, fusion proteins containing antibodies or antibody portions, and any other immunoglobulin molecule containing an antigen-recognition site. Furthermore, "monoclonal" antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof produced by any method, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0087] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequences are derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of an antibody or antigen-binding fragment thereof from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and function, while the constant regions are homologous to the sequences of an antibody or antigen-binding fragment thereof from another species (usually human) to avoid eliciting an immune response in that species.

[0088] The term "humanized" antibody or antigen-binding fragment thereof refers to a non-human (e.g., murine) antibody or antigen-binding fragment that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof that contains minimal non-human (e.g., murine) sequence. Typically, humanized antibodies or antigen-binding fragments thereof are human immunoglobulins in which residues from their complementarity-determining regions (CDRs) have been replaced ("CDR-grafted") by residues from a CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and function (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). Humanized antibodies or antigen-binding fragments thereof can be further modified by substitution of additional residues within the Fv framework regions and / or the replaced non-human residues to improve and optimize the specificity, affinity, and / or function of the antibody or antigen-binding fragment. Generally, a humanized antibody or antigen-binding fragment thereof comprises a VH and VL region that contains substantially all of at least one, and usually two or three, of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are those of a human immunoglobulin consensus region. A humanized antibody or antigen-binding fragment thereof can also comprise at least a portion of an immunoglobulin constant region or Fc region, typically that of a human immunoglobulin. Examples of methods used to make humanized antibodies are described in U.S. Patent No. 5,225,539, Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904(1996). In some embodiments, the antibody is a resurfaced antibody.

[0089] The term "human" antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin locus, and such antibodies or antigen-binding fragments are made using any technique known in the art. This definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0090] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as herein defined.

[0091] As used herein, an "acceptor human framework" refers to a V-type nucleotide sequence derived from a human immunoglobulin framework or a human consensus framework. L Framework or V H A framework comprising the amino acid sequence of a framework. An acceptor human framework "derived" from a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. If pre-existing amino acid changes are present in the VH, in some embodiments, those changes occur at only three, two, or one of positions 71H, 73H, and 78H, e.g., the amino acid residues at those positions may be 71A, 73T, and / or 78A. In some embodiments, a VL acceptor human framework is derived from a VL. L The sequence is identical to a human immunoglobulin framework sequence or a human consensus framework sequence.

[0092] The "human consensus framework" is based on human immunoglobulin V L or V H A framework that represents the most commonly occurring amino acid residues in a selection of framework sequences. Generally, human immunoglobulin V L or V HThe selection of sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). For example, V L For example, the subgroup may be subgroup kappa I, kappa II, kappa III, or kappa IV, such as those of Kabat et al., supra. H For example, the subgroup may be subgroup I, subgroup II, or subgroup III, such as in Kabat et al., supra.

[0093] An "amino acid modification" at a specified position (e.g., of an antibody of the present disclosure) refers to a substitution or deletion of the specified residue, or an insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent to" a particular residue means an insertion within 1-2 residues thereof. The insertion can be N-terminal or C-terminal to the particular residue. In some embodiments, the amino acid modification is a substitution.

[0094] Antibody "effector functions" refer to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or amino acid sequence variant Fc region), and vary with the antibody isotype.

[0095] The term "Fc region" or "fragment crystallizable region" is used herein to define the C-terminal region of an immunoglobulin heavy chain and includes native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from Pro230 to the carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, an intact antibody composition can include an antibody population in which all K447 residues have been removed, an antibody population in which the K447 residue has not been removed, and an antibody population having a mixture of antibodies with and without the K447 residue. Native-sequence Fc regions suitable for use in the antibodies of the present disclosure include human IgG1, IgG2, IgG3, and IgG4.

[0096] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes), native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0097] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, and in some embodiments, one or more amino acid substitution(s). In some embodiments, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about 1 to about 10 amino acid substitutions in the native-sequence Fc region or in the Fc region of the parent polypeptide, and in some embodiments, preferably about 1 to about 5 amino acid substitutions. In some embodiments, the variant Fc region has at least 80% homology, at least 90% homology, or at least 95% homology to the native-sequence Fc region and / or the Fc region of the parent polypeptide.

[0098] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native-sequence human FcR. In some embodiments, the FcR binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses (including allelic variants and alternatively spliced ​​forms of these receptors). FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences but differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif ("ITAM") in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. FcRs can also extend the serum half-life of antibodies.

[0099] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or its antigen-binding fragment) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity," as used herein, refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or its antigen-binding fragment and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant ( KD Affinity can be expressed by, but not limited to, the equilibrium dissociation constant (K D ) and the equilibrium binding constant (K A ) can be measured and / or expressed in many ways well known in the art. D is k off / k on It is calculated from the quotient of K A is k on / k off It is calculated from the quotient of k on refers to, for example, the binding rate constant of an antibody or its antigen-binding fragment with an antigen, and k off k refers to, for example, the dissociation rate constant of an antibody or antigen-binding fragment thereof from an antigen. on and k off can be determined by techniques well known to those skilled in the art, such as BIAcore® or KinExA.

[0100] With respect to the binding of an antibody to a target molecule, the terms "specific binding" or "specifically binds to" or "is specific for" a particular polypeptide or epitope on a particular polypeptide target refer to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target, e.g., an excess of unlabeled target. In this case, specific binding is indicated when binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. The terms "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular polypeptide target, as used herein, refer to, for example, binding of an antibody to a target having a KD of about 10 -4 M or less, 10 -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less or KD is 10 -4 M~10 -6 M or 10 -6 M~10 -10 M or 10 -7 M~10 -9 M. As will be appreciated by those skilled in the art, affinity and Kd value are inversely proportional. High affinity for an antigen is measured by a low KD value. In some embodiments, the term "specific binding" refers to the binding of a molecule to a specific polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0101] The term "linker" or "linked" refers to a covalent bond between two polypeptides or two heterologous molecules. In some embodiments, the linker is a chemical linker. In some embodiments, the linker comprises a peptide bond, and the two polypeptides or two heterologous molecules are linked to each other directly or via one or more additional amino acids. A glycine linker is one that contains one or more glycines but no other amino acids, e.g., GGGG (SEQ ID NO: 351). A glycine-rich linker is one that contains one or more glycines and can contain other amino acids as long as glycine is the predominant species of the linker, e.g., GGGNGG (where N is any amino acid) (SEQ ID NO: 352). A glycine-serine linker is a linker that contains both glycine and serine in any proportion, e.g., GGGS (SEQ ID NO: 353). Similarly, a proline linker is one that contains one or more prolines but no other amino acids. A proline-rich linker is one that contains one or more prolines and can contain other amino acids as long as proline is the predominant species of the linker.

[0102] As used herein, "percent amino acid sequence identity" and "homology," with respect to peptide, polypeptide, or antibody sequences, are defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a particular peptide or polypeptide sequence after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of those in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art that are required to achieve maximal alignment over the full length of the sequences being compared.

[0103] The term "epitope" includes any determinant capable of binding by an antibody. An epitope is the region of an antigen bound by an antibody targeting that antigen, and, if the antigen is a polypeptide, includes specific amino acids that make direct contact with the antibody. In most cases, epitopes reside on polypeptides, but in some cases, they may reside on other types of molecules, such as nucleic acids. Epitopes may have chemically active molecular surface groups, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen selectively recognize epitopes on the target antigen in a complex mixture of polypeptides and / or macromolecules.

[0104] An antibody that "binds to the same epitope" as a reference antibody refers to an antibody that contacts the same amino acid residues on the antigen as the reference antibody. The ability of an antibody to bind to the same epitope as a reference antibody can be determined using peptide scanning mutagenesis or high-throughput alanine scanning mutagenesis. In the latter method, a comprehensive mutation library of an antigen or a portion thereof (e.g., the extracellular domain) can be generated by mutating each individual amino acid residue to alanine (or, if the amino acid residue is alanine, to another residue such as serine) and testing each mutant for binding to the target antibody or its antigen-binding fragment.

[0105] An antibody is said to "competitively inhibit" the binding of a reference antibody to a particular epitope if the antibody selectively binds to that epitope or an overlapping epitope, thereby blocking the binding of the reference antibody to that epitope to some extent. Competitive inhibition can be measured by any method known in the art, such as, for example, a competitive ELISA assay. An antibody can be said to competitively inhibit the binding of the reference antibody to a particular epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0106] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are not in a form in which they are found in nature. In some aspects, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0107] As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0108] The term "expression system" refers to one or more nucleic acid molecules comprising operably linked coding and control sequence(s), and a host cell and / or other in vitro transcription and translation machinery, such that one or more proteins encoded by the nucleic acid molecule(s) are produced.

[0109] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, into which additional DNA segments can be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" or simply, "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Since the plasmid is the most commonly used form of vector, the terms "plasmid" and "vector" may be used interchangeably herein.

[0110] "Polynucleotide" or "nucleic acid," used interchangeably herein, refer to a polymer of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or a synthetic reaction.

[0111] A "host cell" includes an individual cell or cell culture that can be or has been the recipient of a vector(s) for incorporating a polynucleotide insert. A host cell includes the progeny of a single host cell, although the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of the invention.

[0112] As used herein, a "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed at the dosages and concentrations employed.

[0113] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of a treated individual's clinical pathology. Desirable effects of treatment include a reduction in the rate of progression, an improvement or alleviation of the pathology, and an improvement in the remission or prognosis of a particular disease, disorder, or condition. For example, an individual is effectively "treated" if one or more symptoms associated with a particular disease, disorder, or condition are reduced or eliminated.

[0114] As used herein, the terms "administer," "administering," "administration," and the like refer to methods that can be used to deliver an agent, e.g., an anti-human antibody or antigen-binding fragment thereof, to a desired site of biological action.

[0115] The term "effective amount" refers to an amount effective, at least at the dosage and for the duration necessary, to achieve a desired therapeutic result. An effective amount may be provided in one or more administrations. An effective amount is also an amount in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. In therapeutic applications, beneficial or desired results include clinical results, such as a reduction in one or more symptoms attributable to the disease, an improvement in the quality of life of the person suffering from the disease, a reduction in the dosage of other drugs required to treat the disease, e.g., augmentation of other drug therapies by targeting, a delay in disease progression, and / or an extension of survival. An effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve a therapeutic treatment directly or indirectly. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of the administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result can be or is achieved.

[0116] As used herein, the terms "subject" and "patient" are used interchangeably. A subject can be a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey, or other primate, etc.). In some embodiments, the subject is a cynomolgus monkey. In some embodiments, the subject is a human.

[0117] As used herein, administration "in conjunction with" or "in combination with" another compound or composition includes simultaneous administration and / or administration at different times. Co-administration also encompasses administration as a combined drug or as separate compositions, including at different dosing frequencies or intervals, and using the same or different routes of administration. In some embodiments, co-administration is administration as part of the same treatment regimen.

[0118] As used herein, "neurological disease" refers to a disease or disorder that affects and / or has an etiology in the CNS. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, eye disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, stroke, behavioral disorder, and lysosomal storage disease.

[0119] As used herein, "lysosomal storage disease" or (LSD) refers to an inherited metabolic disorder characterized by the excessive accumulation of undigested or partially digested substrates, such as macromolecules, in various cells of organs, ultimately resulting in cellular dysfunction and clinical abnormalities. LSDs are defined as defects in lysosomal function, generally classified by the accumulated substrate, and include sphingolipidoses, oligosaccharidoses, mucolipidoses, mucopolysaccharidoses, lipoprotein storage disorders, and neuronal ceroid lipofuscinoses. LSDs can also include other deficiencies or defects in proteins that result in the accumulation of macromolecules, such as proteins required for normal post-translational modification of lysosomal enzymes or proteins important for proper lysosomal trafficking. LSDs are diseases caused by defects in a single gene. Enzyme defects cause approximately 70 percent of LSDs, with the remainder being defects in enzyme activators or related proteins.

[0120] "Protein replacement therapy" or "PRT" refers to a medical procedure that supplements or replaces a specific protein in a patient in whom that protein is deficient or absent.

[0121] "Enzyme replacement therapy enzyme" or "ERT enzyme" refers to an enzyme deficient in a lysosomal storage disease. "ERT enzyme variant" refers to a functional variant, including allelic and splice variants, of a wild-type ERT enzyme or a fragment thereof, which, for example, has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding wild-type ERT enzyme or fragment thereof when assayed under identical conditions. A "catalytically active fragment" of an ERT enzyme refers to a portion of a full-length ERT enzyme or a variant thereof, which, for example, has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length ERT enzyme or variant thereof when assayed under identical conditions.

[0122] As used herein, the terms "about" and "approximately," when used to modify a numerical value or numerical range, indicate that deviations of up to 10% above and below that value or range remain within the intended meaning of the stated value or range. When embodiments are described herein in terms of "about" or "approximately" (a numerical value or range), it is understood that otherwise similar embodiments that refer to that particular numerical value or range are also provided.

[0123] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "an antibody" refers to a number of antibodies, from one to molar amounts, and equivalents thereof known to those skilled in the art.

[0124] Whenever an embodiment is described herein using the term "comprising," it should be understood that other similar embodiments are also provided that are described using the terms "consisting of" and / or "consisting essentially of." In this disclosure, the terms "comprises," "comprising," "containing," and "having" and similar terms may mean "including" and similar terms. "Consisting of" or "consisting essentially of" is open-ended, allowing for the presence of more than what is recited, but excluding prior art embodiments, so long as basic or novel characteristics of what is recited are not altered by the presence of more than what is recited.

[0125] Anti-CD98hc antigen-binding domain Provided herein is an antigen-binding domain that specifically binds to human CD98hc.

[0126] Such antigen-binding domains can cross the blood-brain barrier (BBB) ​​and transport other agents (e.g., therapeutically active agents) bound to the antigen-binding domain across the BBB. Thus, in some embodiments, provided herein are antigen-binding domains that specifically bind to human CD98hc and are capable of internalization into epithelial cells of the BBB, such as HCMEC / D3 cells.

[0127] In some embodiments, an antigen-binding domain that specifically binds to human CD98hc comprises the six CDRs of an antibody listed in Tables 9 and 10 (i.e., the three VH CDRs of an antibody listed in Table 9 and the three VL CDRs of the same antibody listed in Table 10), or the six CDRs of an antibody listed in Tables 14 and 15 (i.e., the three VH CDRs of an antibody listed in Table 14 and the three VL CDRs of the same antibody listed in Table 15), or the six CDRs of an antibody listed in Table 23 or 26.

[0128] In some embodiments, an antigen-binding domain that specifically binds to human CD98hc comprises the six CDRs of an antibody listed in Table 8, Table 13, Table 22, or Table 25. In some embodiments, the CDRs of such an antigen-binding domain can be determined according to the Chothia numbering scheme, which refers to the location of the structural loops of an immunoglobulin (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226). Typically, using the Kabat numbering convention, the Chothia CDR-H1 loop is located at amino acids 26-32, 33, or 34 in the heavy chain; the Chothia CDR-H2 loop is located at amino acids 52-56 in the heavy chain; the Chothia CDR-H3 loop is located at amino acids 95-102 in the heavy chain; the Chothia CDR-L1 loop is located at amino acids 24-34 in the light chain; the Chothia CDR-L2 loop is located at amino acids 50-56 in the light chain; and the Chothia CDR-L3 loop is located at amino acids 89-97 in the light chain. The ends of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, vary between H32 and H34 depending on the length of the loop (this is due to the insertions at H35A and H35B in the Kabat numbering scheme; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34).

[0129] In some embodiments, an antigen binding domain that specifically binds to human CD98hc comprises the six Chothia CDRs of an antibody listed in Table 8, Table 13, Table 22, or Table 25. In some embodiments, such an antigen binding domain that specifically binds to human CD98hc comprises one or more CDRs, wherein the Chothia and Kabat CDRs have the same amino acid sequence. In some embodiments, provided herein is an antigen binding domain that specifically binds to human CD98hc, comprising a combination of Kabat and Chothia CDRs.

[0130] In some embodiments, the CDRs of an antigen-binding domain that specifically binds to human CD98hc can be determined according to MacCallum RM et al., (1996) J Mol Biol 262: 732-745. See also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In some embodiments, provided herein is an antigen-binding domain that specifically binds to human CD98hc, comprising the VH and VL CDRs of an antibody listed in Table 8, Table 13, Table 22, or Table 25, as determined by the method of MacCallum RM et al.

[0131] In some aspects, the CDRs of an antigen-binding domain that specifically binds to human CD98hc are a compromise between Kabat CDRs and Chothia structural loops, and can be determined according to the AbM numbering scheme, which refers to AbM hypervariable regions, as used in Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In some aspects, provided herein is an antigen-binding domain that specifically binds to human CD98hc, comprising the VH and VL CDRs of an antibody listed in Table 8, Table 13, Table 22, or Table 25, as determined by the AbM numbering scheme.

[0132] In some embodiments, an antigen-binding domain that specifically binds to human CD98hc comprises the six IMGT CDRs of an antibody listed in Table 8, Table 13, Table 22, or Table 25 according to the IMGT numbering scheme described in Lefranc MP, (1999) The Immunologist 7: 132-136 and Lefranc MP et al., (1999) Nucleic Acids Res 27: 209-212. According to the IMGT numbering scheme, VH-CDR1 is located at positions 26-35, VH-CDR2 is located at positions 51-57, VH-CDR3 is located at positions 93-102, VL-CDR1 is located at positions 27-32, VL-CDR2 is located at positions 50-52, and VL-CDR3 is located at positions 89-97.

[0133] In some embodiments, the antigen-binding domains provided herein that specifically bind to human CD98hc are described by their VL domain alone, their VH domain alone, their three VL CDRs alone, or their three VH CDRs alone. See, e.g., Rader C et al., (1998) PNAS 95:8910-8915, the entire contents of which are incorporated herein by reference. This document describes the humanization of a murine anti-αvβ3 antibody, obtaining humanized antibody variants with similar or higher affinity to the original antibody by identifying complementary light or heavy chains from a human light or heavy chain library, respectively. See also Clackson T et al., (1991) Nature 352:624-628, the entire contents of which are incorporated herein by reference. This document describes a method for generating antibodies that bind to a specific antigen by using a particular VL domain (or VH domain) to screen a library for complementary variable domains. This screening yielded 14 new partners for a particular VH domain and 13 new partners for a particular VL domain that were shown to be strong binders by ELISA. See also Kim SJ & Hong HJ, (2007) J Microbiol 45: 572-577, which is incorporated herein by reference in its entirety. This document describes a method for generating antibodies that bind to a specific antigen by using a particular VH domain to screen a library (e.g., a human VL library) for complementary VL domains, and the selected VL domain can then be used to select additional complementary (e.g., human) VH domains.

[0134] In some embodiments, an antigen-binding domain that specifically binds to human CD98hc comprises the VH of an antibody listed in Table 8, Table 13, Table 22, or Table 25.

[0135] In some embodiments, an antigen-binding domain that specifically binds to human CD98hc comprises the VL of an antibody listed in Table 8, Table 13, Table 22, or Table 25.

[0136] In some embodiments, an antigen-binding domain that specifically binds to human CD98hc comprises the VH and VL of an antibody listed in Table 8 (i.e., the VH of an antibody listed in Table 8 and the VL of the same antibody listed in Table 8), or the VH and VL of an antibody listed in Table 13 (i.e., the VH of an antibody listed in Table 13 and the VL of the same antibody listed in Table 13), or the VH and VL of an antibody listed in Table 22 or Table 25.

[0137] In some embodiments, an antigen binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 80% identical to a VH amino acid sequence of an antibody in Table 8, Table 13, Table 22, or Table 25, and (ii) a VL comprising an amino acid sequence that is at least 80% identical to a VL amino acid sequence of the same antibody in Table 8, Table 13, Table 22, or Table 25. In some embodiments, an antigen binding domain that specifically binds to human CD98hc also comprises the CDRs of an antibody in Table 8, Table 13, Table 22, or Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0138] In some embodiments, an antigen binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 85% identical to a VH amino acid sequence of an antibody in Table 8, Table 13, Table 22, or Table 25, and (ii) a VL comprising an amino acid sequence that is at least 85% identical to a VL amino acid sequence of the same antibody in Table 8, Table 13, Table 22, or Table 25. In some embodiments, an antigen binding domain that specifically binds to human CD98hc also comprises the CDRs of an antibody in Table 8, Table 13, Table 22, or Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0139] In some embodiments, an antigen binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 90% identical to a VH amino acid sequence of an antibody in Table 8, Table 13, Table 22, or Table 25, and (ii) a VL comprising an amino acid sequence that is at least 90% identical to a VL amino acid sequence of the same antibody in Table 8, Table 13, Table 22, or Table 25. In some embodiments, an antigen binding domain that specifically binds to human CD98hc also comprises the CDRs of an antibody in Table 8, Table 13, Table 22, or Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0140] In some embodiments, an antigen binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 95% identical to a VH amino acid sequence of an antibody in Table 8, Table 13, Table 22, or Table 25, and (ii) a VL comprising an amino acid sequence that is at least 95% identical to a VL amino acid sequence of the same antibody in Table 8, Table 13, Table 22, or Table 25. In some embodiments, an antigen binding domain that specifically binds to human CD98hc also comprises the CDRs of an antibody in Table 8, Table 13, Table 22, or Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0141] In some embodiments, an antigen binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 96% identical to a VH amino acid sequence of an antibody in Table 8, Table 13, Table 22, or Table 25, and (ii) a VL comprising an amino acid sequence that is at least 96% identical to a VL amino acid sequence of the same antibody in Table 8, Table 13, Table 22, or Table 25. In some embodiments, an antigen binding domain that specifically binds to human CD98hc also comprises the CDRs of an antibody in Table 8, Table 13, Table 22, or Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0142] In some embodiments, an antigen binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 97% identical to a VH amino acid sequence of an antibody in Table 8, Table 13, Table 22, or Table 25, and (ii) a VL comprising an amino acid sequence that is at least 97% identical to a VL amino acid sequence of the same antibody in Table 8, Table 13, Table 22, or Table 25. In some embodiments, an antigen binding domain that specifically binds to human CD98hc also comprises the CDRs of an antibody in Table 8, Table 13, Table 22, or Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0143] In some embodiments, an antigen binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 98% identical to a VH amino acid sequence of an antibody in Table 8, Table 13, Table 22, or Table 25, and (ii) a VL comprising an amino acid sequence that is at least 98% identical to a VL amino acid sequence of the same antibody in Table 8, Table 13, Table 22, or Table 25. In some embodiments, an antigen binding domain that specifically binds to human CD98hc also comprises the CDRs of an antibody in Table 8, Table 13, Table 22, or Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0144] In some embodiments, an antigen binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 99% identical to a VH amino acid sequence of an antibody in Table 8, Table 13, Table 22, or Table 25, and (ii) a VL comprising an amino acid sequence that is at least 99% identical to a VL amino acid sequence of the same antibody in Table 8, Table 13, Table 22, or Table 25. In some embodiments, an antigen binding domain that specifically binds to human CD98hc also comprises the CDRs of an antibody in Table 8, Table 13, Table 22, or Table 25 (e.g., the non-identical amino acids in the VH and / or VL are outside the CDRs).

