CD98HC antigen binding domains and uses thereof

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 369,885, filed on July 29, 2022; U.S. Provisional Application No. 63 / 489,675, filed on March 10, 2023; and U.S. Provisional Application No. 63 / 495,514, filed on April 11, 2023; each of which is incorporated herein by reference in its entirety.

[0003] Reference to a sequence listing submitted electronically

[0004] The contents of the electronically submitted Sequence Listing (Name: 4503_021PC03_SequenceListing_ST26.xml; Size: 498,296 bytes; and Creation Date: July 27, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure relates to antigen binding domains that specifically bind to human CD98 heavy chain (CD98hc). These antigen binding domains can cross the blood-brain barrier, and can transport other agents (e.g., therapeutically active agents) associated with the antigen binding domain across the blood-brain barrier. Background Art

[0006] The passive transfer of substances from blood to brain is limited by the blood-brain barrier (BBB). BBB provides precise control of the central nervous system (CNS) homeostasis, allowing proper neuronal function and also protecting neural tissue from toxins and pathogens. Changes in BBB are important components of the pathology and progression of different nervous system diseases. However, BBB has problems in delivering therapeutic agents to CNS. Although recombinant protein and antibody therapy have shown great success outside CNS, such biologics cannot effectively pass through BBB. Therefore, the delivery of some therapeutic agents to CNS depends on injecting the therapeutic agent directly into CNS. However, such injections are invasive procedures, and their efficacy is limited by the rapid output of cerebrospinal fluid (CSF) containing therapeutic agents from brain to blood. Alternatively, therapeutic agents for CNS can be systemically administered at high doses to allow the therapeutic agent to fully penetrate BBB. However, this method may have unexpected effects due to peripheral high doses or to increase manufacturing and formulation burdens to achieve high doses. Therefore, it is necessary to deliver therapeutic agents through BBB Improved products and methods. Summary of the invention

[0007] Provided herein are antigen binding domains, fusion proteins, antibodies, and multispecific proteins that specifically bind to human CD98 heavy chain (CD98hc), and methods of making and using the same.

[0008] In some aspects, provided herein is an antigen binding domain that specifically binds to a human CD98 heavy chain (CD98hc), wherein the antigen binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequence comprising the following amino acid sequences: SEQ ID NOs: 413, 414, 112, and 176-178, respectively; SEQ ID NOs: 50-52 and 116-118, respectively; SEQ ID NOs: 53-55 and 119-121, respectively; SEQ ID NOs: 56-58 and 122-124, respectively; SEQ ID NOs: 59-61 and 125-127, respectively; SEQ ID NOs: 62-64 and 128-130, respectively; SEQ ID NOs: 65-67 and 131-133, respectively; SEQ ID NOs: NO:68-70 and 134-136, respectively; SEQ ID NO:71-73 and 137-139, respectively; SEQ ID NO:74-76 and 140-142, respectively; SEQ ID NO:77-79 and 143-145, respectively; SEQ ID NO:80-82 and 146-148, respectively; SEQ ID NO:83-85 and 149-151, respectively; SEQ ID NO:86-88 and 152-154, respectively; SEQ ID NO:89-91 and 155-157, respectively; SEQ ID NO:92-94 and 158-160, respectively; SEQ ID NO:95-97 and 161-163, respectively; SEQ ID NO:98-100 and 164-166, respectively; SEQ ID NO:101-103 and 167-169, respectively; SEQ ID NO:107-110 and 111-112, respectively; NO:104-106 and 170-172, respectively; SEQ ID NO:107-109 and 173-175, respectively; SEQ ID NO:110-112 and 176-178, respectively; SEQ ID NO:113-115 and 179-181, respectively; SEQ ID NO:226-228 and 273-275, respectively; SEQ ID NO:229-231 and 276-278, respectively; SEQ ID NO:232-234 and 279-281, respectively; SEQ ID NO:235-237 and 282-284, respectively; SEQ ID NO:238-240 and 285-287, respectively; SEQ ID NO:241-243 and 288-290, respectively; SEQ ID NO:244-246, 273, 274 and 291, respectively;SEQ ID NOs: 247-249, 292, 274 and 293, respectively; SEQ ID NOs: 250-252, 294, 274 and 291, respectively; SEQ ID NOs: 253-255 and 295-297, respectively; SEQ ID NOs: 256-258, 298, 274 and 299, respectively; SEQ ID NOs: 259-261 and 300-302, respectively; SEQ ID NOs: 262-264, 303, 274 and 304, respectively; SEQ ID NOs: 265-267, 305, 306 and 291, respectively; SEQ ID NOs: 268-270 and 307-309, respectively; SEQ ID NOs: 265, 271, 272, 310, 274 and 299, respectively; SEQ ID NOs: NO:110, 414, 112 and 176-178, respectively; SEQ ID NO:110, 414, 112 and 176-178, respectively; SEQ ID NO:413, 415, 112 and 176-178, respectively; SEQ ID NO:110, 415, 112 and 176-178, respectively; SEQ ID NO:110, 416, 112 and 176-178, respectively; SEQ ID NO:413, 416, 112 and 176-178, respectively; SEQ ID NO:413, 415, 112, 417, 418 and 178, respectively; SEQ ID NO:419, 422, 112 and 176-178, respectively; SEQ ID NO:419, 423, 112 and 176-178, respectively; SEQ ID NO:419, 424, 112 and 176-178, respectively; SEQ ID NO:419, 425, 112 and 176-178, respectively; SEQ ID NO:419, 426, 112 and 176-178, respectively; SEQ ID NO:419, 422, 112, 427, 177 and 178, respectively; SEQ ID NO:419, 422, 112, 428, 177 and 178, respectively; SEQ ID NO:419, 422, 112, 429, 177 and 178, respectively; SEQ ID NO:419, 422, 112, 430, 177 and 178, respectively; SEQ ID NO:419, 422, 112, 431, 177 and 178, respectively; SEQ ID NO:419, 422, 112, 431, 177 and 178, respectively NO:419, 422, 112, 432, 177 and 178; SEQ ID NO:419, 422, 112, 433, 177 and 178, respectively; SEQ ID NO:420, 422, 112, 427 and 176-178, respectively;SEQ ID NOs: 421, 422, 112, 427 and 176-178, respectively; or SEQ ID NOs: 421, 426, 112, 434, 177 and 178, respectively. ;

[0009] In some aspects, the antigen binding domain comprises a VH and a VL, wherein the VH and the VL comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the following amino acid sequence: 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. NO:34 and 35, respectively; SEQ ID NO:36 and 37, respectively; SEQ ID NO:38 and 39, respectively; SEQ ID NO:40 and 41, respectively; SEQ ID NO:42 and 43, respectively; SEQ ID NO:44 and 45, respectively; SEQ ID NO:46 and 47, respectively; SEQ ID NO:48 and 49, respectively; SEQ ID NO:194 and 195, respectively; SEQ ID NO:196 and 197, respectively; SEQ ID NO:198 and 199, respectively; SEQ ID NO:200 and 201, respectively; SEQ ID NO:202 and 203, respectively; SEQ ID NO:204 and 205, respectively; SEQ ID NO:206 and 207, respectively; SEQ ID NO:208 and 209, respectively; SEQ ID NO:210 and 211, respectively; SEQ ID NO:212 and 213, respectively; SEQ ID NO:214 and 215, respectively. NO:214 and 215, respectively; SEQ ID NO:216 and 217, respectively; SEQ ID NO:218 and 219, respectively; SEQ ID NO:220 and 221, respectively; SEQ ID NO:222 and 223, respectively; SEQ ID NO:224 and 225, respectively; SEQ ID NO:355 and 356, respectively; SEQ ID NO:357 and 358, respectively; SEQ ID NO:359 and 360, respectively; SEQ ID NO:361 and 362, respectively; SEQ ID NO:365 and 366, respectively; SEQ ID NO:367 and 368, respectively; SEQ ID NO: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. ;

[0010] In some aspects, provided herein is an antigen binding domain that specifically binds to human CD98hc, wherein the antigen binding domain comprises VH and VL, wherein VH comprises the following amino acid sequence: SEQ ID NO: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, 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.

[0011] In some aspects, provided herein is an antigen binding domain that specifically binds to human CD98hc, wherein the antigen binding domain comprises VH and VL, wherein VL comprises the following amino acid sequence: SEQ ID NO:364,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,356,358,360,362,366,368,370,372,374,376,378,380,382,384,386,388,400,402 or 404.