[0145] In some aspects, provided herein is an antigen-binding domain that binds to the same CD98hc epitope as an antibody comprising the VH amino acid sequence of an antibody in Table 8, Table 13, Table 22, or Table 25 and the VL amino acid sequence of the same antibody in Table 8, Table 13, Table 22, or Table 25.

[0146] In some aspects, provided herein is an antigen-binding domain that competitively inhibits the binding to CD98hc of an antibody comprising the VH amino acid sequence of an antibody in Table 8, Table 13, Table 22, or Table 25 and the VL amino acid sequence of the same antibody in Table 8, Table 13, Table 22, or Table 25.

[0147] In some embodiments, an antigen-binding domain that specifically binds to human CD98hc comprises a VH and a VL (e.g., a VH and a VL in Table 8, Table 13, Table 22, or Table 25) on a single polypeptide chain. In some embodiments, the antigen-binding domain comprises an scFv. The scFv may comprise a VH on the N-terminal side of the VL, or a VL on the N-terminal side of the VH. The scFv may comprise a linker, for example, between the VH and VL. Thus, the scFv may be oriented as VH-linker-VL or VL-linker-VH. Such a linker may be about 5 to 25 amino acids in length. Such a linker may be about 5 to 20 amino acids in length. Such a linker may be about 10 to 25 amino acids in length. Such a linker may be, for example, a glycine linker, a glycine-rich linker, or a glycine-serine linker. Such a linker may comprise the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 182). Such a linker may comprise the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 337).

[0148] In some embodiments, an antigen-binding domain that specifically binds to human CD98hc comprises a VH on a first polypeptide and a VL on a second polypeptide (eg, a Fab).

[0149] In some embodiments, an antigen-binding domain that specifically binds to human CD98hc comprises an antigen-binding fragment of a heavy chain-only antibody (e.g., a VHH or nanobody).

[0150] In some embodiments, the antigen-binding domain that specifically binds to human CD98hc is a mouse antigen-binding domain. In some embodiments, the antigen-binding domain that specifically binds to human CD98hc is a chimeric antigen-binding domain. In some embodiments, the antigen-binding domain that specifically binds to human CD98hc is a humanized antigen-binding domain. In some embodiments, the antigen-binding domain that specifically binds to human CD98hc is a human antigen-binding domain.

[0151] In some embodiments, the antigen binding domains provided herein that specifically bind to human CD98hc also bind to cynomolgus monkey CD98hc.

[0152] In some embodiments, the antigen-binding domains provided herein bind to human CD98hc with an affinity of 250 nM or less (e.g., 10 μM to 250 nM, 5 μM to 250 nM, 1 μM to 250 nM, 1 nM to 250 nM, or 3 nM to 250 nM), an affinity of 200 nM or less (e.g., 10 μM to 200 nM, 5 μM to 200 nM, 1 μM to 200 nM, 1 nM to 200 nM, or 3 nM to 200 nM), or an affinity of 150 nM or less (e.g., 10 μM to 150 nM, 5 μM to 150 nM, 1 μM to 150 nM, 1 nM to 150 nM, or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, for example, using a Carterra LSA platform. In some embodiments, the antigen-binding domains provided herein specifically bind to human CD98hc 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.

[0153] In some embodiments, the antigen-binding domains provided herein that specifically bind to human CD98hc have an ELISA OD of at least 0.45. 450 In some embodiments, the antigen-binding domains provided herein that specifically bind to human CD98hc have an ELISA OD of at least 0.45. 450 In some embodiments, the antigen-binding domains provided herein that specifically bind to human CD98hc have an ELISA OD of at least 0.45. 450 binds to human CD98hc with an ELISA OD of at least 0.45 450 It binds to cynomolgus monkey CD98hc.

[0154] In some embodiments, antigen-binding domains provided herein that specifically bind to human CD98hc bind to cynomolgus monkey CD98hc with an affinity of 250 nM or less (e.g., 10 μM to 250 nM, 5 μM to 250 nM, 1 μM to 250 nM, 10 μM to 250 nM, 1 nM to 250 nM, or 3 nM to 250 nM), or 200 nM or less (e.g., 10 μM to 200 nM, 5 μM to 20 The antibody binds with an affinity of 0 nM, 1 μM to 200 nM, 10 μM to 250 nM, 1 nM to 200 nM, or 3 nM to 200 nM), or with an affinity of 150 nM or less (e.g., 10 μM to 150 nM, 5 μM to 150 nM, 1 μM to 150 nM, 10 μM to 250 nM, 1 nM to 150 nM, or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, for example, using a Carterra LSA platform. In some embodiments, an antigen binding domain provided herein that specifically binds to cynomolgus CD98hc binds to cynomolgus CD98hc with an affinity of between 1 μM and 100 μM, between 1 μM and 1 nM, between 1 μM and 10 nM, between 1 μM and 100 nM, between 1 μM and 150 nM, or between 1 μM and 250 nM.

[0155] In some embodiments, antigen-binding domains provided herein that specifically bind to human CD98hc bind to human CD98hc and cynomolgus monkey CD98hc, respectively, with an affinity of 250 nM or less (e.g., 1 μM to 250 nM, 1 nM to 250 nM, or 3 nM to 250 nM), 200 nM or less (e.g., 1 μM to 200 nM, 1 nM to 200 nM, or 3 nM to 200 nM), or 150 nM or less (e.g., 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 embodiments, antigen-binding domains provided herein that specifically bind to human CD98hc bind to human CD98hc and cynomolgus monkey CD98hc with an affinity of 1 μM to 100 μM, 1 μM to 1 nM, 1 μM to 10 nM, 1 μM to 100 nM, 1 μM to 150 nM, or 1 μM to 250 nM, respectively.

[0156] In some embodiments, antigen-binding domains provided herein that specifically bind to human CD98hc bind to human CD98hc with an affinity of 3.1 nM to 210 nM. In some embodiments, antigen-binding domains provided herein that specifically bind to human CD98hc bind to human CD98hc with an affinity of 18 nM to 35 nM. In some embodiments, antigen-binding domains provided herein that specifically bind to human CD98hc bind to human CD98hc with an affinity of 12 nM to 34 nM. In some embodiments, antigen-binding domains provided herein that specifically bind to human CD98hc bind to cynomolgus monkey CD98hc with an affinity of 3.2 to 145 nM. In some embodiments, antigen-binding domains provided herein that specifically bind to human CD98hc bind to cynomolgus monkey CD98hc with an affinity of 340 nM to 1.5 μM. In some embodiments, antigen-binding domains provided herein that specifically bind to human CD98hc bind to cynomolgus monkey CD98hc with an affinity of 120 nM to 880 μM. In some embodiments, antigen-binding domains provided herein that specifically bind to human CD98hc bind to human CD98hc with an affinity of 3.1 nM to 210 nM and to cynomolgus monkey CD98hc with an affinity of 3.2 nM to 145 nM, optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, for example, using a Carterra LSA platform.

[0157] In some embodiments, antigen binding domains provided herein that specifically bind to human CD98hc do not reduce cell surface expression of CD98hc on HCMED / D3 cells by more than 20% compared to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, for example, using Western blot or FACS.

[0158] In some embodiments, antigen binding domains provided herein that specifically bind to human CD98hc do not increase cell surface expression of CD98hc on HCMED / D3 cells by more than 50% compared to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, for example, using Western blot or FACS.

[0159] In some embodiments, antigen-binding domains provided herein that specifically bind to human CD98hc do not decrease cell surface expression of CD98hc on HCMED / D3 cells by more than 20% compared to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control, and do not increase cell surface expression of CD98hc on HCMED / D3 cells by more than 50% compared to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, for example, using Western blot or FACS.

[0160] In some embodiments, an antigen-binding domain provided herein that specifically binds to human CD98hc accumulates in the brain of a vascular-depleted human CD98hc knock-in mouse at least 1.5-fold more than an isotype control after peripheral injection. In some embodiments, an antigen-binding domain provided herein that specifically binds to human CD98hc accumulates in the brain of a vascular-depleted mouse at least 1-fold more than an isotype control.

[0161] In some embodiments, an antigen binding domain provided herein that specifically binds to human CD98hc results in at least a 5-fold increase in brain:serum concentration ratio compared to an isotype control 24 hours after administration to mice.

[0162] Also provided herein are antigen-binding domains that bind to the same epitope on CD98hc as the CD98hc antigen-binding domains provided herein. Also provided herein are antigen-binding domains that competitively inhibit the binding of the CD98hc antigen-binding domains provided herein to CD98hc.

[0163] Drugs containing anti-CD98hc antigen-binding domain Provided herein are agents (e.g., fusion proteins, multispecific (e.g., bispecific) proteins, antibodies, antigen-binding fragments thereof, etc.) that comprise an antigen-binding domain that specifically binds to human CD98hc.

[0164] fusion proteins In some embodiments, the fusion proteins provided herein comprise an antigen-binding domain that specifically binds to human CD98hc and a heterologous protein or polypeptide. In some embodiments, the heterologous protein is a protein or polypeptide or a fragment thereof useful in protein replacement therapy (PRT). In some embodiments, the heterologous polypeptide is an enzyme (e.g., an enzyme for use in enzyme replacement therapy (ERT)) or a catalytically active fragment thereof. In some embodiments, the heterologous polypeptide is an ERT enzyme or an ERT enzyme variant, or a catalytically active fragment thereof. In some embodiments, the heterologous polypeptide in the fusion proteins provided herein is a growth factor. In some embodiments, the heterologous polypeptide in the fusion proteins provided herein is a decoy receptor. In some embodiments, the heterologous polypeptide in the fusion proteins provided herein is progranulin (PGRN), prosaposin (PSAP), or motor neuron survival protein (SMN). In some embodiments, the heterologous protein is selected from the group consisting of ubiquitin protein ligase E3A (UBE3A), α-L iduronidase (IDUA), iduronate-2-sulfatase (IDS), N-acetylgalactosamine-6-sulfatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramide The enzyme is selected from beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta-hexosaminidase B, arylsulfatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme substitute thereof, such as larosinase, idursulfase, elosulfase alpha or galsulfase, or a variant or catalytically active fragment thereof.In some embodiments, the heterologous protein is selected from the group consisting of clusterin (APOJ), reelin, tripeptidyl peptidase 1 (CLN2 / TPP1), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), α-L iduronidase (IDUA), iduronate-2-sulfatase (IDS), N-acetylgalactosamine-6-sulfatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramide The heterologous protein or enzyme is selected from beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta-hexosaminidase B, arylsulfatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme substitute thereof, such as larodinase, idursulfase, elosulfase alpha, or galsulfase, or a variant or catalytically active fragment thereof. In some embodiments, the heterologous protein in the fusion protein is located N-terminal to the antigen-binding domain that specifically binds human CD98hc. In some embodiments, the heterologous protein or polypeptide in the fusion protein is located C-terminal to the antigen-binding domain that specifically binds human CD98hc. In some embodiments, the heterologous protein or polypeptide and the antigen-binding domain that specifically binds human CD98hc are directly linked via a peptide bond. In some embodiments, the heterologous fusion protein and the antigen-binding domain that specifically binds to human CD98hc are linked via a linker (e.g., a peptide linker). In some embodiments, the fusion protein comprises the antigen-binding domain, a heterologous protein or polypeptide, and an Fc portion. In some embodiments, the antigen-binding domain and the heterologous protein or polypeptide are linked to the N-terminus of the Fc portion of the fusion protein.In other embodiments, the antigen-binding domain is linked to the N-terminus of the Fc portion and the heterologous protein or polypeptide is linked to the C-terminus of the Fc portion of the fusion protein. In other embodiments, the antigen-binding domain is linked to the C-terminus of the Fc portion and the heterologous protein or polypeptide is linked to the N-terminus of the Fc portion of the fusion protein.

[0165] Bispecific and multispecific proteins In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise an antigen-binding domain that specifically binds to human CD98hc. In some embodiments, the antibodies or antigen-binding fragments thereof comprise an antigen-binding domain that specifically binds to human CD98hc and an antigen-binding domain that specifically binds to a CNS antigen or brain antigen. In some embodiments, the CNS antigen or brain antigen is not CD98hc. Also provided herein are antibodies or antigen-binding fragments thereof that bind to the same epitope on CD98hc as the CD98hc antigen-binding domains provided herein. Also provided herein are antibodies or antigen-binding fragments thereof that competitively inhibit the binding of the CD98hc antigen-binding domains provided herein to CD98hc.

[0166] In some embodiments, the multispecific proteins provided herein comprise a first antigen-binding domain that binds to human CD98hc and a second antigen-binding domain. The first antigen-binding domain that binds to human CD98hc can be any antigen-binding domain that binds to human CD98hc provided herein. The second antigen-binding domain can be an antigen-binding domain that specifically binds to a CNS antigen or a brain antigen. In some embodiments, the CNS antigen or brain antigen is not CD98hc.

[0167] In some embodiments, the multispecific proteins provided herein comprise an antigen-binding domain that binds to human CD98hc linked to an antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof can bind to a CNS antigen or a brain antigen. In some embodiments, the CNS antigen or brain antigen is not CD98hc. In some embodiments, such multispecific proteins can be in a 2+1 antibody format (shown in FIG. 1A) or a 2+2 antibody format (FIGS. 1B and 1C).

[0168] In some embodiments, the multispecific proteins provided herein comprise a CD98hc antigen-binding domain that is an scFv linked to an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains. In some embodiments, the scFv is linked to the C-terminus of one of the two antibody heavy chains, e.g., via a protein linker.

[0169] In some embodiments, a multispecific protein comprises 1) an antigen-binding domain that binds to CD98hc, 2) a second antigen-binding domain that binds a different CNS or brain antigen, and 3) an Fc region, wherein the CD98hc antigen-binding domain and the second antigen-binding domain are connected or linked to the Fc region of the multispecific protein. In other embodiments, a multispecific protein comprises 1) an antigen-binding domain that comprises a heavy chain variable region and binds to CD98hc, 2) a second antigen-binding domain that comprises a heavy chain variable region and binds a different CNS or brain antigen, and 3) an Fc region, wherein the CD98hc antigen-binding domain and the second antigen-binding domain are connected or linked to the Fc region of the multispecific protein. In some embodiments, a multispecific protein comprises an antigen-binding domain that binds to CD98hc, a second antigen-binding domain that binds a different CNS or brain antigen, and an Fc region. In some embodiments, the CD98hc antigen-binding domain and the second antigen-binding domain are connected or linked to the N-terminus of the Fc portion of the multispecific protein. In other embodiments, the CD98hc antigen binding domain is connected or linked to the N-terminus of the Fc portion of the multispecific protein and the second antigen binding domain is linked to the C-terminus of the Fc portion of the multispecific protein. In other embodiments, the CD98hc antigen binding domain is connected or linked to the C-terminus of the Fc portion of the multispecific protein and the second antigen binding domain is linked to the N-terminus of the Fc portion of the multispecific protein.

[0170] In some embodiments, the multispecific proteins provided herein comprise two copies of a CD98hc antigen-binding domain that is an scFv and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, and one of the two copies of the antigen-binding domain is linked to the C-terminus of one of the antibody heavy chains and the other copy of the antigen-binding domain is linked to the C-terminus of the other antibody heavy chain. In some embodiments, each scFv is linked to each heavy chain via a protein linker.

[0171] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein can be multispecific, e.g., bispecific. Many different formats and uses of bispecific binding molecules are known in the art (e.g., as reviewed in Kontermann; Drug Discovery Today, 2015 July;20(7):838-47; MAbs, 2012 March-April;4(2):182-97). Bispecific proteins according to the present invention are not limited to any particular bispecific format or method of production. Thus, bispecific proteins of the present disclosure can include various configurations having a first antigen-binding domain that binds to human CD98hc and a second antigen-binding domain that binds, for example, to a CNS antigen or a brain antigen.

[0172] Examples of bispecific molecules that can be used in the present disclosure include, for example, (i) a single antibody with two arms containing different antigen-binding domains; (ii) a single-chain antibody with specificity for two different epitopes, for example, via two scFvs linked in tandem by an additional peptide linker; and (iii) a dual variable domain antibody (DVD-Ig) in which each light and heavy chain contains two variable domains in tandem via a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig.™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)), (iv) chemically linked bispecific (Fab') fragments, (v) Tandabs, which are fusions of two single-chain diabodies that give tetravalent bispecific antibodies with two binding sites for each target antigen, (vi) Flexibodies, which are combinations of scFvs and diabodies that give multivalent molecules, (vii) so-called "dock-and-lock" molecules based on the "dimerization and docking domain" of protein kinase A, which, when applied to Fabs, can give trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments, (viii) so-called Scorpion molecules, which, for example, contain two scFvs fused to either end of a human Fab arm, and (ix) diabodies. Other examples of antibody structures are described in WO2019 / 246288, which is incorporated by reference.

[0173] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are multivalent (e.g., bivalent). In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are trivalent (e.g., 2+1 antibody format). In some embodiments, the trivalent format comprises a single CD98hc antigen-binding domain provided herein and two antigen-binding domains that bind to CNS or brain antigens. The two antigen-binding domains that bind to CNS or brain antigens may comprise the same amino acid sequence or different amino acid sequences. In some embodiments, the CD98hc antigen-binding domain is an scFv. In some embodiments, the CD98hc antigen-binding domain is a VHH.

[0174] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are tetravalent (e.g., 2+2 antibody format). In some embodiments, the tetravalent format comprises two CD98hc antigen-binding domains provided herein and two antigen-binding domains that bind to CNS or brain antigens. The two CD98hc antigen-binding domains may comprise the same amino acid sequence or different amino acid sequences. In some embodiments, the two CD98hc antigen-binding domains comprise the same amino acid sequence. In some embodiments, one or both of the CD98hc antigen-binding domains is an scFv. The two antigen-binding domains that bind to CNS or brain antigens may comprise the same amino acid sequence or different amino acid sequences.

[0175] The fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein can include, for example, a linker linking the CD98hc antigen-binding domain to a heterologous protein, antibody or antigen-binding fragment thereof, or other antigen-binding domain. The linker can be, for example, a glycine linker, a glycine-rich linker, or a glycine-serine linker. The linker can include the amino acid sequence of (GGGGS)x3 (SEQ ID NO: 183). The linker can include the amino acid sequence of (GGSGG)x3 (SEQ ID NO: 338). The linker can include the amino acid sequence of GGSGG (no repeats) (SEQ ID NO: 354). The linker can be from 1 to about 20 amino acids in length.

[0176] The fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein may comprise a constant region. In some embodiments, the CD98hc antigen-binding domain provided herein is linked to a constant region, e.g., the C-terminus of a constant region. In some embodiments, the constant domain is a human constant domain. In some embodiments, the constant domain is a mouse, rat, rabbit, or monkey (e.g., cynomolgus monkey) constant domain. The constant region may be a heavy chain constant region. The constant region may be a human constant region. The constant region may be a human heavy chain constant region. The constant region may be an IgG constant region. The constant region may be an IgG1 constant region. The constant region may be an IgG2 constant region. The constant region may be an IgG4 constant region. The constant region may be a human IgG constant region. The constant region may be a human IgG1 constant region. The constant region may be a human IgG2 constant region. The constant region may be a human IgG4 constant region.

[0177] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein comprise a heavy chain and a light chain. With respect to the heavy chain, in some embodiments, the heavy chain of the antigen-binding proteins described herein can be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain can comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain comprises a human gamma (γ) heavy chain constant region. In some embodiments, the heavy chain comprises the amino acid sequence of an IgG1 heavy chain constant region. In some embodiments, the heavy chain comprises the amino acid sequence of an IgG2 (IgG2a or IgG2b) heavy chain constant region. In some embodiments, the heavy chain comprises the amino acid sequence of an IgG4 heavy chain constant region. With respect to the light chain, in some embodiments, the light chain is a kappa light chain. In some embodiments, the light chain is a lambda light chain. In some aspects, the light chain is a human kappa light chain or a human lambda light chain.

[0178] In some aspects, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein comprise a constant region comprising the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In some aspects, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein comprise a constant region comprising the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (IgG2a and IgG2b) of immunoglobulin molecule. In some embodiments, the constant region comprises the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (IgG2a and IgG2b).

[0179] Non-limiting examples of human constant region sequences are described, for example, in U.S. Pat. No. 5,693,780 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242.

[0180] In some aspects, the constant regions provided herein comprise a knob mutation. In some aspects, the constant regions provided herein comprise a hole mutation. Thus, in some aspects, a fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein can comprise a constant region that comprises a knob mutation and a constant region that comprises a hole mutation.

[0181] FC Domain The fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein can comprise an Fc domain or a fragment thereof. In some embodiments, the Fc domain is of the IgG class, the IgM class, or the IgA class. In some embodiments, the Fc domain or a fragment thereof is an IgG Fc domain or a fragment thereof. In some embodiments, the Fc domain or a fragment thereof is a human IgG Fc domain or a fragment thereof. In some embodiments, the Fc domain or a fragment thereof is a human IgG1 Fc domain or a fragment thereof. In some embodiments, the Fc domain or a fragment thereof is a human IgG2 Fc domain or a fragment thereof. In some embodiments, the Fc domain or a fragment thereof is a human IgG4 Fc domain or a fragment thereof.

[0182] In some embodiments provided herein, the provided fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein comprises a modified Fc domain or fragment thereof. In some embodiments, the modified Fc domain or fragment thereof is a modified IgG1 Fc comprising one or more modifications. For example, in some embodiments, the IgG1 modified Fc comprises one or more amino acid substitutions (e.g., compared to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from the following: N297A (Bolt S et al. (1993) Eur J Immunol 23:403-411), D265A (Shields et al. (2001) RJ Biol. Chem. 276, 6591-6604), L234A, L235A (Hutchins et al. (1995) Proc Natl Acad Sci USA, 92:11980-11984; Alegre et al., (1994) Transplantation 57:1537-1543.31; Xu et al., (2000) Cell Immunol, 200:16-26), G237A (Alegre et al. (1994) Transplantation 57:1537-1543.31; Xu et al. al. (2000) Cell Immunol, 200:16-26), C226S, C229S, E233P, L234V, L234F, L235E (McEarchern et al., (2007) Blood, 109:1185-1192), P331S (Sazinsky et al., (2008) Proc Natl Acad Sci USA 2008, 105:20167-20172), S267E, L328F, A330L, M252Y, S254T, E430G, and / or T256E (amino acid positions according to EU numbering rules). In some embodiments, the bispecific antibody comprises the amino acid substitutions L234A, L235A, and P331S (LALAPS) according to EU numbering. In some embodiments of any of the modified IgG1 Fcs, the Fc comprises N325S and L328F mutations (according to EU numbering). In some embodiments of any of the modified IgG1 Fcs, the Fc comprises P329G or P329S according to EU numbering.

[0183] In some aspects provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are bispecific fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins. Bispecific molecules include, for example, kappa-lambda bodies, dual affinity retargeting molecules (DARTs), knob-in-hole antibodies, strand-exchange engineered domain bodies (SEED bodies), and duobodies. In some aspects, the bispecific fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein comprise a knob mutation and a hole mutation. In some aspects, the knob mutation comprises the amino acid substitution T366W according to EU numbering. In some aspects, the hole mutation comprises the amino acid substitutions T366S, L368A, and Y407V according to EU numbering.