[0012] In some aspects, the antigen binding domains provided herein comprise a VH and a VL 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: 39 and 40, 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. NO:220 and 221, respectively; SEQ ID NO:222 and 223, respectively; SEQ ID NO:224 and 225, respectively; SEQ ID NO:355 and 356, respectively; SEQ ID NO:357 and 358, respectively; SEQ ID NO:359 and 360, respectively; SEQ ID NO:361 and 362, respectively; SEQ ID NO:365 and 366, respectively; SEQ ID NO:367 and 368, respectively; SEQ ID NO:369 and 370, respectively; SEQ ID NO: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. ;

[0013] In some aspects, the antigen binding domain can pass through the blood-brain barrier (BBB). In some aspects, the antigen binding domain is combined with cynomolgus monkey CD98hc. In some aspects, the antigen binding domain is internalized into blood-brain barrier epithelial cells. In some aspects, the blood-brain barrier epithelial cells are HCMEC / D3 cells.

[0014] In some aspects, the antigen binding domain binds to human CD98hc with an affinity between 500nM and 10μM. In some aspects, the antigen binding domain binds to human CD98hc with an affinity between 50nM and 500nM. In some aspects, the antigen binding domain binds to human CD98hc with an affinity between 1nM and 50nM. In some aspects, 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 aspects, the antigen binding domain binds to human CD98hc with an affinity of 3.1nM to 210nM. In some aspects, the antigen binding domain binds to cynomolgus monkey CD98hc with an affinity of 3.2nM to 1.5μM. In some aspects, affinity is measured by high-throughput surface plasmon resonance (SPR) detection.

[0015] In some aspects, the antigen binding domain does not reduce the cell surface expression of CD98hc on HCMEC / D3 cells by more than 20% relative to the cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. In some aspects, the antigen binding domain does not increase the cell surface expression of CD98hc on HCMEC / D3 cells by more than 50% relative to the cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control.

[0016] In some aspects, 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. In some aspects, 24 hours after administration to mice, the brain: serum concentration ratio of the antigen binding domain increases by at least 5-fold compared to the isotype control.

[0017] In some aspects, the antigen binding domain comprises a VH and a VL comprising the following amino acid sequences: 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: 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 aspects, the antigen binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 14 and 15, respectively.

[0018] In some aspects, the antigen binding domain is included in VH and VL on a single polypeptide chain. In some aspects, the antigen binding domain includes a single chain variable fragment (scFv). In some aspects, scFv is in the orientation VH-joint-VL. In some aspects, scFv is in the orientation VL-joint-VH. In some aspects, the joint is about 5 to about 25 amino acids, about 5 to about 20 amino acids, about 10 to about 25 amino acids, or about 10 to about 20 amino acids. In some aspects, the joint includes GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 182) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 337) amino acid sequence. In some aspects, scFv includes SEQ ID NO: 318-336 any one of the amino acid sequence. In some aspects, the antigen binding domain includes VH on the first polypeptide and VL on the second polypeptide. In some aspects, the antigen binding domain is a mouse, chimeric, humanized or human antigen binding domain. In some aspects, the antigen binding domain is a humanized antigen binding domain.

[0019] In some aspects, provided herein is an antigen binding domain that specifically binds to human CD98hc, wherein the antigen binding domain is a VHH comprising (i) a VH CDR1, a VH CDR2, and a VHCDR3 of an antigen binding domain provided herein or (ii) a VH of an antigen binding domain provided herein, optionally wherein the VHH is capable of crossing the blood-brain barrier (BBB).

[0020] In some aspects, provided herein are fusion proteins and heterologous proteins or peptides comprising antigen binding domains provided herein. In some aspects, provided herein are heterologous proteins or peptides comprising the following amino acid sequences: β-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, glucocerebrosidase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), glycoprotein nonmetastatic melanoma protein B (Glycoprotein nonmetastatic protein B) (GPNMB), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor on myeloid cells 2 (TREM2), CD33 or sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4 domain A4A (MS4A4A), transmembrane 4 domain A 6A (MS4A6A) or transmembrane protein 106B (TMEM106b), clusterin (APOJ), reelin, ubiquitin protein ligase E3A (UBE3A), tripeptidyl peptidase 1 (CLN2 / TPP1), α-L-iduronidase (IDUA), iduronate 2-sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-α-glucosamine N-acetyltransferase (HGSNAT) and N-acetyl-α-glucosaminidase (NAGLU), N-sulfoglucosamine sulfohydrolase (SGSH) or parts thereof.

[0021] In some aspects, antibodies comprising antigen binding domains provided herein are provided herein. In some aspects, antibodies or antigen binding fragments thereof that bind to the same epitope of human CD98hc as the antigen binding domains provided herein are provided herein. In some aspects, antibodies or antigen binding fragments thereof that competitively inhibit the binding of antigen binding domains provided herein to human CD98hc are provided herein.

[0022] In some aspects, multispecific proteins are provided herein, comprising a first antigen binding domain, which is an antigen binding domain provided herein that is connected to a second antigen binding domain. In some aspects, the second antigen binding domain specifically binds to CNS antigens. In some aspects, multispecific proteins are provided herein, comprising an antigen binding domain provided herein that is connected to an antibody or its antigen binding fragment. In some aspects, an antibody or its antigen binding fragment specifically binds to a CNS antigen. In some aspects, an antibody or its antigen binding fragment comprises a heavy chain constant region. In some aspects, the antigen binding domain provided herein is optionally connected to the C-terminus of the heavy chain constant region via an amino acid linker. In some aspects, multispecific proteins are bispecific. In some aspects, multispecific proteins are divalent, trivalent or tetravalent. In some aspects, multispecific proteins are divalent.

[0023] In some aspects, the multispecific proteins provided herein are trivalent, optionally wherein the trivalent protein comprises one of the antigen binding domains that binds to human CD98hc and two antigen binding domains that bind to CNS antigens.

[0024] In some aspects, the multispecific proteins provided herein are tetravalent, optionally wherein the tetravalent protein comprises two of the antigen binding domains that bind to human CD98hc and two antigen binding domains that bind to a CNS antigen.

[0025] In some aspects, the multispecific proteins provided herein are trivalent and bispecific and comprise an antigen binding domain provided herein linked to an antibody that binds a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, and wherein the antigen binding domain is a scFv linked to the C-terminus of one of the two antibody heavy chains, optionally via an amino acid linker.

[0026] In certain aspects, the multispecific proteins provided herein are tetravalent and bispecific and comprise two antigen binding domains provided herein, and antibodies that bind to CNS antigens, wherein the antibodies comprise two heavy chains and two light chains, wherein each of the two antigen binding domains is a scFv, Fab, or VHH, wherein one of the two antigen binding domains is linked to the C-terminus of one of the antibody heavy chains, optionally via an amino acid linker, and wherein the other antigen binding domain is linked to the C-terminus of the other antibody heavy chain, optionally via an amino acid linker.

[0027] In some aspects, the antibody or antigen-binding fragment thereof comprises a constant region containing a knob mutation and a constant region containing a hole mutation. In some aspects, the antigen-binding domain is optionally connected to the constant region containing the hole mutation via an amino acid linker. In some aspects, the antigen-binding domain is optionally connected to the constant region containing the knob mutation via an amino acid linker. In some aspects, 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). In some aspects, 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).

[0028] In some aspects, the CNS antigen is a brain antigen. In some aspects, the CNS antigen is not CD98hc.

[0029] In some aspects, 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 aspects, the antibody or antigen-binding fragment thereof comprises a constant region and a mutation that reduces effector function, the constant region comprising a knob mutation, 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 and a mutation that reduces effector function, wherein the constant region comprises a hole mutation, 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.

[0030] In some aspects, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof. In some aspects, the IgG antibody or antigen-binding fragment thereof is an IgG1 antibody or antigen-binding fragment thereof or an IgG4 antibody or antigen-binding fragment thereof.

[0031] In some aspects, the multispecific protein binds human CD98hc with an equilibrium dissociation constant (KD) of about 3 nM to about 225 nM and / or binds cynomolgus monkey CD98hc with a KD of about 3 nM to about 225 nM.

[0032] In some aspects, compared with the internalization of isotype control, the internalization of multispecific protein in blood-brain barrier epithelial cells is more than 10 times higher, and optionally wherein blood-brain barrier epithelial cells are HCMEC / D3 cells. In some aspects, relative to the cell surface expression of CD98hc on HCMEC / D3 cells treated with isotype control, multispecific protein will not reduce the cell surface expression of CD98hc on HCMEC / D3 cells by more than 20%. In some aspects, relative to the cell surface expression of CD98hc on HCMEC / D3 cells treated with isotype control, multispecific protein will not increase the cell surface expression of CD98hc on HCMEC / D3 cells by more than 50%. In some aspects, multispecific protein accumulates at least 1.5 times or at least 2 times more than isotype control in the mouse brain of vascular depletion. In some aspects, 24 hours after being applied to mice, the brain of multispecific protein: serum concentration ratio increases by at least 5 times compared with isotype control.