[0184] In some embodiments provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein comprise mutations to promote heterodimerization of the Fc region. In some embodiments, the bispecific dimerization Fc region provided herein is formed by an Fc region comprising amino acid mutations, substitutions, additions, or deletions that promote heterodimerization, allowing different polypeptides comprising different Fc regions to dimerize to form a heterodimeric form. In some embodiments, the bispecifics of the present disclosure comprise a first Fc sequence comprising a first CH3 region and a second Fc sequence comprising a second CH3 region, wherein the sequences of the first and second CH3 regions are different such that the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the homodimeric interaction between the first CH3 region and the second CH3 region, respectively.

[0185] Methods for promoting heterodimerization between Fc regions include amino acid deletions, additions, or substitutions in the amino acid sequence of the Fc region, for example, by including a group of "knob-into-hole" deletions, additions, or substitutions, or by including amino acid deletions, additions, or substitutions that result in electrostatic guidance of the Fc, which favors attractive interactions between different polypeptide chains. Methods for promoting heterodimerization between complementary Fc polypeptides have been previously described, for example, in Ridgway et al., 1996, Protein Eng, 9:617-621; Merchant et al., 1998, Nature Biotechnol, 16:677-681; Moore et al., 2011, MAbs, 3:546-557; Von Kreudenstein et al., 2013, J Biol Chem, 285:19637-19464; Leaver-Fay et al., 2016, Structure, 24:641-651; Ha et al., 2016, Frontiers in Immunology, 7:1; Davis et al., 2010, Protein Eng Des Sel,23:195-202;WO1996 / 027011;WO1998 / 050431;WO2006 / 028936;WO2009 / 089004;WO2011 / 143545;WO2014 / 067011;WO2012 / 058768;WO2018 / 027025;US2014 / 0363426;US2015 / 03076 28; US2018 / 0016354; US2015 / 0239991; US2017 / 0058054; USPN5731168; USPN7183076; USPN9701759; USPN9605084; USPN9650446; USPN8216805; USPN8765412; and USPN8258268.

[0186] In some embodiments, the complementary Fc polypeptides of an Fc heterodimer contain mutations that alter the charge polarity across the Fc dimer interface, such that coexpression of electrostatically matched Fc regions favors favorable attractive interactions, thereby promoting the formation of desired Fc heterodimers, while unfavorable repulsive charge interactions suppress the formation of undesired Fc homodimers (Guneskaran et al., 2010, J Biol Chem, 285:19637-19646). When coexpressed in cells, association between polypeptide chains is possible, but the chains rarely self-associate due to charge repulsion.

[0187] Additionally, the complementary Fc polypeptides of an Fc heterodimer may contain a "knob-into-hole" structure that promotes heterodimerization between two Fc polypeptides. "Knob-into-hole" technology has been described, for example, in U.S. Patent Nos. 5,731,168, 7,695,936, 8,216,805, and 8,765,412; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protrusion ("knob") into the interface of a first polypeptide and a corresponding hole ("hole") into the interface of a second polypeptide, such that the protrusion lies within the hole, promoting heterodimer formation and preventing homodimer formation. The protrusion is formed by substituting a small amino acid side chain at the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary hole of the same or similar size as the protrusion is formed at the interface of the second polypeptide by substituting a large amino acid side chain with a smaller one (e.g., alanine or threonine). The protrusion and hole can be formed by modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or peptide synthesis. In some embodiments, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In some embodiments, the Fc domain subunit containing the knob modification further comprises the amino acid substitution S354C, and the Fc domain subunit containing the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of disulfide bridges between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).Thus, in such a configuration, a first Fc polypeptide contains amino acid modifications to form the "knob" and a second Fc polypeptide contains amino acid modifications to form the "hole", thereby forming an Fc heterodimer consisting of the complementary Fc polypeptides.

[0188] Exemplary paired amino acid modifications of complementary Fc polypeptides in the Fc heterodimer form are shown in the table below (EU numbering).

[0189] Table 1. Exemplary paired Fc modifications of heterodimeric Fc domains TIFF2025528751000002.tif65165

[0190] Some agents provided herein comprise antigen-binding fragments of antibodies. Antigen-binding fragments of antibodies include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments, and scFv fragments, as well as other fragments described below. A review of specific antibody fragments is provided in Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., WO 93 / 16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and have increased in vivo half-lives, see U.S. Pat. No. 5,869,046.

[0191] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, e.g., EP 404,097, WO 1993 / 01161, Hudson et al. Nat. Med. 9:129-134 (2003). Triabodies and tetrabodies are also described in Hudson et al. Nat. Med. 9:129-134 (2003). Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In some embodiments, single-domain antibodies are human single-domain antibodies (see, e.g., U.S. Pat. No. 6,248,516).

[0192] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0193] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein may be chimeric. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, e.g., a monkey) and a human constant region. In a further example, the chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody.

[0194] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein may be humanized. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the non-human parent antibody. In some aspects, humanized antibodies are substantially non-immunogenic in humans. In some aspects, humanized antibodies have substantially the same affinity for a target as an antibody from another species from which the humanized antibody is derived. See, e.g., U.S. Patent Nos. 5,530,101, 5,693,761, 5,693,762, and 5,585,089. In some aspects, amino acids in antibody variable domains are identified that can be modified to reduce their immunogenicity without losing the native affinity of the antigen-binding domain. See, e.g., U.S. Patent Nos. 5,766,886 and 5,869,619. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the framework regions (FRs) are derived from human antibody sequences. A humanized antibody may also comprise at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues were derived), e.g., to restore or improve antibody specificity or affinity.

[0195] Humanized antibodies and methods for making them are reviewed, for example, in Almagro et al. Front. Biosci. 13:161 9-1633 (2008) and further described, for example, in U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409. Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)), framework regions derived from consensus sequences of human antibodies of specific subpopulations of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions obtained from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0196] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein may be human. Human antibodies can be produced using various techniques well known in the art. Human antibodies are generally described in van Dijk et al. Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg Curr. Opin. Immunol. 20:450-459 (2008).

[0197] Human antibodies can be prepared by administering immunogens to transgenic animals engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Large fragments of human Ig loci can be incorporated into mouse strains deficient in mouse antibody production, with the expectation that such mice will produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can preserve the diversity of large variable genes and the proper regulation of antibody production and expression. By exploiting mouse mechanisms for antibody diversification and selection and the lack of immune tolerance to human proteins, the recapitulated human antibody repertoire in these mouse strains can yield high-affinity, fully human antibodies against any antigen of interest, including human antigens. Hybridoma technology can be used to produce and select antigen-specific human MAbs with desired specificity. Certain exemplary methods are described in U.S. Pat. No. 5,545,807, EP 546073, and EP 546073. See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE™ technology), U.S. Patent No. 5,770,429 (describing HUMAB® technology), U.S. Patent No. 7,041,870 (describing KM MOUSE® technology), and U.S. Patent Application Publication No. 2007 / 0061900 (describing VELOCIMOUSE® technology). The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0198] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol. 133:3001 (1984) and Boerner et al. J. Immunol. 147:86 (1991)). Human antibodies generated via human B cell hybridoma technology are also described in Li et al. Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Pat. No. 7,189,826, which describes the production of monoclonal human IgM antibodies from hybridoma cell lines. Human hybridoma technology is also described in Vollmers et al., Histology and Histopathology, 20(3):927-937 (2005) and Vollmers et al., Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005). Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domains can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0199] In some embodiments provided herein, the antibodies are human antibodies isolated by in vitro methods and / or screening of combinatorial libraries for antibodies with the desired activity. Suitable examples include, but are not limited to, phage display (CAT, Morphosys, Dyax, Biosite / Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosome display (CAT), yeast display (Adimab), and the like. In certain phage display methods, repertoires of VH and VL genes are individually cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phage, as described in Winter et al. Ann. Rev. Immunol. 12:433-455 (1994). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. See also: Sidhu et al. J. Mol. Biol. 338(2):299-310, 2004; Lee et al. J. Mol. Biol. 340(5):1073-1093, 2004; Fellouse Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al. Methods 284(-2):119-132 (2004). Phages typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies against the immunogen without the need to construct hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12:725-734 (1993), naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self antigens without immunization.Finally, naive libraries can also be synthetically generated by cloning unrearranged V gene segments from stem cells, using PCR primers containing random sequences to encode hypervariable CDR3 regions, and achieving in vitro rearrangement, as described in Hoogenboom et al., J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Application Publication Nos. 2007 / 0292936 and 2009 / 0002360. Antibodies isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0200] As provided herein, a fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein can comprise an antigen-binding domain that binds to a CNS or brain antigen. In some embodiments, the CNS or brain antigen is selected from the group consisting of beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretagogue protein (GSP), and leucine-rich repeat kinase 2 (LRRK2). The antigen-binding domain may be an antigen-binding domain that binds to ribosomal kinase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid-binding Ig-like lectin 3 (Siglec3), transmembrane 4-domain A4A (MS4A4A), transmembrane 4-domain A6A (MS4A6A), or transmembrane protein 106B (TMEM106b).In some embodiments, the CNS or brain antigen is beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebroidase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), ubiquitin protein ligase E3A (UBE3A), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33, sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), sialic acid-binding Ig-like lectin 11 (Siglec11), glycoprotein non-transferase protein The protein may be protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4-domain A4A (MS4A4A), transmembrane 4-domain A6A (MS4A6A), MSA4A4E, transmembrane protein 106B (TMEM106b), CR1, ABCA1, ABCA7, HLA-DR1, HLA-DR5, IL1RAP, TREML2, IL-34, SORL1, and ADAM1.

[0201] In some embodiments, the CNS or brain antigen is present on a cancer cell within the central nervous system. In some embodiments, the CNS or brain antigen is a cell surface target on a hematological cancer cell selected from B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b. In some embodiments, the CNS or brain antigen is selected from siglec-3 or CD33, siglec-5, siglec-7, siglec-9, siglec14, PILRA, IL18-BP, MerTK, ACKR1, ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM (CD66E), EGFR, EGFR viii, ETBR, FGFR(1-4), folate receptor alpha, and GAL-3BP. (galectin-binding protein), GD2, GD3, GloboH (globohexacylceramide), gp100, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine-rich repeat-containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4, colon cancer kinase), RON, ROR1, TF (tissue factor), and TROP2.

[0202] As provided herein, a fusion protein, an antibody or antigen-binding fragment thereof, or a multispecific protein can comprise an antigen-binding domain that binds to a CNS antigen or a brain antigen. As provided herein, a multispecific protein can comprise an antigen-binding domain that binds to a CNS antigen or a brain antigen. The antigen-binding domain that binds to a CNS antigen or a brain antigen can comprise a VH and a VL. Exemplary CNS antigen-binding VH and VL sequences are shown below. Additional VH and VL and antigen-binding domain sequences can be found in US2017 / 0224702, US2018 / 0002433, US2021 / 0236634, and US2021 / 0238265, each of which is incorporated by reference in its entirety.

[0203] Table 2: Exemplary CNS antigens TIFF2025528751000003.tif195165

[0204] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are able to cross the BBB as a result of the anti-CD98 antigen-binding domain in the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein being able to cross the BBB.

[0205] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are internalized into blood-brain barrier epithelial cells at a rate greater than 10-fold compared to internalization by an isotype control. The blood-brain barrier endothelial cells may be, for example, HCMEC / D3 cells.

[0206] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein do not reduce cell surface expression of CD98hc on HCMEC / D3 cells by more than 20% compared to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, for example, using Western blot or FACS.

[0207] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein do not increase cell surface expression of CD98hc on HCMEC / D3 cells by more than 50% compared to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, for example, using Western blot or FACS.

[0208] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein do not decrease cell surface expression of CD98hc on HCMEC / D3 cells by more than 20% compared to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control, and do not increase cell surface expression of CD98hc on HCMEC / D3 cells by more than 50% compared to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, for example, using Western blot or FACS.

[0209] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein accumulate in the brains of vascular-depleted mice at least 1.5-fold more than an isotype control. In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein accumulate in the brains of vascular-depleted mice at least 2-fold more than an isotype control.

[0210] In some embodiments, a fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein has at least a 5-fold increase in brain:serum concentration ratio compared to an isotype control 24 hours after administration to mice.

[0211] In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein bind to human CD98hc with an equilibrium dissociation constant (K D In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein bind to cynomolgus monkey CD98hc with a K of about 3 nM to about 225 nM. D In some embodiments, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein bind to human CD98hc with an equilibrium dissociation constant (KD ) and binds to cynomolgus monkey CD98hc with a K of approximately 3 nM to approximately 225 nM. D Combine with.

[0212] Polynucleotide, method for producing anti-CD98hc antigen-binding domain, and drug containing same In some aspects, provided herein are polynucleotides comprising nucleotide sequences encoding an antigen-binding domain that specifically binds to human CD98hc, a fusion protein, an antibody, or an antigen-binding fragment thereof, or a multispecific protein as described herein or a domain thereof as described herein, and vectors (e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells)).

[0213] In some embodiments, the polynucleotides provided herein comprise a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically binds to human CD98hc provided herein. In some embodiments, the polynucleotides provided herein comprise a nucleic acid molecule encoding the light chain of an antigen-binding domain that specifically binds to human CD98hc provided herein. In some embodiments, the polynucleotides provided herein comprise a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically binds to human CD98hc provided herein and a nucleic acid molecule encoding the light chain of an antigen-binding domain that specifically binds to human CD98hc provided herein.

[0214] In some aspects, provided herein are polynucleotide combinations or compositions. In some aspects, the combinations or compositions comprise a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode a multispecific protein provided herein, e.g., the first polynucleotide encodes a first heavy chain, the second polynucleotide encodes a second heavy chain and an antigen-binding domain that specifically binds to human CD98hc provided herein, and the third polynucleotide encodes a light chain. In some aspects, the antigen-binding domain that specifically binds to human CD98hc is an 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.

[0215] In some embodiments, the combination or composition comprises a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides 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 CD98hc, the second polynucleotide encodes a second heavy chain and a second antigen-binding domain that specifically binds to human CD98, and the third polynucleotide encodes a light chain. In some aspects, the first and second antigen-binding domains that bind to human CD98hc comprise the same amino acid sequence. In some aspects, the first and second antigen-binding domains that bind to human CD98hc comprise different amino acid sequences. In some aspects, the first and second antigen-binding domains that bind to human CD98hc are scFvs. In some aspects, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation. In some embodiments, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0216] In some embodiments, the combination or composition comprises a first polynucleotide and a second polynucleotide, wherein the first and second polynucleotides encode a multispecific protein provided herein, and the first polynucleotide encodes a heavy chain and an antigen-binding domain that binds to human CD98hc provided herein, and the second polynucleotide encodes a light chain.

[0217] Also provided herein are antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human CD98hc as described herein, or polynucleotides comprising nucleotide sequences encoding such domains as described herein, that have been optimized, for example, by codon / RNA optimization, substitution with a heterologous signal sequence, and / or removal of mRNA instability elements. Methods for generating nucleic acids optimized for recombinant expression by introducing codon changes (e.g., codon changes that encode the same amino acid due to the degeneracy of the genetic code) and / or removing inhibitory regions within the mRNA can be performed by applying the optimization methods described, for example, in U.S. Patent Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498.

[0218] Polynucleotides comprising nucleotide sequences encoding antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human CD98hc described herein, or domains thereof described herein, can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the nucleic acid art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Using such PCR amplification methods, for example, nucleic acids comprising sequences encoding the light chain and / or heavy chain of an antigen-binding domain, antibody, or antigen-binding fragment thereof can be obtained. The amplified nucleic acid can be cloned into a vector for expression in a host cell or for further cloning, for example, to generate antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human CD98hc described herein, or domains thereof described herein.

[0219] The polynucleotides provided herein may be, for example, in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and the DNA may be double-stranded or single-stranded. If single-stranded, the DNA may be the coding strand or the non-coding (antisense) strand. In some embodiments, the polynucleotide is a cDNA or DNA lacking one or more endogenous introns. In some embodiments, the polynucleotide is a non-naturally occurring polynucleotide. In some embodiments, the polynucleotide is recombinantly produced. In some embodiments, the polynucleotide is isolated. In some embodiments, the polynucleotide is substantially pure.

[0220] In some embodiments, the polynucleotides provided herein are in the form of RNA. In some embodiments, the polynucleotides provided herein are in the form of RNA encoding the fusion proteins provided herein. In some embodiments, the polynucleotides provided herein are synthetic messenger RNA (mRNA). In some embodiments, the synthetic mRNA has at least one nucleoside modification. In some embodiments, the at least one nucleoside modification is pyridin-4-one ribonucleoside, 5-azauridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- Taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydrosh Pseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine cytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-Methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza 8-Aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine Bamoyl adenosine, N6,N6-dimethyl adenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guano In some embodiments, the guanosine is selected from 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0221] In some aspects, provided herein is a polynucleotide encoding a fusion protein comprising an antigen binding protein provided herein and a heterologous polypeptide. In some aspects, the heterologous polypeptide is selected from the group consisting of beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, beta-glucocerebromide (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurite outgrowth factor (ERF), and / or neurite outgrowth factor (NNF). In some embodiments, the heterologous polypeptide comprises an antigen-binding domain that binds to amylotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33, or sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), glycoprotein non-transferase B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4-domain A4A (MS4A4A), transmembrane 4-domain A6A (MS4A6A), or transmembrane protein 106B (TMEM106b), or portions thereof. In some embodiments, the heterologous polypeptide comprises an antigen-binding domain that binds to ubiquitin protein ligase E3A (UBE3A). In some aspects, provided herein is a polynucleotide encoding a fusion protein comprising an antigen-binding domain provided herein and a heterologous polypeptide.In some embodiments, the heterologous polypeptide is selected from the group consisting of beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, beta-glucocerebrosidase (GBA), progranulin (PGRN), prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurulin, and the like. In some embodiments, the multispecific protein comprises an amino acid sequence of a ubiquitin-binding domain that binds to ubiquitin protein ligase E3A (UBE3A), or a portion thereof. In some embodiments, the polynucleotide is mRNA (eg, synthetic mRNA).

[0222] In some embodiments, the present disclosure provides a polynucleotide encoding a fusion protein disclosed herein that comprises a heterologous polypeptide. In some aspects, the heterologous polypeptide is an ERT enzyme or an ERT enzyme variant, or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide comprises β-glucocerebrosidase (GBA), progranulin (PGRN), prosaposin (PSAP), or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide in the fusion protein provided herein is a growth factor. In some aspects, the heterologous polypeptide in the fusion protein provided herein is a decoy receptor. In some aspects, the heterologous polypeptide in the fusion protein provided herein is progranulin (PGRN), prosaposin (PSAP), or motor neuron survival protein (SMN). In some embodiments, the heterologous protein is selected from the group consisting of α-L iduronidase (IDUA), iduronate-2-sulfatase (IDS), N-acetylgalactosamine-6-sulfatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramide The enzyme is selected from beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta-hexosaminidase B, arylsulfatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme substitute thereof, such as larosinase, idursulfase, elosulfase alpha or galsulfase, or a variant thereof, or a catalytically active fragment thereof.In some embodiments, the heterologous protein is selected from the group consisting of clusterin (APOJ), reelin, ubiquitin protein ligase E3A (UBE3A), tripeptidyl peptidase 1 (CLN2 / TPP1), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), α- L-iduronidase (IDUA), iduronate-2-sulfatase (IDS), N-acetylgalactosamine-6-sulfatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramide beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta-hexosaminidase B, arylsulfatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, synthetic enzyme substitutes thereof, such as larosinase, idursulfase, elosulfase alpha or galsulfase, or variants thereof, catalytically active fragments thereof. α-L iduronidase (IDUA), iduronate-2-sulfatase (IDS), N-acetylgalactosamine-6-sulfatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramide The polynucleotide is an enzyme selected from beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta-hexosaminidase B, arylsulfatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme substitute thereof, such as larodinase, idursulfase, elosulfase alpha, or galsulfase, or a variant or catalytically active fragment thereof. In some embodiments, the polynucleotide is an mRNA (e.g., a synthetic mRNA).

[0223] In certain aspects, provided herein are vectors (e.g., expression vectors) for recombinant expression in a host cell (e.g., a mammalian host cell), comprising a polynucleotide comprising a nucleotide sequence encoding an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human CD98hc described herein, or a domain thereof described herein. Vectors for producing an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human CD98hc described herein, or a domain thereof described herein, can be made, for example, by recombinant DNA technology using techniques well known in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors operably linked to a promoter, comprising a nucleotide sequence encoding an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human CD98hc described herein, or a domain thereof described herein. Such vectors can include, for example, nucleotide sequences encoding an antigen-binding domain, a constant region of an antibody or antigen-binding fragment thereof (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036, and U.S. Pat. No. 5,122,464), and a variable domain of an antigen-binding domain, an antibody or antigen-binding fragment thereof; such nucleotide sequences can be cloned into such vectors to express an entire heavy chain, an entire light chain, or an entire heavy and light chain. In some embodiments, the vector is a gene therapy vector (e.g., an AAV or lentiviral vector).

[0224] In certain aspects, provided herein are expression systems comprising a polynucleotide comprising a nucleotide sequence encoding an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human CD98hc as described herein, or a domain thereof as described herein. The expression system can be contained in a vector. The expression system can also be integrated into the chromosome of a host cell. In some aspects, the expression system is a cell-free expression system. In some aspects, the expression system comprises a host cell comprising a polynucleotide and / or vector provided herein.

[0225] Accordingly, also provided herein are cells, e.g., host cells, comprising polynucleotides and / or vectors for recombinantly expressing an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human CD98hc as described herein, or a domain thereof as described herein. In some embodiments, to express a two-chain antigen-binding protein, vectors encoding the heavy and light chains separately can be co-expressed in the host cell to express the entire immunoglobulin. In some embodiments, the host cell comprises two different vectors: a first vector comprising a polynucleotide encoding the heavy chain of an antigen-binding protein described herein, and a second vector comprising a polynucleotide encoding the light chain of the antigen-binding protein. In some embodiments, a first host cell comprises a first vector comprising a polynucleotide encoding the heavy chain, and a second host cell comprises a second vector comprising a polynucleotide encoding the light chain. In some embodiments, a population of host cells comprising such a first host cell and such a second host cell is provided herein.

[0226] In some embodiments, provided herein are methods for producing an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human CD98hc as described herein, or a domain thereof as described herein, in a host cell. In some aspects, provided herein are methods for producing a single-chain antigen-binding domain that specifically binds to human CD98hc, an Fc domain, and a heterologous protein or polypeptide as described herein, in a host cell. In some aspects, provided herein are methods for producing a single-chain antigen-binding domain that specifically binds to human CD98hc, an Fc domain, and a second antigen-binding domain as described herein, in a host cell. The expression vector can be introduced into cells (e.g., host cells) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human CD98hc as described herein, or a domain thereof as described herein.