[0033] In some aspects, the CNS antigen is β-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), Parkin, presenilin 1, presenilin 2, β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotransmitter. trophic factor receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor 2 on myeloid cells (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-metastatic melanoma protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4 domain A4A (MS4A4A), transmembrane 4 domain A 6A (MS4A6A), MS4A4E, transmembrane protein 106B (TMEM106b), ubiquitin protein ligase E3A (UBE3A), CR1, ABCA1, ABCA7, HLA-DR1, HLA-DR5, IL1RAP, TREML2, IL-34, SORL1 or ADAM1. In some aspects, the CNS antigen is MS4A4A, optionally wherein (i) the antigen binding domain, antibody or antigen binding domain that binds to MS4A4A comprises a VH comprising the amino acid sequence of SEQ ID NO: 407 and / or a VL comprising the amino acid 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.

[0034] In some aspects, the multispecific protein comprises the amino acid sequence of SEQ ID NOs: 405-410.

[0035] In some aspects, the fusion proteins provided herein, the antibodies or antigen-binding fragments thereof provided herein, or the multi-specific proteins provided herein are capable of crossing the BBB. In some aspects, the fusion proteins, antibodies or antigen-binding fragments thereof, or the multi-specific proteins provided herein are connected to an imaging agent.

[0036] In some aspects, provided herein is a composition 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.

[0037] In some aspects, provided herein is a composition 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, optionally wherein the first and second antigen-binding domains that bind to human CD98hc comprise the same amino acid sequence.

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

[0039] In some aspects, the ratio of the first, second, and third polynucleotides is about 1:3:6.

[0040] In some aspects, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0041] In some aspects, provided herein is a composition 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 wherein the second polynucleotide encodes a light chain.

[0042] In some aspects, provided herein are host cells comprising a composition provided herein.

[0043] In some aspects, provided herein are isolated polynucleotides comprising nucleic acid molecules encoding the heavy chain of the antigen binding domains provided herein. In some aspects, provided herein are isolated polynucleotides comprising nucleic acid molecules encoding the light chain variable region of the antigen binding domains provided herein.

[0044] In some aspects, provided herein are isolated vectors comprising polynucleotides provided herein. In some aspects, provided herein are isolated vectors comprising nucleic acid molecules encoding the heavy chain variable region of the antigen binding domains provided herein and nucleic acid molecules encoding the light chain variable region provided herein.

[0045] In some respects, provided herein is a host cell comprising a polynucleotide provided herein or a carrier provided herein. In some respects, the host cell is selected from the group consisting of: Escherichia coli (E.coli), Pseudomonas (Pseudomonas), Bacillus (Bacillus), Streptomyces (Streptomyces), yeast, CHO, YB / 20, NSO, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells and human cells in tissue culture.

[0046] In some aspects, provided herein are methods for producing an antigen binding domain or a multispecific protein, comprising culturing a host cell provided herein, thereby producing the antigen binding domain or the multispecific protein, optionally wherein the method further comprises isolating the antigen binding domain or the multispecific protein from the culture.

[0047] In some aspects, provided herein are isolated antigen binding domains or multispecific proteins thereof produced by the methods provided herein.

[0048] In some aspects, provided herein is a pharmaceutical composition comprising (i) an antigen binding domain, a fusion protein, an antibody or its antigen binding fragment or a multi-specific protein provided herein, and (ii) a pharmaceutically acceptable carrier. In some aspects, compared with an isotype control, the concentration of the fusion protein, antibody or its antigen binding fragment or multi-specific protein in the brain increases after being administered to a subject. In some aspects, compared with an isotype control, administration increases the delivery of the fusion protein, antibody or its antigen binding fragment, multi-specific protein or pharmaceutical composition to the brain by at least 50%, at least 100%, at least 200%, at least 500% or at least 1000%.