[0227] A variety of host-expression vector systems can be utilized to express the antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human CD98hc described herein, or domains thereof described herein (see, e.g., U.S. Patent No. 5,807,715). Such host-expression systems represent not only carriers in which the coding sequences of interest can be produced and subsequently purified, but also cells that, when transformed or transfected with the appropriate nucleotide coding sequences, are capable of expressing the antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human CD98hc described herein, or domains thereof described herein. These host expression systems include, but are not limited to, bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems (e.g., Chlamydomonas) infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the coding sequences. reinhardtii); or mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells) harboring recombinant expression constructs containing promoters derived from the genomes of mammalian cells (e.g., metallothionein promoters) or mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters).In some embodiments, cells for expressing antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human CD98hc described herein, or domains thereof described herein, are CHO cells, e.g., CHO cells derived from CHO GS System™ (Lonza). In some embodiments, cells for expressing antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human CD98hc described herein, or domains thereof described herein, are human cells, e.g., human cell lines. In some aspects, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In some aspects, bacterial cells such as E. 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 that specifically bind to human CD98hc described herein, or domains thereof described herein. For example, mammalian cells such as Chinese hamster ovary cells (CHO) in combination with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus are effective expression systems for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-105; and Cockett MI et al., (1990) Biotechnology 8: 662-667). In some embodiments, an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human CD98hc described herein, or a domain thereof described herein, is produced by CHO cells or NS0 cells.

[0228] In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product may contribute to the function of the protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells.

[0229] Once an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human CD98hc as described herein, or domains thereof as described herein, is produced by recombinant expression, it can be purified by any purification method known in the art, such as chromatography (e.g., ion exchange, affinity, particularly affinity for a particular antigen following Protein A, and size exclusion column chromatography), centrifugation, differential solubility, or other standard methods for purifying proteins. Additionally, purification can be facilitated by fusing an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human CD98hc as described herein, or domains thereof as described herein, to a heterologous polypeptide sequence.

[0230] In some embodiments, antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human CD98hc described herein are isolated or purified. Generally, isolated antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human CD98hc described herein are substantially free of other proteins. For example, in some embodiments, preparations of antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins that specifically bind to human CD98hc described herein are substantially free of cellular material and / or chemical precursors.

[0231] Pharmaceutical Composition Provided herein are compositions comprising an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human CD98hc described herein. In some embodiments, the antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human CD98hc with the desired degree of purification is present in a formulation containing, for example, a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed. Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives.

[0232] In some embodiments, the pharmaceutical composition comprises an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to CD98hc described herein and a pharmaceutically acceptable carrier (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). In some embodiments, the pharmaceutical compositions described herein are used as medicaments. Compositions used for in vivo administration can be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes.

[0233] Also provided herein are pharmaceutical compositions comprising a polynucleotide encoding an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein that specifically binds to human CD98hc as described herein. In some embodiments, the polynucleotide is RNA. In some embodiments, the polynucleotide is synthetic mRNA. In some embodiments, the pharmaceutical composition comprising the polynucleotide further comprises a lipid-based transfection reagent.

[0234] The pharmaceutical compositions described herein can be used to produce a biological effect(s) in vivo or in vitro, for example, to cross the blood-brain barrier in a subject.

[0235] In some embodiments, the pharmaceutical compositions provided herein are used to treat diseases or conditions such as neurological disorders, neurodegenerative diseases, cancer, ophthalmic disorders, seizure disorders, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, and CNS inflammation. In some embodiments, the pharmaceutical compositions provided herein are used to treat diseases or conditions such as Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, and traumatic brain injury. In some embodiments, the pharmaceutical compositions provided herein are used to treat frontotemporal dementia.

[0236] 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.

[0237] Anti-CD98hc antigen-binding domain and method of use of drugs containing same The antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, and multispecific proteins comprising such antigen-binding domains provided herein can advantageously be transported across the blood-brain barrier. Accordingly, provided herein are methods for administering or transporting an antigen-binding protein that specifically binds to human CD98hc, or a fusion protein comprising an antigen-binding protein that specifically binds to human CD98hc, an antibody, antigen-binding fragment thereof, or a multispecific protein across the blood-brain barrier in a subject, the method comprising administering to the subject an antigen-binding protein that specifically binds to human CD98hc, or a fusion protein comprising an antigen-binding protein that specifically binds to human CD98hc, an antibody, antigen-binding fragment thereof, or a multispecific protein.

[0238] Given the ability of the antigen-binding domains and fusion proteins, antibodies, antigen-binding fragments thereof, and multispecific proteins comprising such antigen-binding domains provided herein to be transported across the blood-brain barrier, they can be used to treat neurological diseases or disorders. In some embodiments, a method of treating a neurological disease or disorder in a subject comprises administering to the subject an antigen-binding protein that specifically binds human CD98hc, or a fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein comprising an antigen-binding protein that specifically binds human CD98hc. In some embodiments, the neurological disease or disorder can be a disease or condition such as, for example, a neuropathic disorder, a neurodegenerative disease, cancer, an ocular disorder, a seizure disorder, a lysosomal storage disorder, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and inflammation of the CNS. Neurological diseases or disorders include, for example, neurodegenerative diseases (Lewy body disease, post-poliomyelitis syndrome, Shy-Drager syndrome, oligocerebellar atrophy, Parkinson's disease, Gaucher disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), tauopathies (such as Alzheimer's disease and supranuclear palsy), prion diseases (bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), ophthalmoplegia, motor neuron diseases, heterogeneous degenerative disorders of the nervous system (e.g., Canavan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette's syndrome, Meningitis, and the like), and The cause may be dementia (such as Pick's disease, spinocerebellar ataxia), cancer of the CNS and / or brain (such as glioblastoma or brain metastases resulting from cancer elsewhere in the body), Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), limbic-predominant age-related TDP-43 encephalopathy (LATE), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, or traumatic brain injury.In some aspects, the neurological disease or disorder is dementia. In some aspects, the neurological disease or disorder is frontotemporal dementia. In some embodiments, the neurological disease or disorder is Alzheimer's disease. In some embodiments, the neurological disease or disorder is Parkinson's disease. In some aspects, the neurological disease or disorder is frontotemporal epilepsy. In some embodiments, the neurological disease or disorder is autism. In some aspects, the neurological disease or disorder is lissencephaly.

[0239] In some aspects, provided herein are methods of treating a lysosomal storage disease with the fusion proteins disclosed herein, hi some aspects, the lysosomal storage disease is selected from Gaucher disease, ceroid lipofuscinosis (Batten disease), mucopolysaccharidosis (MPS) type I, MPS type II, and MPS type III.

[0240] The antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, and multispecific proteins comprising such antigen-binding domains provided herein that specifically bind to human CD98hc can be used to detect antigens (e.g., CNS antigens or brain antigens). For such purposes, the antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, and multispecific proteins comprising such antigen-binding domains that specifically bind to human CD98hc can be labeled. Exemplary labels include, for example, radioisotopes (e.g., 64CU) and fluorescent labels. Thus, provided are methods of detecting an antigen using an antigen binding protein that specifically binds to human CD98hc, or a fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein comprising an antigen binding protein that specifically binds to human CD98hc. In some aspects, a method of detecting an antigen in the CNS (e.g., brain) of a subject comprises administering an antigen binding protein that specifically binds to human CD98hc, or a fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein comprising an antigen binding protein that specifically binds to human CD98hc, to the antigen in the CNS (e.g., brain). Such methods may further comprise, for example, performing positron emission tomography (PET) imaging on the subject. In some aspects, disclosed herein are methods of detecting a CNS antigen in vitro, comprising contacting a sample in vitro with a fusion protein, antibody, or multispecific protein disclosed herein and locating an imaging agent in the sample.

[0241] The antigen-binding domains that specifically bind to human CD98hc described herein, as well as fusion proteins, antibodies, antigen-binding fragments thereof, and multispecific proteins comprising such antigen-binding domains, can be used for prognostic, diagnostic, monitoring, and / or screening applications, including in vivo applications that are well known and standard to those of skill in the art, including applications based on the present specification. In some aspects, provided herein are antigen-binding proteins that specifically bind to human CD98hc, or fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins comprising antigen-binding proteins that specifically bind to human CD98hc for use in diagnosis. In some aspects, the antigen-binding proteins that specifically bind to human CD98hc, or fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins comprising antigen-binding proteins that specifically bind to human CD98hc, comprise a detectable label.

[0242] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0243] The present disclosure will be better understood by reference to the following examples, which should not be construed as limiting the scope of the disclosure. All citations throughout this disclosure are expressly incorporated herein by reference. [Example]

[0244] Example 1. Generation of Avi-His-tagged variants of CD98 heavy chain (CD98hc) The following method was used to generate Avi-His-tagged variants of the extracellular domain (ECD) of human, cynomolgus monkey (cyno), and mouse CD98 heavy chain (CD98hc). Mammalian expression of human, cynomolgus monkey (cyno), and mouse variants of BBB receptor antigens (SEQ ID NOS: 1-3) was achieved by cloning cDNA-based synthetic genes into mammalian expression vectors followed by transient transfection and expression in Expi293 cells. The constructs included a heterologous signal peptide and a C-terminal Avi-His tag to enable purification and biotinylation. Briefly, antigen-encoding plasmids were transfected using the Expifectamine 293 Transfection Kit (ThermoFisher A14524) according to the manufacturer's specifications. Five days after transfection, culture supernatants were harvested, clarified by centrifugation, and purified in a drip column format using HisPur Ni-NTA resin (Thermo Scientific 88223). 200 mL of culture supernatant was filtered using a 0.2 μm filtration unit, and 3 mL of resin slurry in PBS was added to the filtered supernatant. The sample was incubated overnight with shaking at 4 °C. The next day, the beads were loaded onto a 20 mL drip column, washed with 10 mL of His-Select wash buffer (Millipore Sigma H5288), and eluted with 5 mL of His-Pur elution buffer (Millipore Sigma H5413). The eluate was buffer-exchanged into PBS using an Amicon Ultra-15 centrifugal filter unit (Millipore UFC9010). Antibody concentration was quantified by measuring absorbance at 280 nm using a Nanolop 8000 (ThermoFisher). Antigen purity was measured by SDS-PAGE. The antigen was analyzed for aggregation by size-exclusion chromatography (SEC). Some antigens were biotinylated using the BirA Biotin-Protein Ligase Kit (AVIDITY) according to the manufacturer's instructions.

[0245] Table 3. Avi-His-tagged variants of CD98hc ECD TIFF2025528751000004.tif170165

[0246] Example 2. Generation of a CHO cell line overexpressing CD98hc As described herein, several cell lines were generated for screening the binding of anti-CD98hc antibodies. Briefly, CHO cells stably expressing human CD98hc and mouse CD98hc were generated using the pLenti-EF1a construct, which expresses full-length human CD98hc and mouse CD98hc (SEQ ID NOs: 4 and 5, respectively). Lentivirus constructs (Genecopoia) were used to express human CD98hc with puromycin selection and mouse CD98hc containing GFP as a reporter. Lentivirus was generated by transfection of 293T cells using the ViraSafe™ Lentiviral Packaging System (CellBiolabs VPK-206). The lentivirus-containing supernatant was then used to transduce Chinese hamster ovary (CHO) cells. Two days after transduction, puromycin was added to the medium as selection pressure for human CD98hc. Mouse CD98hc was FACS-sorted for GFP-positive cells using a FACS Aria system (BD Biosciences). The resulting CHO cells stably expressing human and mouse CD98hc were analyzed for cell surface expression by flow cytometry.

[0247] Table 4. Sequence of full-length CD98hc TIFF2025528751000005.tif144165

[0248] Example 3. Generation of CD98hc humanized mice We generated a humanized mouse line expressing the human extracellular domain of CD98hc (Taconic Biosciences GmbH, Germany). We used CRISPR to replace the mouse ECD with a human one, while retaining the mouse intracellular and transmembrane portions under the control of a mouse promoter. Brain sections from these mice were evaluated by IHC and / or Western blot to confirm the expression and localization of the human ECD in vivo (data not shown).

[0249] Example 4. Production of anti-CD98hc hybridoma antibody To obtain antibodies against CD98hc, hybridomas were generated using the following procedure: BALB / c mice or Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) were immunized twice weekly by subcutaneous or intraperitoneal injection of purified extracellular domain polypeptides of human, cynomolgus monkey, and / or mouse CD98hc (obtained as described above in Example 1) with or without adjuvant. After a total of 6 to 8 injections and 3 days after the final boost, lymph nodes were harvested from the mice for hybridoma cell line generation.

[0250] Sera from animals were analyzed for reactivity to CD98hc by FACS against CHO cells overexpressing human or mouse CD98hc and by ELISA against the Avi-His polypeptides of human, cynomolgus monkey, and mouse CD98hc (described below). Lymphocytes from animals whose sera showed strong binding to CHO cells overexpressing human or mouse CD98hc were isolated and fused with myeloma fusion partners by electrofusion (Hybrimune, BTX, Holliston, MA) to generate mouse myeloma cells. The cells were incubated overnight at 37°C and 5% CO in Clonacell-HY Medium C (Stemcell Technologies, Vancouver, BC, Canada, catalog number 03803).

[0251] The next day, the fused cells were centrifuged and resuspended in Clonacell-HY Medium E (Stemcell Technologies, Catalog No. 03805) with the addition of HAT (Sigma Aldrich, Catalog No. H0262). Cells were seeded into T225 flasks and grown at 37°C and 5% CO2 for 6 days, after which the hybridoma library was cryopreserved. After thawing the mouse hybridoma library, the cells were resuspended in 10 ml of ClonaCell-HY Medium C supplemented with anti-mouse IgG Fc-FITC (Jackson ImmunoResearch, West Grove, PA) and then gently mixed with 90 ml of methylcellulose-based ClonaCell-HY Medium D containing the HAT component (Stemcell Technologies, Catalog No. 03804). Cells were seeded into Nunc OmniTrays (Thermo Fisher Scientific, Rochester, NY) and grown at 37°C and 5% CO2 for 11 days. IgG-positive colonies were selected using the Clonepix2 (Molecular Devices, Sunnyvale, CA) system and transferred to 96-well plates containing high-glucose DMEM medium supplemented with 10% Fetal Cone II serum (Hyclone SH30066.03, Cytiva, Malborough, MA), 1x GlutaMAX (Gibco 35050061, Thermo Fisher Scientific, Waltham, MA), and 20% Clonacell-HY medium E (Stemcell Technologies, catalog no. 03805). This procedure was performed for two hybridoma campaigns (numbers 3 and 4).

[0252] Alternatively, fused hybridomas were allowed to recover overnight, resuspended in ClonaCell-HY Medium C containing anti-mouse IgG Fc-FITC, and mixed with ClonaCell-HY Medium D. Cells were seeded, expanded, and picked using the Clonepix 2 system as described above. This method was repeated for two additional hybridoma campaigns (numbers 1 and 2).

[0253] A total of four hybridoma campaigns were performed, and a total of 898 IgG-positive hybridoma clones were isolated. After 6 days of culture, tissue culture supernatants from the hybridomas were screened by FACS on CHO cells overexpressing human or mouse CD98hc (described below).

[0254] Example 5. Screening of anti-CD98hc antibody hybridoma supernatants by FACS IgG-positive hybridoma supernatants (898 total) were screened by FACS for their ability to differentially bind to CHO cells overexpressing human or mouse CD98hc cells compared to parental CHO cells. Overexpressing cells were harvested, washed, and labeled with various concentrations and combinations of CellTrace cell proliferation dyes CFSE and Violet (ThermoFisher, Cat. Nos. C34554 and 34557, respectively) to generate unique barcoded cell populations. Barcoded cells (5 × 10 of each cell population) were collected, washed, and labeled with various concentrations and combinations of CellTrace cell proliferation dyes CFSE and Violet (ThermoFisher, Cat. Nos. C34554 and 34557, respectively) to generate unique barcoded cell populations. 4The cells were dispensed into a 96-well U-bottom plate and incubated with 50 μl of hybridoma cell culture supernatant or 1 μg / ml of commercially available purified mouse anti-human CD98hc monoclonal antibody (Sigma Aldrich, catalog number SAB 4700503) and rat anti-mouse CD98hc antibody (io-Rad Laboratories, Hercules, CA, catalog number MCA2684) on ice for 30 minutes. After this primary incubation, the supernatant was removed by centrifugation, and the cells were washed twice with 175 μl of ice-cold FACS buffer (PBS + 1% FBS + 2 mM EDTA). The cells were then incubated on ice for 20 minutes with anti-mouse IgG Fc-allophycocyanin (APC) or anti-rat IgG Fc-APC (Jackson Labs, Cat. Nos. 115-136-071 and 112-136-071, respectively) diluted 1:1000 in FACS buffer. After this secondary antibody incubation, the cells were washed twice again with ice-cold FACS buffer and resuspended in a final volume of 50 μl of FACS buffer containing 0.25 μl / well of propidium iodide (BD Biosciences, Cat. No. 556463). Binding intensity on cells was analyzed using a FACS Canto system (BD Biosciences) with a sorting gate set to exclude dead (i.e., propidium iodide-positive) cells. For each barcoded cell population, the ratio of the mean fluorescence intensity (MFI) of the APC divided by the MFI of the secondary antibody was calculated for each anti-CD98hc hybridoma supernatant tested and is shown in Table 5.

[0255] A total of 121 hybridoma clones from the four hybridoma campaigns showed MFI ratios greater than 2x for binding to CHO cells stably overexpressing human CD98hc. 103 of these clones also bound to HCMEC / D3 endothelial cells, which endogenously express human CD98hc, with MFI ratios greater than 5x. None of the hybridoma clones showed binding to CHO cells stably overexpressing mouse CD98hc. Thus, 121 of the 898 clones were determined to be "FACS positive," and 104 of these FACS-positive clones possessed unique CD98hc sequences. Data for a subset of FACS-positive clones are shown in Table 3.

[0256] Table 5. FACS MFI values ​​of unique anti-CD98hc antibodies identified from the hybridoma campaign TIFF2025528751000006.tif198165

[0257] Example 6. Screening of anti-CD98hc antibody hybridoma supernatants by recombinant anti-CD98hc protein binding assay Hybridoma culture supernatants (obtained in Example 5) from 121 hybridomas were screened for their ability to bind to Avi-His-tagged human, cynomolgus monkey, and mouse CD98hc (prepared as described in Example 1) and compared with binding to Avi-His-tagged human TfR as a 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 1 hour. The blocking buffer was removed, and human, cynomolgus monkey, or mouse Avi-His-tagged CD98hc and Avi-His-tagged human TfR proteins were added at 1 μg / ml in ELISA diluent and captured for 1 hour at room temperature. After washing three times with 300 μl of PBST (PBS + 0.05% Tween 20, Thermo Scientific 28352), hybridoma cell culture supernatant or 1 μg / ml of commercially available purified mouse anti-human CD98hc monoclonal antibody (Sigma-Aldrich, Cat. No. SAB4700503) and rat anti-mouse CD98hc antibody (Bio-Rad Laboratories, Hercules, CA, Cat. No. MCA2684) were added to each well (50 μl / well). After a 30-minute incubation at room temperature, each plate was washed three times with 300 μl of PBST. Anti-mouse IgG Fc-HRP or anti-rat IgG Fc-HRP (Jackson Immunoresearch, Cat. Nos. 115-035-071 and 112-036-071, respectively) secondary antibodies were diluted 1:5000 in ELISA diluent and added to each well at 50 μl / well. The wells were incubated for 30 minutes at room temperature with shaking. After a final set of washes (3 × 300 μl in PBST), 50 μl / well of BioFx TMB substrate (Surmodics, Eden Prairie, MN, Cat. No. TMBW-1000-01) was added to the wells.The reaction was then quenched after 5-10 minutes with 50 μl / well of 2N sulfuric acid. The absorbance of each plate at 450 nm was read on a SpectraMax M5 (Molecular Devices, Sunnyvale, CA) using SoftMax Pro software. ELISA data for selected antibodies are shown in Table 4 below.

[0258] From this screening of hybridoma supernatants, 120 of 121 anti-CD98hc hybridoma clones showed OD values ​​of more than 0.45 against human and cynomolgus monkey CD98hc. 450 One hybridoma clone, CD98HC.02.020, bound to mouse CD98hc.

[0259] Table 6. ELISA of unique anti-CD98hc antibodies identified from hybridoma campaigns 3 and 4 OD450 value TIFF2025528751000007.tif200165

[0260] Example 7. In vitro internalization of anti-CD98hc antibody into blood-brain barrier endothelial cell lines Supernatants from 121 anti-CD98hc hybridoma clones were purified using a ProPlus Phylip column (Biotage, Uppsala, Sweden, catalog number PTH 91-20-07) on a Hamilton STAR platform (Hamilton Company, Reno, NV). Briefly, antibodies from the supernatants were captured by protein A coupled to a resin-packed tip, washed twice with PBS, eluted with Pierce IgG elution buffer (ThermoFisher, catalog number 21004), and neutralized to a final pH of 6.0 with 1 M Tris-HCl pH 8. The concentration of the purified antibodies was determined by measuring absorbance at 280 nm using a Nanolop 8000 (ThermoFisher). Hybridoma-purified antibodies were then tested for their ability to internalize into endothelial cells.

[0261] Internalization into blood-brain barrier endothelial cells is the first step in transcytosis, leading to translocation across the BBB into the brain. To identify anti-CD98hc antibodies with internalization capacity, HCMEC / D3 cells were seeded at 2.5 × 10^4 cells / well in black-walled, clear-bottom 96-well plates (#3904, Corning). The next day, cells were treated with 6 μg / ml of anti-CD98hc antibody pre-bound to an equivalent concentration of pHrodo-Red labeling reagent (Z25612, Invitrogen) in 100 μl of culture medium (EBM2, Lonza). Anti-CD98hc reference antibodies of human IgG isotype and known internalization capacity were included in the assay as negative and positive controls, respectively.

[0262] Each plate was then placed in an IncuCyte machine (live cell analysis system) and images were taken every 2 hours for 24 hours. Images were then processed and analyzed using IncuCyte software. Internalization data (pHrodo Red-positive area μm) at the 24 hour time point were 2 / image) are shown in Table 5 as relative fold change to isotype hIgG1.

[0263] Table 7. Fold change in internalization of purified specific anti-CD98hc antibodies identified from the hybridoma campaign relative to isotype hIgG1. TIFF2025528751000008.tif173165

[0264] Example 8. Molecular cloning of anti-CD98hc antibodies Anti-CD98hc antibodies from the 121 hybridomas described above were cloned as follows: Anti-CD98hc antibodies were selected based on positive FACS data (described above in Example 5). 1-2 × 10 5Hybridoma cells were harvested, washed with PBS, and resuspended in 200 μl of RNAlater (Invitrogen, catalog no. AM7021). Samples were stored at -80°C and sent to Abterra Biosciences (San Diego, CA) for sequencing. Briefly, RNA was extracted and cDNA synthesis was performed. The variable regions of IgG / IgM, IgK, and IgL were amplified using proprietary primers in a 5' RACE method. Hybridoma variable region amplicons were sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA). Reads from the hybridomas were processed through Abterra's Reptor analysis pipeline. Exemplary unique amino acid sequences of the variable heavy and variable light chains of anti-CD98hc antibodies are shown in Table 8 below. Tables 9 and 10 show the CDR sequences (Kabat) for each of the unique VH and VL sequences in Table 8.

[0265] Table 8. Unique VH / VL sequences derived from hybridoma campaigns TIFF2025528751000009.tif120165TIFF2025528751000010.tif222165TIFF2025528751 000011.tif226165TIFF2025528751000012.tif198165TIFF2025528751000013.tif87165

[0266] Table 9: CDR heavy chain sequences derived from hybridoma campaigns TIFF2025528751000014.tif99165TIFF2025528751000015.tif214165TIFF2025528751000016.tif141165

[0267] Table 10: CDR light chain sequences derived from hybridoma campaigns TIFF2025528751000017.tif65165TIFF2025528751000018.tif224165TIFF2025528751000019.tif157165

[0268] Example 9. Reformatting of Hybridoma Antibodies as 2+1 Bispecific Antibodies Antibodies for formatting into a 2+1 bispecific format were selected based on several criteria: 1) antibodies covering a wide range of affinities based on ELISA and FACS binding assays, 2) antibodies covering a wide range of internalization activities, 3) no particularly high-risk modification sites were identified in the CDRs, and 4) phylogenetic diversity within the resulting hybridoma sequences. Antibody cross-reactivity between human and cynomolgus CD98hc was also used as a selection criterion. From 121 anti-CD98hc antibodies, a panel of 24 anti-CD98hc hybridoma antibodies was selected based on these criteria for reformatting into scFvs in a VH-linker-VL format.

[0269] A non-target specific IgG isotype antibody ("inactive isotype control antibody") with knob-and-hole mutations in the heavy chain constant region was used to format a 2+1 bispecific antibody. The anti-CD98hc scFv (VH and VL) was attached to the IgG isotype antibody via a linker at the C-terminus of the constant region with the "hole" mutation (the "hole" side of the IgG isotype antibody). The IgG isotype antibody conjugated to the scFv is referred to as a 2+1 bispecific antibody, as shown in Figure 1A. The 20-amino acid linker connecting the VH and VL domains within the scFv was GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 182) (Bird et al., Science 1988. Oct 21;242(4877):423-6), and the linker connecting the C-terminus of the Fc "hole" domain to the scFv was (GGGGS)x3 (SEQ ID NO: 183). DNA encoding the 2+1 bispecific antibody was prepared by gene synthesis and cloned into the expression vector pcDNA3.4 (ThermoFisher).

[0270] The scFv (VH and VL) sequences were formatted into IgG heavy chain expression constructs with "hole" mutations ("heavy chain hole" constructs) using the framework of SEQ ID NO: 184. Corresponding heavy chain knob and light chain expression constructs were also generated (SEQ ID NOs: 186-187). In some cases, the "heavy chain hole" construct contained a series of mutations (H435R, Y436F) to minimize binding to Protein A (Tustian et al., MAbs. May-Jun 2016;8(4):828-38) (SEQ ID NO: 185). In some cases, the heavy chain hole and heavy chain knob also contained mutations that reduced effector function, such as L234A / L235A / P331S (LALAPS) or N325S / L328F (NSLF) (SEQ ID NOs: 188-193).

[0271] Exemplary structures of 2+1 bispecific antibodies included the following elements: 1) an isotype control hIgG1 wild-type antibody with knob (T366W) and hole (T366S_L368A_Y407V) mutations 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. Table 11 shows exemplary heavy and light chain sequences with different knob or hole mutations.

[0272] Table 11. Sequences of 2+1 bispecific antibodies TIFF2025528751000020.tif175165TIFF2025528751000021.tif218165TIFF2025528751000022.tif173165 TIFF2025528751000023.tif213165TIFF2025528751000024.tif162165TIFF2025528751000025.tif212165

[0273] To generate the 2+1 bispecific antibody, transient transfection of Expi293 cells (Invitrogen) was performed according to the manufacturer's instructions. For a 20 mL culture, 20 μg of total DNA consisting of three expression plasmids (heavy chain-hole-scFv sequence, heavy chain-knob sequence, and light chain sequence) was used. The molar ratio of heavy chain-knob, heavy chain-hole-scFv, and light chain plasmids was optimized to 1:3:6 to obtain high purity of the 2+1 bispecific antibody. Five days after transfection, cell culture supernatant was collected. The clarified supernatant was purified using a drip column containing MabSelect Sure resin (Cytiva), washed with PBS, eluted with pH 3.5 elution buffer, and neutralized with Tris-HCl to a final pH of 5.5-6.0. The neutralized eluate containing the antibody was dialyzed into PBS. Antibody concentration was quantified by measuring absorbance at 280 nm using a Nanodrop 8000 (ThermoFisher) or Lunatic (Unchained Labs). Purity of the 2+1 bispecific antibodies was determined by SDS-PAGE. The 2+1 bispecific antibodies were analyzed for aggregation by size-exclusion chromatography (SEC). Twenty-two of the 24 antibodies were then produced as 2+1 bispecific antibodies, and 22 of the 2+1 bispecific antibodies were analyzed by ELISA, FACS, and internalization to confirm that binding in the 2+1 format was retained (data not shown).

[0274] Example 10. Generation and screening of anti-CD98hc antibodies using single B cell cloning Anti-CD98hc antibodies were also generated by B cell cloning technology. BALB / c mice (Charles River Laboratories, Wilmington, MA) were immunized twice weekly by subcutaneous or intraperitoneal injection of purified extracellular domain polypeptides of human, cynomolgus monkey, and / or mouse CD98hc (obtained as described above in Example 1) with or without adjuvant. After a total of 14 injections, lymph nodes and spleens were harvested from mice whose sera showed strong binding to CHO cells overexpressing human or mouse CD98hc by FACS 3 days after the final boost.

[0275] These tissues were processed into single-cell suspensions and enriched for B cells using a negative selection magnetic bead kit (Stemcell Technologies, Vancouver, BC, Canada, Cat. #19844). Cells were blocked with anti-mouse CD16 / CD32 (2.4G2, Cat. #553142, BD Biosciences, San Jose, CA) and an unlabeled, irrelevant Avi-His-tagged protein. Next, cells were stained with AF488- and AF647-tagged huCD98hc Avi-His proteins using the Lightening Link kit (Novus Biologics, Cat. #ab236553, 336-0010, Littletown, CO), anti-mouse IgG BV605 (Poly4053, Biolegend, Cat. #405327, San Diego, CA), and anti-mouse IgM BV786 (II / 41, BD Bioscience, Cat. #743328). After washing, cells were stained with anti-mouse CD19 BV421 (6D5, Biolegend, catalog no. 115537), anti-mouse CD138 BV421 (281-2, Biolegend, catalog no. 142508), and anti-human CD3 PE (UCHT1, Biolegend, catalog no. 100206). Finally, the viability dye 7-AAD (BD Biosciences, catalog no. 559925) was added prior to sorting. CD19- and / or CD138-positive plasma blast and memory B cell populations that were IgM-negative, IgG-positive, and human CD98hc-positive were bulk-sorted using a FACS ARIA II cell sorter (BD Biosciences).

[0276] Sorted cells were processed for single-cell B cell receptor sequencing using Chromium Single Cell 5' Gel Beads v2 and loaded into three wells of a K-chip for gel bead-in-emulsion (GEM) formation using a Chromium controller (10X Genomics, Pleasanton, CA, catalog numbers 1000266 and 1000287). After GEM cleanup, the GEM-RT product was purified, and cDNA was amplified for the antibody heavy and light chains using the Chromium Single Cell Mouse BCR Amplification Kit (10X Genomics, catalog number 1000255). GEX and VDJ libraries were prepared according to the manufacturer's instructions, evaluated with the Agilent BioAnalyzer High Sensitivity DNA Kit, and quantified by qPCR (KAPA Library Quantification Kit, catalog number 07960140001, Roche Sequencing, Wilmington, MA). Libraries were dual-index sequenced using an Illumina 150-cycle kit on an Illumina MiSeq or NovaSeq (Illumina, San Diego, CA). Sequencing data were processed with the 10x Genomics Cell ranger mkfastq, count, and vdj pipelines. The resulting VDJ sequences were post-processed to identify unique IgG pairs.

[0277] 450 unique sequences were identified, from which a panel of 94 sequences with diverse CDRH3 sequences were selected, synthesized, and expressed in a 2+1 bispecific antibody format.

[0278] To generate the 2+1 bispecific antibodies, transient transfection of Expi293 cells (Invitrogen) was performed in a 96-well deep-well block according to the manufacturer's instructions. For each 0.8 mL culture in each well of the 96-well deep-well block, 0.8 μg of total DNA consisting of three expression plasmids (heavy chain-hole-scFv, heavy chain-knob, and light chain sequences) was used. The molar ratio of the three plasmids (heavy chain-knob, heavy chain-hole-scFv, and light chain plasmids) was optimized to 1:3:6 to obtain high purity of the 2+1 bispecific antibodies. Five days after transfection, cell culture supernatants were harvested. The clarified supernatants were purified using ProPlus Phylip columns on a Hamilton STAR platform as described in Example 7. The concentration of the purified antibodies was determined by measuring absorbance at 280 nm using a Nanolop 8000 (ThermoFisher).

[0279] Purified 2+1 anti-CD98hc bispecific antibodies were screened for binding to human and cynomolgus CD98hc by ELISA as described in Example 6. Purified 2+1 bispecific antibodies were screened at concentrations ranging from 1 to 30 μg / mL. ELISA binding to human and cynomolgus CD98 and a control cynomolgus CD98hc AVI-His-tagged protein was calculated as a ratio to the secondary antibody alone, and the values ​​are listed in Table 10A. Purified 2+1 bispecific antibodies were also screened by FACS for their ability to bind to CHO cells overexpressing human or mouse CD98hc or cells endogenously expressing human CD98hc. Antibodies were incubated with transfected CHO, parental CHO, or HCMEC / D3 cells, washed, and then stained with goat anti-human IgG Fc (Jackson Immunoresearch, 109-136-098) and labeled with fixable viability eFluor 450 (eBioscience, catalog 65-0863-14). Data were acquired using a FACS Canto (BD Biosciences). The ratio of mean fluorescence intensity binding to transfected cell lines relative to parental CHO is shown in Table 12B. The ratio of binding to endogenously expressing human brain endothelial cells relative to the secondary antibody alone is also shown in Table 12B. Of the 94 2+1 anti-CD98hc bispecific antibodies screened, 47 were positive by ELISA, showing signals at least 5-fold higher than the secondary antibody alone, and 16 were positive by ELISA and FACS, with FACS binding increased by 3-fold or more compared to non-expressing cells. The sequences of the 2+1 anti-CD98hc anti-CD98hc bispecific antibodies that showed binding by ELISA and FACS are shown in Table 13. The CDR sequences (Kabat) are shown in Tables 14 and 15.

[0280] Table 12A. ELISA characterization of 2+1 anti-CD98hc bispecific antibodies derived from single B cell cloning TIFF2025528751000026.tif140165

[0281] Table 12B. FACS characterization of anti-CD98hc 2+1 bispecific antibodies derived from single B cell cloning TIFF2025528751000027.tif146165

[0282] Table 13. Antibody sequences of anti-CD98hc 2+1 bispecific antibodies derived from single B cell cloning TIFF2025528751000028.tif207165TIFF2025528751000029.tif211165TIFF2025528751000030.tif145165

[0283] Table 14. Heavy chain CDR sequences of anti-CD98hc 2+1 bispecific antibodies derived from single B cell cloning TIFF2025528751000031.tif187164

[0284] Table 15. Light chain CDR sequences of anti-CD98hc 2+1 bispecific antibodies derived from single B cell cloning TIFF2025528751000032.tif211165

[0285] Example 11. ELISA binding of 2+1 anti-CD98hc bispecific antibodies To measure the binding retention of the reformatted 2+1 anti-CD98hc antibody by ELISA, high-binding ELISA plates (Thermo Fisher) were coated with streptavidin (1 μg / ml, Thermo Fisher) overnight at 4°C. Plates were then washed three times with phosphate-buffered saline containing 0.05% Tween-20 (PBST) and incubated with blocking buffer (PBS + 1% BSA, 1 h, RT). Plates were washed three times again with PBST and incubated with biotinylated human CD98hc, cynomolgus monkey CD98hc, and mouse CD98hc, as well as the irrelevant antigen, human transferrin receptor (1 μg / ml, 1 h, RT). Plates were washed three times again with PBST and incubated with anti-CD98hc antibody (1 μg / ml, 1 h, RT). Plates were washed again three times with PBST and incubated with a secondary detection antibody, anti-human horseradish peroxidase-conjugated antibody (dilution 1:5000, 30 min, room temperature). Plates were washed three times for the final time and incubated with TMB-ELISA substrate solution (Thermo Fisher) followed by quenching with H2SO4. Anti-CD98hc binding was then measured using a SpectraMax M5 (Molecular Devices) at an optical density of 450 nm (OD 450 ) were analyzed. The binding characteristics of various 2+1 anti-CD98hc bispecific antibodies to CD98hc (human, cynomolgus monkey, mouse) and control human CD98hc, shown as fold change compared to isotype hIgG, are summarized in Table 16 below. All 2+1 anti-CD98hc antibodies demonstrated binding specificity for human CD98hc compared to mouse CD98hc and the control, and showed varying degrees of cross-reactive binding to cynomolgus monkey CD98hc.

[0286] Table 16. ELISA OD of reformatted 2+1 anti-CD98hc bispecific antibodies 450 value TIFF2025528751000033.tif160165

[0287] Example 12. Binding of 2+1 anti-CD98hc bispecific antibodies by FACS To determine the binding retention of the 2+1 anti-CD98hc antibody by FACS, the antibody was evaluated for binding to HCMEC / D3 cells (a cell line derived from human brain endothelial cells), CHO cell lines overexpressing human and mouse CD98hc, and parental CHO cells for nonspecific binding. Cells were seeded (50,000 cells / well) in tissue culture plates, washed, and resuspended in FACS buffer (PBS + 2% BSA + 1 mM EDTA). The 2+1 anti-CD98hc antibody was then incubated with the cells (5 μg / ml, 1 hour on ice) and washed twice with FACS buffer. The cells were then incubated with allophycocyanin-conjugated anti-mouse secondary antibody (Jackson Immunoresearch) (1:1000, 30 minutes on ice) and then washed twice with FACS buffer. The cells were then resuspended in FACS buffer supplemented with 0.5% propidium iodide (Thermo Fisher). Anti-CD98hc binding was then analyzed by measuring mean fluorescence intensity (MFI) on an iQue flow cytometer (IntelliCyt). FACS data are summarized in Table 17, presented as fold change in MFI compared to an isotype hIgG1 control. Both 2+1 anti-CD98hc antibodies demonstrated binding specificity to hCMEC / D3 cells and, to a lesser extent, to the CHO-huCD98hc cell line.

[0288] Table 17. Fold change in FACS MFI of reformatted 2+1 anti-CD98hc bispecific antibodies TIFF2025528751000034.tif148165

[0289] Example 13. In vitro internalization of 2+1 anti-CD98hc bispecific antibodies into blood-brain barrier endothelial cell lines Internalization of 2+1 anti-CD98hc bispecific antibodies into hCMEC / D3 cells was measured as described above in Example 7. An anti-CD98hc hIgG1 antibody was included as a positive control, and an isotype hIgG1 antibody was included as a negative control. The fold change in total integrated intensity of the pHrodo-red signal per well compared to isotype hIgG1 was calculated and is summarized in Table 18 below. Table 18 shows that 22 of the 2+1 anti-CD98hc bispecific antibodies that retained target binding also demonstrated internalization. All 2+1 anti-CD98hc antibodies were internalized into hCMEC / D3 cells to varying degrees.

[0290] Table 18. Internalization values ​​of 2+1 anti-CD98hc bispecific antibodies TIFF2025528751000035.tif164165

[0291] Example 14. Epitope binning of 2+1 anti-CD98hc bispecific antibodies Epitope binning analysis was performed with a 2+1 anti-CD98hc bispecific antibody using a classic sandwich experiment using a Carterra LSA instrument (Carterra, Salt Lake City, UT). Briefly, HC200M was used for kinetic evaluation and then tested in a binning assay to test the immobilized antibody for its ability to form a sandwich pair with recombinant huCD98hc Avi-His and the injected antibody. In each cycle, 200 nM huCD98hc Avi-His was injected over the chip for 5 min, followed by a 5 min injection of the test antibody (diluted to 40 μg / ml in running buffer), followed by regeneration with two 30-s injections of Pierce™ IgG elution buffer (ThermoScientific). Data were processed and analyzed using Epitope high-throughput binning analysis software (Carterra). Antibodies that were able to bind to the antigen captured by the immobilized antibody were designated as "sandwich" or "paired" antibodies, and these antibodies were assigned to an epitope bin distinct from that of the immobilized antibody. A matrix of paired and unpaired antibodies was constructed from the binding results of these experiments, allowing the generation of an epitope bin landscape for the 2+1 anti-CD98hc bispecific antibody. Some antibodies had overlapping binning profiles, suggesting that these antibodies could recognize adjacent, but not completely overlapping, epitopes. Slight heterogeneity within each bin is indicated by assigning letters such as a, b, c, and d. For antibodies that appear to overlap in two epitope bins, the numbers are underlined to indicate the overlapping bin. Each epitope bin is summarized in Table 19. "Unique" means that the 2+1 anti-CD98hc bispecific antibody did not share an epitope with any other antibody (tested in the panel).

[0292] Table 19. Epitope bins of 2+1 anti-CD98hc bispecific antibodies TIFF2025528751000036.tif83165

[0293] Example 15. Binding Kinetics of 2+1 Anti-CD98hc Bispecific Antibody The binding kinetics of the humanized 2+1 anti-CD98hc bispecific antibody to human and cynomolgus monkey CD98hc Avi-His was evaluated using a Carterra LSA instrument (Carterra, Salt Lake City, UT). Briefly, the 2+1 anti-CD98hc bispecific antibody was prepared in duplicate by 50-fold dilution in 10 mM acetic acid, pH 5.0 (Carterra) to a final concentration of 20 μg / mL. Using a single-channel flow cell, an HC200M sensor chip (Carterra) was activated with a 7-minute injection of a 1:1:1 mixture of 100 mM MES pH 5.5, 100 mM sulfo-NHS, and 400 mM EDC (all reconstituted in MES pH 5.5; 100 μl of each was mixed in a vial immediately before the assay). After switching to the multichannel array flow cell, each antibody was injected for 15 minutes over four 96-spot arrays of the activated chip. The remaining unbound active groups on the chip were then blocked by injecting 1 M ethanolamine, pH 8.5 (Carterra) for 7 min using a single-channel flow cell. The resulting sensor chip contained eight spots for each antibody at four different densities. Two independent experiments were performed as follows, yielding an N of 1 to 8 determinations for each antibody. Spots with analyte binding of less than 20 RU were excluded from further analysis.

[0294] After priming with running buffer HBS-EP+ (Teknova) containing 0.5 mg / ml BSA (MP Biomedicals), the immobilized 2+1 anti-CD98hc bispecific antibodies were examined for their ability to bind several forms of CD98hc Avi-His, including the human and cynomolgus monkey orthologs described above. Affinity estimates were generated by injecting each analyte across the antibody array using a single-channel flow cell. CD98hc Avi-His analytes were diluted in running buffer in six 3-fold serial dilutions starting at 600 nM for human and cynomolgus monkey CD98hc Avi-His. Analytes were injected for 5 min, and dissociation was followed for 10 min. After each analyte injection, the antibodies were regenerated with Pierce IgG Elution Buffer (ThermoScientific). Data were processed and analyzed using NextGenKIT high-throughput kinetic analysis software (Carterra).

[0295] Equilibrium dissociation constants (KD) were calculated from the fitted on and off rate constants (k-on and k-off) for the anti-CD98hc Avi-His antibodies of the present disclosure. These values ​​were combined, the mean and standard deviation calculated, and graphs were generated using GraphPad Prism. The KD values ​​are summarized in Table 20 below. As shown in the table, some antibodies exhibited heterogeneous binding profiles that did not fit the 1:1 binding model, and therefore their rate constants could not be determined.

[0296] Table 20. Equilibrium dissociation constants (K D ) TIFF2025528751000037.tif45165TIFF2025528751000038.tif204165

[0297] These results demonstrate that the 2+1 anti-CD98hc bispecific antibodies exhibit a wide range of affinities for CD98hc Avi-His, ranging from approximately 3 nM to 200 nM. Specifically, the affinities of the 2+1 anti-CD98hc bispecific antibodies binding to human CD98hc Avi-His ranged from 3.1 nM to 210 μM, while the affinities of the anti-CD98hc antibodies of the present disclosure binding to cynomolgus monkey CD98hc Avi-His ranged from 3.2 nM to 145 nM.

[0298] Example 16. Ability of 2+1 anti-CD98hc bispecific antibodies to affect CD98hc levels or localization Binding of 2+1 anti-CD98hc bispecific antibodies to their receptors on target cells may result in dysregulation of their cell surface expression or recycling. To assess this, HCMEC / D3 cells (a cell line derived from human brain endothelial cells, Weksler B. et al., FASEB J. 2005;19(13):1872-4) were plated at 7.5 x 10^4 cells / cm in 100 μL of medium (EBM2, Lonza) in tissue culture plates. 2After 24 hours, cells were treated with 20 μg / ml of the 2+1 anti-CD98hc bispecific antibody for 24 hours and then analyzed by either flow cytometry (FACS) or Western blot. Quantitative FACS data revealed no decrease in cell surface expression of CD98hc after treatment with the 2+1 bispecific antibody (Figure 2). Similarly, quantitative Western blot analysis revealed that the total amount of CD98hc protein in antibody-treated cells did not change by more than 50% compared to the isotype control (Figure 3). Notably, cell surface expression of CD98hc increased upon treatment with some of these 2+1 anti-CD98hc bispecific antibodies, likely due to a feedback effect induced in cells in response to amino acid depletion. This phenotype has been reported in cells treated with JPH203 and BCH, which inhibit the activity of the LAT1-CD98hc complex (Maimaiiti M. et al., Scientific Reports. (2020) 10:1292; Haefliger et al., Journal of Experimental & Clinical Cancer Research (2018) 37:234). Therefore, 2+1 anti-CD98hc bispecific antibodies that significantly increased (>2-fold) CD98hc cell surface levels were excluded from further development (except CD98hc.04.003 due to other desirable properties (see other examples)).

[0299] Example 17. In vivo PK study of 2+1 anti-CD98hc bispecific antibodies in huCD98hc ECD mice Antibody Production Method: Antibodies for in vivo PK studies were generated by transient transfection of ExpiCHO cells (Invitrogen) according to the manufacturer's instructions. 320 μg of total DNA consisting of three expression plasmids (heavy chain-hole-scFv, heavy chain-knob, and light chain sequences) was used in a 400 mL culture. The molar ratio of the three plasmids was optimized to obtain a highly pure 2+1 anti-hCD98hc bispecific antibody. Cell culture supernatants were harvested 10 days after transfection. Protein A affinity chromatography was followed by ion exchange chromatography on an AKTA Avant 25 (Cytiva) to purify the antibody and remove product-related impurities. The purified antibody was dialyzed into PBS. Analytical characterization was performed by absorbance at 280 nm, CE-SDS, size exclusion chromatography, and endotoxin measurement.

[0300] Study Design: To establish in vivo proof-of-concept for brain penetration of the 2+1 anti-CD98hc bispecific antibody, knock-in (KI) mice were generated in which the ectodomain region of mouse CD98hc was replaced with that of human CD98hc in one of two alleles. This was done because the 2+1 anti-CD98hc bispecific antibody specifically binds to primate CD98hc but not mouse CD98hc. A two-dose approach was used to determine the brain uptake and peripheral clearance of the injected antibody over time. Briefly, groups of heterozygous (huCD98hc+ / -KI) mice (N=3) were administered 10 mg / kg of either isotype IgG or the 2+1 anti-CD98hc bispecific antibody on days 1 and 15. Blood samples were then collected from antibody-treated animals at different time points into serum separator tubes, allowed to clot at room temperature (RT), and then centrifuged. The resulting supernatant, which corresponds to the serum, was then transferred to a new tube and stored at −80° C. until analysis.

[0301] Brain sample preparation: Brain tissue was collected from antibody-treated mice on day 15 (i.e., 24 hours after secondary antibody injection). Prior to this, mice were anesthetized and cardiac perfused with 15 ml of PBS to flush out the blood vessels. Brain tissue was then crushed and homogenized in 1 ml of HBSS buffer (MilliporeSigma, No. 55037C) containing 10 mM HEPES (Gibco, No. 15630130) using a manual grinder. The sample was then centrifuged at 1000 g for 5 minutes to pellet the blood vessels. The supernatant, corresponding to the parenchyma, was then transferred to a new tube and mixed with 10x RIPA buffer (final concentration 1x) containing a protease and phosphatase inhibitor cocktail (cOmplete™ Mini Protase Inhibitor Cocktail, Roche, No. 11836153001). After 20 minutes of incubation at 4°C on a nutator, the samples were frozen on dry ice and stored at -80°C until analysis. After centrifugation through 18% dextran (70 kDa, product code 31390, Sigma-Aldrich), the vascular fraction was cleared from myelin debris by washing twice in HBSS buffer. The samples were then lysed in RIPA buffer (R0278, Sigma-Aldrich) and stored at -80°C until analysis. The validity of this method was confirmed using WB analysis, which revealed the absence of brain endothelial cell markers (e.g., CD31 and claudin-5) in the brain parenchyma (data not shown).

[0302] Pharmacokinetic (PK) analysis: Antibody concentrations in serum and brain samples were measured using the MSD (Meso Scale Discovery) method. Briefly, 50 μl / well of goat anti-human IgG1 Ab (Jackson ImmunoResearch, no. 109-005-097) was added to MSD plates at 1 μg / ml in PBS and incubated overnight at 4°C. Each plate was then washed three times with wash buffer (1x PBS, 0.05% Tween-20) and then incubated with blocking buffer (wash buffer containing 3% BSA) for 1 hour at room temperature. Serum (diluted 1:10,000 in PBS) and brain (diluted 1:2 in PBS) samples were then added in duplicate to each plate and incubated for 1 hour on a shaker at 500 RPM. Known concentrations of human IgG1 isotype Ab were included in the assay to generate a standard curve. Next, each plate was washed three times with wash buffer, and then 30 μl / well of 0.2 μg / ml Sulfo-Tag labeled goat anti-human antibody (R32AJ-5, Meso Scale Discovery) in PBS was added and incubated for 1 hour on a shaker at 500 RPM. Finally, after washing three times with wash buffer, 150 μl / well of 1× READ buffer (R92PC-2, Meso Scale Discovery) was added to each plate, and then they were read using a Sector Imager S600 instrument.

[0303] We measured antibody concentrations in the vascular-depleted brain and found that the levels of the 2+1 anti-CD98hc bispecific antibody in HET huCD98hc KI mice were up to two-fold higher than those of isotype IgG (Figure 4). Peripheral PK was determined by measuring antibody concentrations in plasma samples from antibody-treated mice (Figure 5). All 2+1 anti-CD98hc bispecific antibodies tested showed higher plasma clearance compared to isotype IgG in HET huCD98hc KI mice, consistent with CD98hc-mediated behavior in the periphery. Three of the 2+1 anti-CD98hc bispecific antibodies tested (CD98hc.04.062, CD98hc.04.063, and CD98hc.04.064) showed significant (5- to 17-fold) changes in brain / serum ratios at the 24-hour time point tested (Figure 6).

[0304] Example 18. Humanization of 2+1 anti-CD98hc bispecific antibodies Based on in vivo mouse data, the 2+1 anti-CD98hc bispecific antibody CD98hc.04.064 was selected for humanization and further development. CD98hc.04.064 was selected because it demonstrated the best brain penetration 24 hours after injection. One of the most common methods for humanizing non-human antibodies is to embed the CDRs of the non-human antibody into a human antibody acceptor framework. Often, such CDR embedding leads to reduced or complete loss of affinity of the humanized antibody due to framework perturbation. Therefore, to restore reduced or lost affinity, it may be necessary to retain specific residues in the mouse framework and replace them with human residues at the corresponding positions (backmutation). Therefore, it is important to accurately predict the residues to be retained in the context of the selected human antibody germline acceptor framework, which will preserve the function and paratope of the humanized antibody. Furthermore, maintaining or improving thermal stability and solubility is desirable for good manufacturability and downstream development.

[0305] Structure-based antibody modeling using the BioMOE module of MOE (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada) was applied to the humanization process of an anti-CD98hc mouse monoclonal antibody (mAb). Briefly, the VH and VL sequences of the mouse mAb to be humanized were compared with functional human VL, VH, LJ, and HJ germline sequences obtained from IMGT (http: / / www.imgt.org / ). Pseudogenes and ORFs were excluded. For each mouse mAb (query), the five most similar VL germline sequences and the five most similar VH germline sequences were selected and combined with the most similar VJ and HJ genes to generate 25 humanized sequences. CDRs embedded in the human frameworks were defined according to the AbM definition (http: / / www.bioinf.org.uk / abs / #cdrdef).

[0306] Two humanized sequences were selected based on the frequency of their VH and VL frameworks in the human repertoire. An Fv homology model was constructed using the query and humanized sequences. The Fv homology model was constructed using the BioMOE or Antibody Modeler modules of MOE (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada). The AMBER10:EHT force field was used for energy minimization throughout the antibody homology modeling process. Based on the Fv homology model, molecular descriptors, such as the interaction energy between the VL and VH, coordinate-based isoelectric point (3DpI), hydrophobic patches, and charged surface area, were calculated and analyzed and sorted using the scoring matrix provided by MOE. These molecular descriptors were used to prioritize humanized mAbs for downstream experimental procedures, such as protein expression, purification, and binding affinity testing, as well as functional assays.

[0307] The BioMOE module of MOE provides Mutation Site Properties, a tool to visualize and classify potential backmutation residues. A backmutation is defined as an amino acid substitution that replaces the humanized sequence back to the original query sequence. Using this tool, the original query (reference) was individually compared with the selected humanized variants in both the primary amino acid sequence and the 3D structure of the 3D Fv homology model.

[0308] Changes between the reference and humanized variants were classified based on differences in amino acid type, potential interactions with residues in the CDRs, potential effects on VL / VH pairing, and potential changes in hydrophobic and charged surface area in and near the CDRs.

[0309] Mutations close to the CDRs or VL / VH interface, with large charge differences, or involving strong hydrogen-bonding interactions were evaluated individually, and significant perturbing mutations were reverted to the original query residues. As a result, the humanized sequence may contain up to five backmutations. The query mouse mAb (mouse CD98hc.04.064) and humanized mAb are listed below as scFvs in Table 21.

[0310] Table 21. Humanized anti-CD98hc scFv sequences TIFF2025528751000039.tif166165TIFF2025528751000040.tif208165

[0311] Table 22: Humanized anti-CD98hc sequences (VH and VL of scFv sequences in Table 21) TIFF2025528751000041.tif178165TIFF2025528751000042.tif119165

[0312] Table 23. Humanized anti-CD98hc CDR sequences (Kabat) TIFF2025528751000043.tif191165TIFF2025528751000044.tif145165

[0313] Example 19. Affinity optimization and liability elimination of 2+1 anti-CD98hc antibodies Based on the characterization of the humanized variants, CD98hc.04.064.4a was selected for further engineering. Affinity optimization of the CD98hc.04.04.064.4a variant was designed by removing liability sequences, such as high-risk isomerization and oxidation sites, and replacing them with residues with high frequency at that position in the immune repertoire. Additionally, residues within the CDR regions predicted to have strong antigen-binding interactions were selected for mutational analysis and evaluation for further affinity maturation. The variants selected for production and further screening are shown in Table 24 below. Selected clones were generated as 2+1 anti-CD98hc bispecific antibodies in the format described above (Example 9).

[0314] Table 24. Affinity optimized 2+1 bispecific antibody variant sequences TIFF2025528751000045.tif220165TIFF2025528751000046.tif227165TIFF2025528751000047.tif77165

[0315] Table 25: Sequences of affinity optimized 2+1 bispecific antibody variants (VH and VL of sequences in Table 24) TIFF2025528751000048.tif221165TIFF2025528751000049.tif230165TIFF2025528751000050.tif87165

[0316] Table 26. CDR sequences of affinity optimized 2+1 bispecific antibodies (Kabat) TIFF2025528751000051.tif197165TIFF2025528751000052.tif220165TIFF2025528751000053.tif64165

[0317] Example 20. Evaluation of in vitro effector functions (CDC and ADCC) To investigate the mechanism of reticulocyte depletion seen with the 2+1 anti-CD98hc bispecific antibodies, we assessed the ability of these same antibodies to induce in vitro effector responses, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC).

[0318] Methods and Results for CDC: The ability of the 2+1 anti-CD98hc bispecific antibody to induce complement deposition was measured in the hCMEC / D3 cell line, which expresses high levels of both receptors. Target cells were detached, washed once in PBS, and diluted to 2 x 10^6 cells / mL in RPMI 1640 medium. 50 μL of target cells (1 x 10^5 cells per well) were dispensed into a round-bottom 96-well plate (Falcon #351177). 25 μL of 4x antibody diluted in the same medium was added to these cells. After incubating the cells and antibody at 37°C for 15 minutes, 25 μL of pooled complement human serum (Innovative Research, IPLA-CSER) was added per well as a complement source, and each plate was incubated for an additional 2 hours at 37°C. Cells were then washed twice with FACS buffer (PBS + 2% FBS + 1 mM EDTA) and resuspended in 50 μL of FACS buffer + 0.25 μL / well of propidium iodide (Fischer Scientific, BD556463) before analysis on an iQue flow cytometer (IntelliCyt). CDC was analyzed as the percentage of PI+ single cells in each well. None of the 2+1 anti-CD98hc bispecific antibodies induced a significant CDC response in this assay (data not shown).

[0319] ADCC Methods and Results: The ability of the 2+1 bispecific antibodies to induce antibody-dependent cellular cytotoxicity (ADCC) was assessed using the Promega ADCC Reporter Bioassay System (#G7010). This system relies on an engineered Jurkat T cell line stably expressing the FcgRIIIa receptor (V158 variant) and an NFAT response element to drive the expression of firefly luciferase. Target cells were diluted to a concentration of 1.2 x 10^6 per mL in assay buffer (RPMI + 4% low IgG serum), and 25 μL of cells (30,000 per well) were dispensed into appropriate wells of a 96-well white assay plate. 25 μL of 3x antibody, also diluted in assay buffer, was added to the wells containing the cells. After antibody addition to the target cells, the provided effector cells (frozen at 2 x 10^7 / mL) were thawed at 37°C, and 630 μL was added to 3.6 mL of warmed (37°C) assay buffer, gently mixed, and 25 μL of effector cells (75,000 cells per well, for an E:T ratio of 2.5) was immediately added to the wells containing the target cells and antibody. The plate was then incubated at 37°C and 5% CO2 for 6 hours to activate the receptor cells and allow luciferase expression. After 6 hours, the plate was equilibrated to room temperature (15 minutes) while Bio-Glo Luciferase Assay Reagent was prepared. 75 μL of luciferase assay reagent was added to each well, the plate was incubated for 10 minutes, and luminescence was measured using a BioTek plate reader.

[0320] In the wild-type human IgG1 format, certain of the 2+1 bispecific antibodies against CD98hc (CD98hc.04.064 and CD98hc.04.003) were able to induce significant ADCC signals (Figure 7), demonstrating the advantage of using an inactive Fc.

[0321] Example 21.04.064 Humanized Panel Affinity Data The binding kinetics of humanized anti-CD98hc antibodies were assessed using a Biacore T200 (Cytiva). Briefly, IgG was diluted to 10 μg / mL and captured using a Protein A / G (ThermoFisher, no. 21186) surface on a CM5 chip prepared by amine coupling according to the instrument manufacturer's recommendations. The captured antibodies were tested for their ability to bind to human and cynomolgus CD98hc.

[0322] Recombinant human and cynomolgus CD98hc analytes, as described in the methods above, were diluted to a concentration of 1000 nM in running buffer (HBS-EP+, Teknova, #8022, 0.5 mg / mL BSA, MP Biomedicals LLC, #820451), followed by three-fold serial dilutions to 250, 62.5, and 15.6 nM. Each sample was injected for 2 min to allow binding, followed by 10 min of dissociation with buffer alone. After each sample injection, the chip was regenerated three times with 30-second injections of 10 mM glycine, pH 1.7. Fresh antibody was captured at the start of each cycle.

[0323] Data was analyzed using Biacore evaluation software to generate kinetic constants. Equilibrium dissociation constants (KD) were calculated from the fitted on- and off-rate constants (k-on and k-off) for each of the anti-CD98hc bispecific antibodies. These values ​​are summarized in Table 27 below.

[0324] Table 27. Equilibrium dissociation constants (K D ) TIFF2025528751000054.tif36165

[0325] The data show that the 4.064 humanized panel bound to human CD98hc in the range of approximately 18 nM to 35 nM and to cynomolgus CD98hc in the range of approximately 340 nM to 1.5 μM.

[0326] Example 22. Serum and brain PK data of CD98hc.04.064 humanized panel after intravenous injection into hCD98hc+ / +KI mice Antibody production methods: The 2+1 antibody for in vivo PK studies as well as a matched control antibody (with the same Fab and Fc domains but lacking the scFv targeting CD98hc) were generated and purified by transient transfection of ExpiCHO cells (Invitrogen) according to the manufacturer's instructions, as described above in Example 17.

[0327] Study Design: This study used homozygous knock-in (KI) mice (described in Example 17) in which the ectodomain region of mouse CD98hc in both alleles was replaced with that of human CD98hc (huCD98hc+ / +). A two-dose study was performed to determine the time course of brain uptake and peripheral clearance of injected Abs. Briefly, groups of homozygous (huCD98hc+ / -KI) mice (N=3) were intravenously administered 10 mg / kg of either isotype IgG or 2+1 anti-CD98hc bispecific antibody on days 1 and 15. Blood samples were then collected from antibody-treated animals at different time points into serum separator tubes, allowed to clot at room temperature (RT), and then centrifuged. The resulting supernatant, corresponding to serum, was then transferred to new tubes and stored at -80°C until analysis.

[0328] Samples from blood vessel-depleted brain and serum were processed and analyzed as described in Example 17.

[0329] Figure 8 shows antibody levels in the blood-depleted brain fraction of huCD98hc KI+ / + mice 24 hours after a second 10 mg / kg dose of 2+1 anti-CD98hc and control antibodies. Antibody levels are shown as fold change relative to a matched control with the same Fab and Fc but lacking the scFv binding domain for CD98hc. As shown in Figure 8, levels of the 2+1 anti-CD98hc bispecific CD98hc.04.064 humanized lead antibody were determined to be 3-4.5-fold higher than TD1 IgG in the blood-depleted brain fraction of huCD98hc+ / + KI mice. Peripheral PK was determined by measuring antibody concentrations in plasma samples from antibody-treated mice.

[0330] Figure 9 shows antibody levels in the serum of huCD98hcKI+ / + mice 24 hours after a second 10 mg / kg dose of 2+1 anti-CD98hc and control antibodies. Antibody levels in the vascular-depleted brain fraction of huCD98hcKI+ / + mice 24 hours after a second 10 mg / kg dose of 2+1 anti-CD98hc and control antibodies. As shown in Figure 9, all tested 2+1 anti-CD98hc bispecific 04.064 humanized antibodies showed similar clearance rates in huCD98hc+ / +KI mice compared to the control IgG. CD98hc.04.064.1e was further engineered.

[0331] Example 23. Affinity data for engineered variants of CD98hc.04.064 The binding kinetics of the 2+1 anti-CD98hc bispecific antibody to recombinant human and cynomolgus monkey CD98hc ECD Avi-His was evaluated using a Biacore T200 instrument (Global Life Sciences Solutions USA LLC, Marlborough, MA). The 2+1 anti-CD98hc bispecific antibody was prepared by diluting it to 50 μg / mL in running buffer HBS-EP+ (Teknova) containing 0.5 mg / mL BSA (MP Biomedicals) and captured on a goat anti-human kappa polyclonal antibody (Southern Biotech) surface prepared according to the instrument manufacturer's recommendations. Two independent experiments were performed as follows, yielding an N between 1 and 3 determinations for each antibody. Affinity estimates were obtained by injecting each analyte over the antibody captured on flow cell 2, 3, or 4. The CD98hc ECD Avi-His analyte was diluted in running buffer in five 3-fold serial dilutions starting at 600 nM. Analytes were injected from lowest to highest concentration without regeneration for 5 min each, and dissociation was followed for 15 min. After each series of analyte injections, each antibody was regenerated with 10 mM glycine pH 2.0 buffer (Carterra). Data were processed and analyzed using Biacore T200 BiaEvaluation software (Cytiva).

[0332] The equilibrium dissociation constant (K) was calculated from the fitted on and off rate constants (k-on and k-off) for the 2+1 anti-CD98hc bispecific antibodies of the present disclosure that bind to human and cynomolgus monkey CD98hc ECD Avi-His. D For N>1 measurements, the mean and standard deviation were calculated, and measurements from all samples were combined and graphed using GraphPad Prism. D The values ​​are summarized in Table 28 below.

[0333] Table 28. Equilibrium dissociation constants (K) of the 2+1 humanized variants of CD98hc.04.064 in a 2+1 format D ) TIFF2025528751000055.tif113165

[0334] These results demonstrate that the 2+1 anti-CD98hc bispecific antibody exhibits a wide range of affinities for human and cynomolgus monkey CD98hc ECD Avi-His, ranging from approximately 13 nM to 880 nM. Specifically, the affinities of the 2+1 anti-CD98hc bispecific antibody for binding to human CD98hc ECD Avi-His ranged from 12 nM to 34 nM, whereas the affinities for binding to cynomolgus monkey CD98hc ECD Avi-His ranged from 120 nM to 880 nM.

[0335] Example 24. In vitro sTREM2 assay of TD1-CD98hc bispecific antibody To test the function of the TD1-CD98hc antibody (used as a PK / PD model for CD98hc-mediated antibody delivery into the brain), an sTREM2 assay was performed. Human monocytes were isolated from whole blood using RosetteSep Human Monocyte Enrichment Cocktail (Stemcell Technologies) and Ficoll centrifugation according to the manufacturer's protocol. After erythrocyte lysis with ACK lysis buffer, monocytes were resuspended in complete medium (RPMI, 10% FBS, penicillin / streptomycin, L-glutamine, HEPES, non-essential amino acids, and sodium pyruvate). To obtain macrophages from these isolated monocytes, 50 ng / ml human M-CSF and 8% v / v human serum were added to the cells for 5–7 days.

[0336] Macrophages were differentiated from four human donors in two independent experiments. 10 per well 5 Macrophages were stimulated with 1 μg / mL of test antibody (huIgG1, TD1, or TD1-CD98hc) for 48 hours. Duplicate wells were processed as technical replicates. Each supernatant was tested for sTREM2 using the MSD platform.

[0337] Technical replicates for each donor were averaged, and then the fold change compared to the NSLF isotype control was calculated and graphed. Each point on the graph in Figure 10 represents an individual donor. A one-way paired analysis of variance was used to calculate statistical significance.

[0338] TD1-CD98hc was found to have activity in an in vitro sTREM2 assay compared to hIgG1, as shown in Figure 10. TD1-CD98hc also elevated sTREM2 to a similar or greater extent than TD1.

[0339] Example 25. TD1-CD98hc bispecific antibody and method for intravenous administration to non-human primates A bispecific antibody was generated in a 2+1 (whole) format containing (i) CD98hc.04.064.1e scFV and (ii) an antibody specific for human MSA4A (TD1) (termed "TD1-CD98hc"). The complete amino acid sequence of TD1-CD98hc is as follows:

[0340] Light chain variable region: DVVMTQSPLSLPVTLGQPASISCKSSRSLLYSAGKTYLSWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGIDFHQTFGGGTKVEIK (SEQ ID NO: 405).

[0341] Light chain constant region: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 406).

[0342] Heavy chain variable region: QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGVPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLVTVSS (SEQ ID NO: 407).

[0343] CH+Fc #1 (Knob, T366W): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 408).

[0344] CH+Fc #2 (Whole, T366S, L368A, Y407V): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 409).

[0345] Linker targeting domain: GGGGSGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGIIDPSDSETHYAQKFQGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARASYGKGYFDYWGQGTLVTVSSGGSEGKSSGSGSESKSTGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLNNINQKNYLAWYQQKPGQPPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSSPFTFGGGTKVEIK (SEQ ID NO: 410).

[0346] The complete amino acid sequence of an antibody (TD1) specific for human MSA4A alone is as follows:

[0347] TD1 heavy chain hIgG1 NSLF (N325S, L328F) QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGVPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 411).

[0348] TD1 light chain DVVMTQSPLSLPVTLGQPASISCKSSRSLLYSAGKTYLSWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGIDFHQTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 412).

[0349] PK / PD studies were conducted in naive male cynomolgus monkeys (n=9) after intravenous (iv) administration of 20 mg / kg of each of the three antibodies on days 1 and 29 (total of two doses) (n=3).

[0350] Table 29. In vivo administration of anti-TD1-CD98hc bispecific antibodies TIFF2025528751000056.tif46165

[0351] The intravenous dose was administered via the saphenous vein as a slow push injection. The target dose level (mg / kg), target dose concentration (mg / mL), and target dose volume (mL / kg) were consistent across groups 1 to 3.

[0352] Example 26. Hematological parameters after intravenous administration of TD1-CD98hc bispecific antibody to non-human primates Blood samples (approximately 2 mL) for hematological analysis were collected from the femoral vein into tubes containing potassium EDTA pre-dose and post-dose on days 3, 8, 15, 29, and 31. The parameters listed in Table 30 below were tested.

[0353] Table 30. Hematological parameters tested after intravenous administration of anti-TD1-CD98hc bispecific antibody TIFF2025528751000057.tif48165

[0354] FIG. 11 shows the absolute reticulocyte counts over the course of the study.

[0355] Overall, no significant changes were observed in hematological parameters, and no consistent reduction in reticulocytes was observed throughout the study.

[0356] Example 27. CSF penetration of TD1-CD98hc bispecific antibody after IV administration in non-human primates Cerebrospinal fluid (CSF) and serum were collected from NHPs at different times after the first and second doses as described in Example 25. Serum and CSF were tested as described below.

[0357] Sandwich ELISA: 100 μL / well of goat anti-human IgG monkey-adsorbed biotin (Southern Biotech; catalog no. 2049-08) working solution (0.5 μg / mL) was added to a 96-well Pierce™ streptavidin-coated high-binding plate with SuperBlock™ blocking buffer (ThermoScientific, reference no. 15500) and incubated for 1 hour ± 10 minutes at room temperature (RT) on a plate shaker at 350 revolutions per minute (RPM). Serum or CSF quality control (QC) and test samples were diluted to the minimum required dilution (MRD) in assay buffer (1× TBS, 0.05% Tween-20, and 0.1% BSA) before loading onto the assay plate. Standard calibration curves and assay buffer QCs (AB-QC) were added directly to the assay plate. After incubation was complete, the plate was washed three times with wash buffer (1x PBS and 0.05% Tween-20) using a plate washer and 100 μL / well of calibration standards, QCs, and MRD-diluted serum or CSF QCs. Test samples were then added to the plate and incubated for 2 hours ± 15 minutes on a plate shaker at 350 RPM at room temperature. The plate was then washed, and 100 μL / well of goat anti-human IgG monkey-adsorbed-HRP (Southern Biotech, catalog no. 2049-05) working solution (0.08 μg / mL) was added to the plate and incubated for 1 hour ± 10 minutes on a plate shaker at 350 RPM at room temperature. After the HRP incubation, the plate was washed, and 100 μL / well of tetramethylbenzidine (TMB) substrate solution (Surmodics Product No. TMBS-1000-01) was added and incubated for 15±10 minutes at room temperature (RT), protected from light by covering with aluminum foil or placing in a drawer. After the incubation was complete, the reaction was stopped by adding 100 μL / well of stop solution (Surmodics Product No. NSTP-1000-01) to the plate. The plate was then placed on a plate shaker at 350 RPM for at least 2 minutes at room temperature.Plates were then read on a Molecular Devices SpectraMax M5 instrument, and raw optical density (OD450-540) signals were generated from each well. The concentration of analyte in each unknown sample was determined by interpolation of concentrations using a calibration standard curve, using a weighting factor of 1 / Y, by SoftMax Pro (Molecular Devices / version 7.1). 2 Standard regression was performed using a four-parameter logistic (4-PL) model with:

[0358] Figure 12 shows that TD1-CD98hc showed increased serum clearance in NHPs compared to TD1 alone and isotype control. Furthermore, as shown in Figure 12, an increase in CSF Cmax was observed in NHPs with TD1-CD98hc administration compared to TD1 alone. This increase was approximately 13-fold at 2 hours, approximately 10-fold at 6 hours, and approximately 4-fold at 24 hours after the initial 10 mg / kg dose, but decreased from the 96 hour time point onwards.

[0359] Example 28. Effect of the CD98hc binding arm on brain uptake and brain pharmacodynamics in non-human primates Brain sample preparation: Brain tissue was collected from antibody-treated cynomolgus monkeys on day 30 (i.e., 48 hours after secondary antibody administration) and then frozen. Prior to this, the animals were anesthetized and cardiac perfused with PBS to wash out the intravascular space. The brain tissue was then thawed, crushed, and homogenized in HBSS buffer (MilliporeSigma, No. 55037C) containing 10 mM HEPES (Gibco, No. 15630130) using a manual grinder. The sample was then centrifuged at 1000 g for 5 minutes to pellet the vascular compartment. The supernatant, corresponding to the parenchyma, was then transferred to a new tube and mixed with 10x RIPA buffer (final concentration 1x) containing a protease and phosphatase inhibitor cocktail (cOmplete™, Mini Protase Inhibitor Cocktail, Roche, No. 11836153001). After incubation on a nutator at 4°C for 45 minutes, the samples were frozen on dry ice and stored at -80°C until analysis. After centrifugation through 18% dextran (70 kDa, product no. 31390, Sigma-Aldrich), the vascular fraction was cleared from myelin debris by washing twice in HBSS buffer. The samples were then lysed in RIPA buffer (R0278, Sigma-Aldrich) and stored at -80°C until analysis. We previously confirmed the validity of this method using Western blot analysis, which showed the absence of brain endothelial cell markers (e.g., CD31 and claudin-5) in the brain parenchyma (data not shown).

[0360] Pharmacokinetic (PK) analysis: Antibody concentrations in brain samples were measured using the MSD (Meso Scale Discovery) method. Briefly, 50 μl / well of goat anti-human IgG1 antibody at 1 μg / ml in PBS was added to MSD plates and incubated overnight at 4°C on a 500 RPM shaker. Each plate was then washed three times with wash buffer (0.05% Tween-20 in 1x PBS) and then incubated with blocking buffer (wash buffer containing 3% BSA) at room temperature for 1 hour. Brain samples (diluted in PBS) were then added in duplicate to each plate and incubated for 2 hours at room temperature on a 500 RPM shaker. Next, each plate was washed three times with wash buffer, followed by the addition of 40 μl / well of 0.5 μg / ml sulfo-tagged goat anti-human Ab (R32AJ-5, Meso Scale Discovery) in PBS and incubation for 1 hour on a 500 RPM shaker. Finally, after three washes with wash buffer, 150 μl / well of 1x READ buffer (R92PC-2, Meso Scale Discovery) was added to each plate, which was then read using a Sector Imager S600 instrument. Eleven two-fold serial dilutions of each antibody (100–0.019 ng / ml) were used to generate standard curves for each treatment. These curves were fitted using a four-parameter logistic regression to calculate the antibody concentrations in the samples.

[0361] Figure 13 shows that the CD98hc BBB-targeting arm was able to increase brain uptake of TD1-CD98hc by up to 3-fold in the frontal cortex and up to 4-fold in the endocrine cortex compared to TD1 alone, with no significant increase observed in the hippocampus.

[0362] Example 29. Pharmacodynamic analysis of TD1-CD98hc bispecific antibody in NHP samples CSF obtained from NHP test subjects as described in Example 27 was further tested for pharmacodynamic purposes as described below.

[0363] Summary of CSF1R Method (Brain Lysate)Colony-stimulating factor 1 receptor (CSF1R) concentrations in cynomolgus monkey brain lysates were measured using an enzyme-linked immunosorbent assay (ELISA) kit (catalog no. DY329) from R&D Systems. Mouse anti-human M-CSF R capture antibody (R&D Systems, part no. 841246) was diluted to a working concentration of 4 μg / mL in 1x PBS (Corning, reference no. 21-040-CM) and coated onto a 96-well microplate (Nunc-Immuno Maxisorp, ThermoScientific, catalog no. 446612) at 100 μL / well and incubated overnight at room temperature without shaking. The next day, the plate was washed three times with wash buffer (1x PBS and 0.05% Tween-20), blocked with 300 μL of reagent diluent (R&D Systems, part no. 841380), and incubated for at least 1 hour without shaking. Human M-CSF R standards and QCs (R&D Systems, part number 841248) were prepared in reagent diluent. Test samples and QCs were diluted to the minimum required dilution (MRD) in assay buffer (1x PBS, 1% BSA, and 0.05% Tween-20). The plate was then washed, and 100 μL / well of human M-CSF R standards, diluted QCs in assay buffer, and diluted samples were added to the appropriate wells and incubated for 2 hours at room temperature. The plate was washed, and 100 μL / well of a 100 ng / mL biotinylated goat anti-human M-CSF R detection antibody (R&D Systems, part number 841247) working solution was added and incubated for 2 hours at room temperature. After incubation was complete, the plate was washed, and then a 1:200 dilution of streptavidin-HRP (R&D Systems, part number 890803) in reagent diluent was added and incubated for 20 minutes at room temperature. The plate was washed a final time, and 100 μL / well of substrate solution (R&D Systems, part numbers 895000 and 895001) was added and incubated for 20 minutes at room temperature. After incubation was complete, 50 μL / well of stop solution (R&D Systems, part number 895032) was added to the plate and mixed thoroughly.The plates were then read on a Molecular Devices SpectraMax M5 instrument, which revealed raw optical density (OD. 450~540 ) signal was generated from each well. The concentration of CSF1R in each unknown sample was determined by interpolation against a calibration standard curve. SoftMax Pro (Molecular Devices / Version 7.1) was used to calculate the CSF1R concentration using a weighting factor of 1 / Y 2 Standard regression was performed using a four-parameter logistic (4-PL) model with:

[0364] Summary of CSF1 Methods (CSF) Colony-stimulating factor 1 (CSF1) concentrations in cynomolgus monkey cerebrospinal fluid (CSF) samples were measured using an electrochemiluminescence (ECL) assay kit from Meso Scale Discovery (MSD, catalog no. K151XRK-4). A 96-well MSD GOLD small spot streptavidin plate (MSD, catalog no. L45SA-1) was coated with 25 μL / well of biotinylated anti-human CSF1 antibody (MSD, catalog no. C21XR-3) diluted 1:17.5 in Diluent 100 (MSD, catalog no. R50AA-4) and incubated for 1 hour at room temperature on a plate shaker set at 700 revolutions per minute (RPM).

[0365] Standards were prepared in Diluent43 (MSD, Cat. No. R50AG-2) and further diluted 1:1 in assay buffer before loading onto the plate. QCs were prepared in Diluent43 and diluted with MRD in assay buffer. The diluted QCs were further diluted 1:1 with Diluent43 before loading onto the plate. Test samples were diluted with MRD in assay buffer. The diluted samples were further diluted 1:1 in Diluent43 before loading onto the plate.

[0366] After washing, 50 μL of standard, QC, and test samples diluted in Diluent 43 were added to the appropriate wells and incubated for 1 hour at room temperature on a shaker set at 700 RPM. The plates were then washed, and 50 μL of detection antibody (SULFO-TAG anti-human CSF1 antibody; MSD, catalog number D21XR-3) diluted 1:100 in Diluent 3 (MSD, catalog number R50AP-2) was added to each well and incubated for 1 hour at room temperature on a plate shaker set at 700 RPM. After incubation was complete, each plate was washed, and then 150 μL / well of MSD GOLD Read Buffer B (MSD, catalog number R60AM-2) was added, and the plates were read on an MSD Sector 600 imager. In the presence of the read buffer, ruthenium produced a chemiluminescent signal when voltage was applied. The intensity of the signal was proportional to the concentration of CSF1 present in the sample. 1 / y 2 CSF1 levels were quantified according to the standard curve using a four-parameter logistic (4-PL) curve fitting equation with weighting of . Data were analyzed using Microsoft Excel and GraphPad Prism 9.0.

[0367] Summary of sTREM2 Methods (Serum, CSF, and Brain Lysate) The concentration of TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) in cynomolgus monkey serum, cerebrospinal fluid (CSF), and brain lysate was measured using an electrochemiluminescence (ECL) assay on the MSD platform. A 96-well standard MSD plate (catalog no. L15XA) was coated overnight with 50 μL / well of capture antibody T2-8F11 (produced by Alector) in 1x PBS (Corning, reference no. 21-040-CM) at 2-8°C on a plate shaker set at 500 revolutions per minute (RPM). After washing with wash buffer (1x PBS and 0.05% Tween-20), the plate was blocked with 150 μL / well of blocking buffer (1% heat-inactivated high-grade BSA in PBS). The plate was then washed, and 50 μL / well of cynomolgus monkey TREM2-Fc standards (made by Alector), QCs, and samples diluted in assay buffer (1× PBS, 0.05% Tween-20, 1% BSA) were added to the appropriate wells and incubated for 1 hour at room temperature (RT) on a shaker set at 500 RPM. TREM2 present in the standards, QCs, and samples was bound by the immobilized T2-8F11 capture antibody. After washing away unbound material, 50 μL / well of biotinylated goat anti-human TREM2 polyclonal antibody (R&D Systems, catalog no. BAF1828) diluted at 100 ng / mL in assay buffer was added to each well of the plate and incubated at room temperature on a shaker set at 500 RPM. After washing the plates, 50 μL of a 0.2 μg / mL solution of Sulfo-Tag labeled streptavidin (MSD, Cat. No. R32AD-1) in assay buffer was added and incubated for 30 minutes at room temperature on a shaker set at 500 RPM. After incubation was complete, each plate was washed, 150 μL / well of 1× read buffer (prepared by diluting 4× read buffer T (with surfactant) (MSD, Cat. No. R92TC-1) with deionized water) was added, and each plate was read on an MSD Sector 600 imager.In the presence of read buffer, ruthenium produced a chemiluminescent signal upon application of voltage. The intensity of the signal was proportional to the concentration of TREM2 present in the sample. The signal was analyzed by SoftMax Pro (Molecular Devices / Version 7.1) using a weighting factor of 1 / Y. 2 Standard regression was performed using a four-parameter logistic (4-PL) model with:

[0368] Figure 14 shows that serum levels of soluble TREM2 (sTREM2) were increased in NHPs treated with TD1-CD98hc after the first and second doses compared with TD1 alone or isotype control. Figure 14 further shows that soluble TREM2 in the CSF of NHPs treated with CD98hc was not elevated compared with TD1 alone, suggesting that TD1-98hc was cleared from the CSF at a faster rate than TD1 alone. Figure 15 shows CSF-1 levels in the CSF from NHPs treated with TD1-CD98hc or TD-1 alone. No consistent increase in CSF-1 levels was observed in either group.

Claims

1. An antigen-binding domain that specifically binds to human CD98hc heavy chain (CD98hc), comprising complementarity-determining regions (CDR) 1, VH CDR2, and VH CDR3 of the heavy chain variable region (VH) that have the following amino acid sequences, respectively, and CDR1, CDR2, and CDR3 sequences of the light chain variable region (VL): (i) SEQ ID NOs: 413, 414, 112, and 176-178, respectively; (ii) SEQ ID NOs: 50-52 and 116-118, respectively; (iii) SEQ ID NOs: 53 to 55 and 119 to 121, respectively; (iv) SEQ ID NOs: 56-58 and 122-124, respectively; (v) SEQ ID NOs: 59-61 and 125-127, respectively; (vi) SEQ ID NOs: 62-64 and 128-130, respectively; (vii) SEQ ID NOs: 65-67 and 131-133, respectively; (viii) SEQ ID NOs: 68-70 and 134-136, respectively; (ix) SEQ ID NOs: 71-73 and 137-139, respectively; (x) SEQ ID NOs: 74 to 76 and 140 to 142, respectively; (xi) SEQ ID NOs: 77-79 and 143-145, respectively; (xii) SEQ ID NOs: 80-82 and 146-148, respectively; (xiii) SEQ ID NOs: 83 to 85 and 149 to 151, respectively; (xiv) SEQ ID NOs: 86-88 and 152-154, respectively; (xv) SEQ ID NOs: 89-91 and 155-157, respectively; (xvi) SEQ ID NOs: 92-94 and 158-160, respectively; (xvii) SEQ ID NOs: 95-97 and 161-163, respectively; (xviii) SEQ ID NOs: 98-100 and 164-166, respectively; (xix) SEQ ID NOs: 101-103 and 167-169, respectively; (xx) SEQ ID NOs: 104 to 106 and 170 to 172, respectively; (xxi) SEQ ID NOs: 107-109 and 173-175, respectively; (xxii) SEQ ID NOs: 110-112 and 176-178, respectively; (xxiii) SEQ ID NOs: 113-115 and 179-181, respectively; (xxiv) SEQ ID NOs: 226-228 and 273-275, respectively; (xxv) SEQ ID NOs: 229-231 and 276-278, respectively; (xxvi) SEQ ID NOs: 232-234 and 279-281, respectively; (xxvii) SEQ ID NOs: 235-237 and 282-284, respectively; (xxviii) SEQ ID NOs: 238-240 and 285-287, respectively; (xxix) SEQ ID NOs: 241-243 and 288-290, respectively; (xxx) SEQ ID NOs: 244-246, 273, 274, and 291, respectively; (xxxi) SEQ ID NOs: 247-249, 292, 274, and 293, respectively; (xxxii) SEQ ID NOs: 250-252, 294, 274, and 291, respectively; (xxxiii) SEQ ID NOs: 253-255 and 295-297, respectively; (xxxiv) SEQ ID NOs: 256-258, 298, 274, and 299, respectively; (xxxv) SEQ ID NOs: 259-261 and 300-302, respectively; (xxxvi) SEQ ID NOs: 262-264, 303, 274, and 304, respectively; (xxxvii) SEQ ID NOs: 265-267, 305, 306, and 291, respectively; (xxxviii) SEQ ID NOs: 268-270 and 307-309, respectively; (xxxix) SEQ ID NOs: 265, 271, 272, 310, 274, and 299, respectively; (xl) SEQ ID NOs: 110, 414, 112, and 176-178, respectively; (xli) SEQ ID NOs: 110, 414, 112, and 176-178, respectively; (xlii) SEQ ID NOs: 413, 415, 112, and 176-178, respectively; (xliii) SEQ ID NOs: 110, 415, 112, and 176-178, respectively; (xliv) SEQ ID NOs: 110, 416, 112, and 176-178, respectively; (xlv) SEQ ID NOs: 413, 416, 112, and 176-178, respectively; (xlvi) SEQ ID NOs: 413, 415, 112, 417, 418, and 178, respectively; (xlvii) SEQ ID NOs: 419, 422, 112, and 176-178, respectively; (xlviii) SEQ ID NOs: 419, 423, 112, and 176-178, respectively; (xlix) SEQ ID NOs: 419, 424, 112, and 176-178, respectively; (l) SEQ ID NOs: 419, 425, 112, and 176-178, respectively; (li) SEQ ID NOs: 419, 426, 112, and 176-178, respectively; (lii) SEQ ID NOs: 419, 422, 112, 427, 177, and 178, respectively; (liii) SEQ ID NOs: 419, 422, 112, 428, 177, and 178, respectively; (liv) SEQ ID NOs: 419, 422, 112, 429, 177, and 178, respectively; (lv) SEQ ID NOs: 419, 422, 112, 430, 177, and 178, respectively; (lvi) SEQ ID NOs: 419, 422, 112, 431, 177, and 178, respectively; (lvii) SEQ ID NOs: 419, 422, 112, 432, 177, and 178, respectively; (lviii) SEQ ID NOs: 419, 422, 112, 433, 177, and 178, respectively; (lix) SEQ ID NOs: 420, 422, 112, and 176-178, respectively; (lx) SEQ ID NOs: 421, 422, 112, and 176-178, respectively; or (lxi) SEQ ID NOs: 421, 426, 112, 434, 177, and 178, respectively.

2. 2. The antigen-binding domain of claim 1, comprising a VH and a VL, wherein the VH and VL each 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 NOs: 363 and 364, respectively; (ii) SEQ ID NOs: 6 and 7, respectively; (iii) SEQ ID NOs: 8 and 9, respectively; (iv) SEQ ID NOs: 10 and 11, respectively; (v) SEQ ID NOs: 12 and 13, respectively; (vi) SEQ ID NOs: 14 and 15, respectively; (vii) SEQ ID NOs: 16 and 17, respectively; (viii) SEQ ID NOs: 18 and 19, respectively; (ix) SEQ ID NOs: 20 and 21, respectively; (x) SEQ ID NOs: 22 and 23, respectively; (xi) SEQ ID NOs: 24 and 25, respectively; (xii) SEQ ID NOs: 26 and 27, respectively; (xiii) SEQ ID NOs: 28 and 29, respectively; (xiv) SEQ ID NOs: 30 and 31, respectively; (xv) SEQ ID NOs: 32 and 33, respectively; (xvi) SEQ ID NOs: 34 and 35, respectively; (xvii) SEQ ID NOs: 36 and 37, respectively; (xviii) SEQ ID NOs: 38 and 39, respectively; (xix) SEQ ID NOs: 40 and 41, respectively; (xx) SEQ ID NOs: 42 and 43, respectively; (xxi) SEQ ID NOs: 44 and 45, respectively; (xxii) SEQ ID NOs: 46 and 47, respectively; (xxiii) SEQ ID NOs: 48 and 49, respectively; (xxiv) SEQ ID NOs: 194 and 195, respectively; (xxv) SEQ ID NOs: 196 and 197, respectively; (xxvi) SEQ ID NOs: 198 and 199, respectively; (xxvii) SEQ ID NOs: 200 and 201, respectively; (xxviii) SEQ ID NOs: 202 and 203, respectively; (xxix) SEQ ID NOs: 204 and 205, respectively; (xxx) SEQ ID NOs: 206 and 207, respectively; (xxxi) SEQ ID NOs: 208 and 209, respectively; (xxxii) SEQ ID NOs: 210 and 211, respectively; (xxxiii) SEQ ID NOs: 212 and 213, respectively; (xxxiv) SEQ ID NOs: 214 and 215, respectively; (xxxv) SEQ ID NOs: 216 and 217, respectively; (xxxvi) SEQ ID NOs: 218 and 219, respectively; (xxxvii) SEQ ID NOs: 220 and 221, respectively; (xxxviii) SEQ ID NOs: 222 and 223, respectively; (xxxix) SEQ ID NOs: 224 and 225, respectively; (xl) SEQ ID NOs: 355 and 356, respectively; (xli) SEQ ID NOs: 357 and 358, respectively; (xlii) SEQ ID NOs: 359 and 360, respectively; (xliii) SEQ ID NOs: 361 and 362, respectively; (xliv) SEQ ID NOs: 365 and 366, respectively; (xlv) SEQ ID NOs: 367 and 368, respectively; (xlvi) SEQ ID NOs: 369 and 370, respectively; (xlvii) SEQ ID NOs: 371 and 372, respectively; (xlviii) SEQ ID NOs: 373 and 374, respectively; (xlix) SEQ ID NOs: 375 and 376, respectively; (l) SEQ ID NOs: 377 and 378, respectively; (li) SEQ ID NOs: 379 and 380, respectively; (lii) SEQ ID NOs: 381 and 382, ​​respectively; (liii) SEQ ID NOs: 383 and 384, respectively; (liv) SEQ ID NOs: 385 and 386, respectively; (lv) SEQ ID NOs: 387 and 388, respectively; (lvi) SEQ ID NOs: 389 and 390, respectively; (lvii) SEQ ID NOs: 391 and 392, respectively; (lviii) SEQ ID NOs: 393 and 394, respectively; (lix) SEQ ID NOs: 395 and 396, respectively; (lx) SEQ ID NOs: 397 and 398, respectively; (lxi) SEQ ID NOs: 399 and 400, respectively; (lxii) SEQ ID NOs: 401 and 402, respectively; or (lxiii) SEQ ID NOs: 403 and 404, respectively.

3. An antigen-binding domain that specifically binds to human CD98hc, comprising a VH and a VL, wherein the VH is selected from the group consisting of SEQ ID NOs: 363, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 194, 196, 198, 200, 202, and 204. , 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 355, 357, 359, 361, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 401, or 403.

4. An antigen-binding domain that specifically binds to human CD98hc, comprising a VH and a VL, wherein the VL is selected from the group consisting of SEQ ID NOs: 364, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 195, 197, 199, 201, 203, and 205. , 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 356, 358, 360, 362, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 400, 402, or 404.

5. The antigen-binding domain of any one of claims 1 to 4, comprising a VH and a VL each comprising the following amino acid sequences: (i) SEQ ID NOs: 363 and 364, respectively; (ii) SEQ ID NOs: 6 and 7, respectively; (iii) SEQ ID NOs: 8 and 9, respectively; (iv) SEQ ID NOs: 10 and 11, respectively; (v) SEQ ID NOs: 12 and 13, respectively; (vi) SEQ ID NOs: 14 and 15, respectively; (vii) SEQ ID NOs: 16 and 17, respectively; (viii) SEQ ID NOs: 18 and 19, respectively; (ix) SEQ ID NOs: 20 and 21, respectively; (x) SEQ ID NOs: 22 and 23, respectively; (xi) SEQ ID NOs: 24 and 25, respectively; (xii) SEQ ID NOs: 26 and 27, respectively; (xiii) SEQ ID NOs: 28 and 29, respectively; (xiv) SEQ ID NOs: 30 and 31, respectively; (xv) SEQ ID NOs: 32 and 33, respectively; (xvi) SEQ ID NOs: 34 and 35, respectively; (xvii) SEQ ID NOs: 36 and 37, respectively; (xviii) SEQ ID NOs: 38 and 39, respectively; (xix) SEQ ID NOs: 40 and 41, respectively; (xx) SEQ ID NOs: 42 and 43, respectively; (xxi) SEQ ID NOs: 44 and 45, respectively; (xxii) SEQ ID NOs: 46 and 47, respectively; (xxiii) SEQ ID NOs: 48 and 49, respectively; (xxiv) SEQ ID NOs: 194 and 195, respectively; (xxv) SEQ ID NOs: 196 and 197, respectively; (xxvi) SEQ ID NOs: 198 and 199, respectively; (xxvii) SEQ ID NOs: 200 and 201, respectively; (xxviii) SEQ ID NOs: 202 and 203, respectively; (xxix) SEQ ID NOs: 204 and 205, respectively; (xxx) SEQ ID NOs: 206 and 207, respectively; (xxxi) SEQ ID NOs: 208 and 209, respectively; (xxxii) SEQ ID NOs: 210 and 211, respectively; (xxxiii) SEQ ID NOs: 212 and 213, respectively; (xxxiv) SEQ ID NOs: 214 and 215, respectively; (xxxv) SEQ ID NOs: 216 and 217, respectively; (xxxvi) SEQ ID NOs: 218 and 219, respectively; (xxxvii) SEQ ID NOs: 220 and 221, respectively; (xxxviii) SEQ ID NOs: 222 and 223, respectively; (xxxix) SEQ ID NOs: 224 and 225, respectively; (xl) SEQ ID NOs: 355 and 356, respectively; (xli) SEQ ID NOs: 357 and 358, respectively; (xlii) SEQ ID NOs: 359 and 360, respectively; (xliii) SEQ ID NOs: 361 and 362, respectively; (xliv) SEQ ID NOs: 365 and 366, respectively; (xlv) SEQ ID NOs: 367 and 368, respectively; (xlvi) SEQ ID NOs: 369 and 370, respectively; (xlvii) SEQ ID NOs: 371 and 372, respectively; (xlviii) SEQ ID NOs: 373 and 374, respectively; (xlix) SEQ ID NOs: 375 and 376, respectively; (l) SEQ ID NOs: 377 and 378, respectively; (li) SEQ ID NOs: 379 and 380, respectively; (lii) SEQ ID NOs: 381 and 382, ​​respectively; (liii) SEQ ID NOs: 383 and 384, respectively; (liv) SEQ ID NOs: 385 and 386, respectively; (lv) SEQ ID NOs: 387 and 388, respectively; (lvi) SEQ ID NOs: 389 and 390, respectively; (lvii) SEQ ID NOs: 391 and 392, respectively; (lviii) SEQ ID NOs: 393 and 394, respectively; (lix) SEQ ID NOs: 395 and 396, respectively; (lx) SEQ ID NOs: 397 and 398, respectively; (lxi) SEQ ID NOs: 399 and 400, respectively; (lxii) SEQ ID NOs: 401 and 402, respectively; or (lxiii) SEQ ID NOs: 403 and 404, respectively.

6. The antigen-binding domain of any one of claims 1 to 5, which is capable of crossing the blood-brain barrier (BBB).

7. The antigen-binding domain of any one of claims 1 to 6, which binds to human CD98hc with an affinity of 500 nM to 10 μM.

8. The antigen-binding domain of any one of claims 1 to 7, which binds to human CD98hc with an affinity of 50 nM to 500 μM.

9. The antigen-binding domain of any one of claims 1 to 8, which binds to human CD98hc with an affinity of 1 nM to 50 nM.

10. The antigen-binding domain of any one of claims 1 to 9, which binds to cynomolgus monkey CD98hc.

11. ELISA OD of at least 0.45 ４５０ and / or an ELISA OD of at least 0.

45. ４５０ The antigen-binding domain of any one of claims 1 to 10, which binds to cynomolgus monkey CD98hc at

12. The antigen-binding domain of any one of claims 1 to 11, wherein the antigen-binding domain is internalized into blood-brain barrier epithelial cells, and optionally the blood-brain barrier epithelial cells are HCMEC / D3 cells.

13. The antigen-binding domain of any one of claims 1 to 12, which binds to human CD98hc with an affinity of 3.1 nM to 210 nM.

14. An antigen-binding domain according to any one of claims 1 to 13, which binds to cynomolgus monkey CD98hc with an affinity of 3.2 nM to 1.5 µM.

15. 15. The antigen-binding domain of claim 13 or 14, wherein the affinity is measured by high-throughput surface plasmon resonance (SPR) detection.

16. The antigen-binding domain of any one of claims 1 to 15, which does not reduce cell surface expression of CD98hc on HCMEC / D3 cells by more than 20% compared to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control.

17. The antigen-binding domain of any one of claims 1 to 16, which does not increase cell surface expression of CD98hc on HCMEC / D3 cells by more than 50% compared to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control.

18. The antigen-binding domain of any one of claims 1 to 17, which accumulates in the brain of vascular-depleted mice at least 1.5-fold or at least 2-fold more than an isotype control.

19. The antigen-binding domain of any one of claims 1 to 18, which increases the brain:serum concentration ratio by at least 5-fold compared to an isotype control 24 hours after administration to mice.

20. 20. The antigen-binding domain of any one of claims 1 to 19, comprising a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 46 and 47, respectively, SEQ ID NOs: 367 and 368, respectively, SEQ ID NOs: 369 and 370, respectively, SEQ ID NOs: 371 and 372, respectively, SEQ ID NOs: 373 and 374, respectively, SEQ ID NOs: 375 and 376, respectively, SEQ ID NOs: 377 and 378, respectively, SEQ ID NOs: 379 and 380, respectively, SEQ ID NOs: 381 and 382, ​​respectively, SEQ ID NOs: 383 and 384, respectively, SEQ ID NOs: 385 and 386, respectively, SEQ ID NOs: 387 and 388, respectively, SEQ ID NOs: 389 and 390, respectively, SEQ ID NOs: 391 and 392, respectively, SEQ ID NOs: 393 and 394, respectively, SEQ ID NOs: 395 and 396, respectively, SEQ ID NOs: 397 and 398, respectively, SEQ ID NOs: 399 and 400, respectively, SEQ ID NOs: 401 and 402, respectively, or SEQ ID NOs: 403 and 404, respectively.

21. The antigen-binding domain of any one of claims 1 to 20, comprising a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 14 and 15, respectively.

22. The antigen-binding domain according to any one of claims 1 to 21, comprising a VH and a VL on a single polypeptide chain.

23. The antigen-binding domain according to any one of claims 1 to 22, comprising an scFv (single-chain fragment variable).

24. The antigen-binding domain of claim 23, wherein the scFv is in a VH-linker-VL orientation.

25. The antigen-binding domain of claim 23, wherein the scFv is in a VL-linker-VH orientation.

26. 26. The antigen-binding domain of claim 24 or 25, wherein the linker is from about 5 to about 25 amino acids, from about 5 to about 20 amino acids, from about 10 to about 25 amino acids, or from about 10 to about 20 amino acids.

27. The antigen-binding domain of any one of claims 24 to 26, wherein the linker comprises the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 182) or GGGGSGGGGGSGGGGGSGGGGS (SEQ ID NO: 337).

28. The antigen-binding domain of claim 23, wherein the scFv comprises the amino acid sequence of any one of SEQ ID NOs: 318 to 336.

29. 22. The antigen-binding domain of any one of claims 1 to 21, comprising a VH on a first polypeptide and a VL on a second polypeptide.

30. 30. An antigen-binding domain according to any one of claims 1 to 29, which is a murine, chimeric, humanised or human antigen-binding domain, optionally a humanised antigen-binding domain.

31. An antigen-binding domain that specifically binds to human CD98hc, comprising: (i) a VH CDR1, a VH CDR2, and a VH CDR3 of the antigen-binding domain of any one of claims 1 to 19 and 27; or (ii) A VHH comprising the VH of the antigen-binding domain of any one of claims 1 to 19 and 27, wherein the VHH is optionally capable of crossing the blood-brain barrier (BBB).

32. A fusion protein comprising the antigen-binding domain of any one of claims 1 to 31 and a heterologous protein or peptide.

33. The heterologous protein or peptide is selected from the group consisting of beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, glucocereb rodase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), glycoprotein non-transferase protein B (GPNMB), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Sialic acid-binding Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4-domain A4A (MS4A4A), transmembrane 4-domain A6A (MS4A6A), or transmembrane protein 106B (TMEM106b), clusterin (APOJ), reelin, ubiquitin protein ligase E3A (UBE3A), tripeptidyl peptidase B (TPB) 33. The fusion protein of claim 32, comprising an amino acid sequence of a protein encoding heparan-alpha-glucosamide N-acetyltransferase 1 (CLN2 / TPP1), alpha-L-iduronidase (IDUA), iduronate 2-sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosamide N-acetyltransferase (HGSNAT), N-acetyl-alpha-glucosaminidase (NAGLU), N-sulfoglucosamine sulfohydrolase (SGSH), or a portion thereof.

34. An antibody comprising an antigen-binding domain according to any one of claims 1 to 31.

35. An antibody or antigen-binding fragment thereof that binds to the same human CD98hc epitope as the antigen-binding domain of any one of claims 1 to 31.

36. An antibody or antigen-binding fragment thereof that competitively inhibits the binding of the antigen-binding domain of any one of claims 1 to 31 to human CD98hc.

37. 32. A multispecific protein comprising a first antigen-binding domain that is the antigen-binding domain of any one of claims 1 to 31 linked to a second antigen-binding domain, optionally wherein said second antigen-binding domain specifically binds to a CNS antigen.

38. 32. A multispecific protein comprising the antigen-binding domain of any one of claims 1 to 31 linked to an antibody or antigen-binding fragment thereof, optionally wherein the antibody or antigen-binding fragment thereof specifically binds to a CNS antigen.

39. 39. The multispecific protein of claim 38, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region.

40. 40. The multispecific protein of claim 39, wherein the antigen-binding domain of any one of claims 1 to 31 is linked, optionally via an amino acid linker, to the C-terminus of the heavy chain constant region.

41. 41. The multispecific protein of any one of claims 37 to 40, which is bispecific.

42. 42. The multispecific protein of any one of claims 37 to 41, which is bivalent, trivalent, or tetravalent.

43. 43. The multispecific protein of claim 42, which is bivalent.

44. The multispecific protein of claim 42, which is trivalent, optionally wherein said trivalent protein comprises one antigen-binding domain that binds to human CD98hc and two antigen-binding domains that bind to CNS antigens.

45. 43. The multispecific protein of claim 42, which is tetravalent, optionally wherein said tetravalent protein comprises two of said antigen binding domains that bind to human CD98hc and two antigen binding domains that bind to a CNS antigen.

46. 32. A multispecific protein comprising the antigen-binding domain of any one of claims 1 to 29 and 31 linked to an antibody that is trivalent and bispecific and binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, and the antigen-binding domain is linked to the C-terminus of one of the two antibody heavy chains by an scFv, optionally via an amino acid linker.

47. 32. A tetravalent and bispecific multispecific protein comprising two antigen-binding domains according to any one of claims 1 to 31 and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, each of the two antigen-binding domains being an scFv, Fab or VHH, and wherein one of the two antigen-binding domains is linked, optionally via an amino acid linker, to the N-terminus of one of the antibody heavy chains, and the other of the antigen-binding domains is linked, optionally via an amino acid linker, to the C-terminus of the other of the antibody heavy chains.

48. 48. The multispecific protein of any one of claims 38 to 47, wherein the antibody or antigen-binding fragment thereof comprises a constant region comprising a knob mutation and a constant region comprising a hole mutation.

49. 49. The multispecific protein of claim 48, wherein the antigen binding domain is linked to the constant region comprising the hole mutation, optionally via an amino acid linker.

50. 49. The multispecific protein of claim 48, wherein said antigen binding domain is linked, optionally via an amino acid linker, to said constant region comprising a knob mutation.

51. 51. The multispecific protein of claims 46-50, wherein the amino acid linker is a glycine-serine linker, optionally wherein the glycine-serine linker comprises the amino acid sequence (GGGGS)x3 (SEQ ID NO: 183).

52. 51. The multispecific protein of claims 46-50, wherein the amino acid linker is a glycine-serine linker, optionally wherein the glycine-serine linker comprises the amino acid sequence (GGSGG)x3 (SEQ ID NO: 338).

53. The multispecific protein of any one of claims 37 to 52, wherein the CNS antigen is a brain antigen.

54. The multispecific protein of any one of claims 37 to 53, wherein the CNS antigen is not CD98hc.

55. 55. The multispecific protein of any one of claims 38 to 54, wherein the antibody or antigen-binding fragment thereof comprises mutations that reduce effector function, optionally wherein the mutations that reduce effector function include (i) L234A, L235A, and / or P331S and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S.

56. 56. The multispecific protein of any one of claims 38 to 55, wherein the antibody or antigen-binding fragment thereof comprises a constant region comprising a knob mutation and a mutation that reduces effector function, 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.

57. 57. The multispecific protein of any one of claims 38 to 56, wherein the antibody or antigen-binding fragment thereof comprises a constant region comprising hole mutations and mutations that reduce effector function, and the mutations that reduce effector function comprise: (i) L234A, L235A, and / or P331S and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S.

58. 58. The multispecific protein of any one of claims 38 to 57, wherein the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

59. 59. The multispecific protein of claim 58, 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.

60. An equilibrium dissociation constant (K Ｄ ) and / or binds to human CD98hc with a K of about 3 nM to about 225 nM Ｄ 60. The multispecific protein of any one of claims 37 to 59, which binds to cynomolgus monkey CD98hc at

61. 61. The multispecific protein of any one of claims 37 to 60, which is internalized into blood-brain barrier epithelial cells at a greater than 10-fold increase compared to internalization by an isotype control, and optionally the blood-brain barrier epithelial cells are HCMEC / D3 cells.

62. 62. The multispecific protein of any one of claims 37 to 61, which does not reduce the cell surface expression of CD98hc on HCMEC / D3 cells by more than 20% compared to the cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control.

63. 63. The multispecific protein of any one of claims 37 to 62, which does not increase cell surface expression of CD98hc on HCMEC / D3 cells by more than 50% compared to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control.

64. 64. The multispecific protein of any one of claims 37 to 63, which accumulates in the vascular-depleted mouse brain at least 1.5-fold or at least 2-fold more than an isotype control.

65. 65. The multispecific protein of any one of claims 37 to 64, which increases the brain:serum concentration ratio by at least 5-fold 24 hours after administration to mice compared to an isotype control.

66. The CNS antigen may 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, β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33, or 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), sialic acid-binding Ig-like lectin 11 (Siglec11), glycoprotein non-transferase B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4 66. The multispecific protein of claims 37 to 65, which is 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.

67. 67. The multispecific protein of claim 66, wherein the CNS antigen is MS4A4A, and optionally (i) the antigen binding domain, antibody, or antigen binding domain that binds to MS4A4A comprises a VH comprising the amino acid sequence of SEQ ID NO: 407 and / or a VL comprising the VL sequence of SEQ ID NO: 405, and / or (ii) the antigen binding domain that binds to human CD98hc comprises the amino acid sequence of SEQ ID NO:

316.

68. 68. The multispecific protein of claim 67, comprising the amino acid sequence of SEQ ID NOs: 405-410.

69. 69. The fusion protein of claim 32 or 33, the antibody or antigen-binding fragment of any one of claims 34 to 36, or the multispecific protein of any one of claims 37 to 68, which is capable of crossing the BBB.

70. 70. The fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein of any one of claims 32 to 69, linked to an imaging agent.

71. 71. A composition comprising a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode the multispecific protein of any one of claims 37 to 45 and 48 to 70, 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 CD98hc, and the third polynucleotide encodes a light chain.

72. 71. A composition comprising a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode the multispecific protein of any one of claims 37 to 44 and 47 to 70, wherein the first polynucleotide encodes a first heavy chain and a first antigen-binding domain that specifically binds to human CD98hc, the second polynucleotide encodes a second heavy chain and a second antigen-binding domain that specifically binds to human CD98, and the third polynucleotide encodes a light chain, and optionally the first and second antigen-binding domains that bind to human CD98hc comprise the same amino acid sequence.

73. 73. The composition of claim 71 or 72, wherein the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation.

74. 74. The composition of any one of claims 71 to 73, wherein the ratio of the first, second, and third polynucleotides is about 1:3:

6.

75. 73. The composition of claim 71 or 72, wherein the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

76. 10. A composition comprising a first polynucleotide and a second polynucleotide, wherein said first and second polynucleotides encode a multispecific protein according to any one of claims 37 to 44 and 47 to 70, wherein said first polynucleotide encodes a heavy chain and the antigen-binding domain that binds to human CD98hc, and said second polynucleotide encodes a light chain.

77. A host cell comprising the composition of any one of claims 71 to 76.

78. An isolated polynucleotide molecule comprising a nucleic acid molecule encoding the heavy chain of the antigen-binding domain of any one of claims 1 to 31.

79. An isolated polynucleotide molecule comprising a nucleic acid molecule encoding the light chain variable region of the antigen-binding domain of any one of claims 1 to 31.

80. 80. An isolated vector comprising the polynucleotide of claim 78 and / or the polynucleotide of claim 79.

81. An isolated vector comprising a nucleic acid molecule encoding a heavy chain variable region of the antigen-binding domain of any one of claims 1 to 31, and a nucleic acid molecule encoding the light chain variable region of the antigen-binding domain.

82. 82. A host cell comprising a polynucleotide according to claim 78 or 79 or a vector according to claim 80 or 81.

83. 83. The host cell of claim 77 or 82, wherein the host cell is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture.

84. 91. A method for producing an antigen-binding domain or a multispecific protein, comprising producing the antigen-binding domain or multispecific protein by culturing the host cell of any one of claims 77, 82, and 83, and optionally further comprising isolating the antigen-binding domain or the multispecific protein from the culture.

85. 85. An isolated antigen-binding domain or multispecific protein thereof produced by the method of claim 84.

86. 71. A pharmaceutical composition comprising: (i) the antigen-binding domain, fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein of any one of claims 1 to 70; and (ii) a pharmaceutically acceptable carrier.

87. 87. The pharmaceutical composition of claim 86, 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.

88. 88. The pharmaceutical composition of claim 86 or 87, wherein said administration increases delivery of the fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition into the brain by at least 50%, at least 100%, at least 200%, at least 500%, or at least 1000% compared to an isotype control.

89. 90. A method of treating a neurological disease or disorder in a subject, comprising administering to the subject a fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition of any one of claims 32-70, 86, and 87.

90. 90. The method of claim 89, wherein said administering increases delivery of the fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition into the brain by at least 50%, at least 100%, at least 200%, at least 500%, or at least 1000% compared to an isotype control.

91. 91. The method of claim 89 or 90, wherein said 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.

92. 92. The method of claims 89-91, wherein the neurological disease or disorder is selected from a neuropathic disorder, a neurodegenerative disorder, a cancer, an ocular disorder, a seizure disorder, a lysosomal storage disorder, an amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and inflammation of the CNS.

93. 93. The method of claim 92, wherein the neurological disease or disorder is selected from Alzheimer's disease (AD), Huntington's disease, dystonia, ataxia, Bell's palsy, stroke, dementia, dementia with Lewy bodies, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, encephalitis, traumatic brain injury, and limbic-predominant age-related TDP-43 encephalopathy (LATE).

94. 94. The method of claim 93, wherein the dementia is frontotemporal dementia (FTD).

95. 94. The method of claim 93, wherein the neurological disease or disorder is Alzheimer's disease.

96. 96. The method of claim 95, 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.

97. 94. The method of claim 93, wherein the neurological disease or disorder is Parkinson's disease.

98. 90. The method of claim 89, wherein the neurological disease or disorder is frontotemporal epilepsy.

99. 90. The method of claim 89, wherein the neurological disease or disorder is autism.

100. 90. The method of claim 89, wherein the neurological disease or disorder is lissencephaly.

101. 34. A method of treating a lysosomal storage disease in a subject, comprising administering to the subject the fusion protein of claim 32 or 33.

102. 102. The method of claim 101, wherein the lysosomal storage disease is selected from Gaucher disease, ceroid lipofuscinosis (Batten disease), mucopolysaccharidosis (MPS) type I, MPS type II, and MPS type III.

103. 100. A method for transporting a fusion protein, an antibody or antigen-binding fragment thereof, or a multispecific protein across the BBB in a subject, the method comprising administering to the subject a fusion protein, an antibody or antigen-binding fragment thereof, a multispecific protein, or a pharmaceutical composition according to any one of claims 32 to 70, 86, and 87.

104. 104. The method of claim 103, wherein the concentration of the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein is increased in the brain after administration compared to an isotype control.

105. 105. The method of claim 103 or 104, 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.

106. 106. The method of claims 103-105, wherein administration of the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein does not result in more than a 10% decrease in reticulocyte count in the subject compared to administration of an isotype control.

107. 107. The method of claim 106, 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.

108. 71. A method of increasing the concentration of a CNS-binding antigen in the CSF of a subject, comprising administering to the subject a multispecific protein of any one of claims 37 to 70, wherein the concentration of the CNS-binding antigen is increased compared to administering the CNS-binding antigen alone to the subject.

109. 68. 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 67, and determining the location of the imaging agent within the subject.

110. 71. A method for detecting a CNS antigen in vitro, comprising contacting an in vitro sample with the fusion protein, antibody, or multispecific protein of claim 70 and determining the location of the imaging agent in the sample.

111. Use of a fusion protein, an antibody or antigen-binding fragment thereof, or a multispecific protein, or a composition according to any one of claims 32 to 70, 88, and 89 in a method according to any one of claims 89 to 109.

112. 110. The fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or composition of any one of claims 32 to 70, 86, and 87 for use in a method according to any one of claims 89 to 109.

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