[0049] In some aspects, provided herein is a method for treating a nervous system disease or disorder of a subject, comprising administering to a subject a fusion protein, an antibody or its antigen-binding fragment, a multi-specific protein or a pharmaceutical composition provided herein. In some aspects, compared to an isotype control, the administration increases the delivery of a fusion protein, an antibody or its antigen-binding fragment, a multi-specific protein or a pharmaceutical composition to the brain by at least 50%, at least 100%, at least 200%, at least 500% or at least 1000%. In some aspects, administration increases the delivery of a fusion protein, an antibody or its antigen-binding fragment, a multi-specific protein or a pharmaceutical composition to the frontal cortex, the entorhinal cortex and / or the hippocampus. In some aspects, a nervous system disease or disorder is selected from a neuropathic disorder, a neurodegenerative disease, a cancer, an ocular disease disorder, an epilepsy, a lysosomal storage disease, an amyloidosis, a viral or microbial disease, an ischemic disorder, a behavioral disorder and CNS inflammation. In some aspects, the neurological disease or disorder is selected from Alzheimer's disease (AD), Huntington's disease, dystonia, ataxia, Bell's palsy, stroke, dementia, Lewy body dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman's syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, encephalitis, traumatic brain injury, and limbic-predominant age-related TDP-43 encephalopathy (LATE). In some aspects, the dementia is frontotemporal dementia (FTD). In some aspects, the neurological disease or condition is Alzheimer's disease. In some aspects, Alzheimer's disease is early-onset Alzheimer's disease, prodromal Alzheimer's disease, mild Alzheimer's disease or late-onset Alzheimer's disease. In some aspects, the neurological disease or condition is Parkinson's disease. In some aspects, the neurological disease or condition is frontotemporal epilepsy. In some aspects, the neurological disease or condition is autism. In some aspects, the neurological disease or condition is anencephaly.

[0050] In some aspects, provided herein is a method for treating a lysosomal storage disease of 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.

[0051] In some aspects, provided herein is a method for transporting fusion protein, antibody or its antigen binding fragment or multi-specific protein through the BBB of a subject, comprising administering to a subject a fusion protein, antibody or its antigen binding fragment, multi-specific protein or pharmaceutical composition provided herein. In some aspects, compared with an isotype control, the concentration of fusion protein, antibody or its antigen binding fragment or multi-specific protein in the brain increases after administration. In some aspects, compared with an isotype control, the concentration of fusion protein, antibody or its antigen binding fragment, multi-specific protein or pharmaceutical composition in the brain increases by at least 50%, at least 100%, at least 200%, at least 500% or at least 1000%. In some aspects, compared with administering an isotype control, administering a fusion protein, antibody or its antigen binding fragment or multi-specific protein does not result in a reduction in the reticulocyte count of a subject by more than 10%. In some aspects, compared with an isotype control, administering a fusion protein, antibody or its antigen binding fragment or multi-specific protein does not result in a reduction in the reticulocyte count of a subject.

[0052] 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.

[0053] In some aspects, provided herein are methods of imaging a CNS antigen in a subject, comprising administering to the subject a fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein, and locating an imaging agent in the subject.

[0054] In some aspects, provided herein are methods for detecting CNS antigens in vitro, comprising contacting an in vitro sample with a fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein, and locating an imaging agent within the sample.

[0055] In some aspects, provided herein are uses of the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins or compositions provided herein in the methods provided herein.

[0056] In some aspects, provided herein are fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins or compositions provided herein for use in the methods provided herein.

[0057] It should be understood that one, some or all of the characteristics of the various aspects described herein can be combined to form other aspects of the present invention. Those skilled in the art will immediately understand these and other aspects of the present disclosure. These and other aspects of the present disclosure are further described by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1A A 2+1 bispecific antibody is shown.

[0059] Figure 1B A 2+2 bispecific antibody with 2 scFvs having the same amino acid sequence is shown.

[0060] Figure 1C A 2+2 bispecific antibody with two scFvs having different amino acid sequences is shown.

[0061] Figure 2 Shown is the fold change in CD98hc surface levels on hCMEC / D3 cells following treatment with 2+1 anti-CD98hc bispecific antibodies compared to untreated cells, as assessed by FACS. See Example 16.

[0062] Figure 3 Shown are total CD98hc protein levels on hCMEC / D3 cells following treatment with 2+1 anti-CD98hc bispecific antibodies as assessed by Western blot. See Example 16.

[0063] Figure 4 The 2+1 anti-CD98hc bispecific antibody is shown in hCD98hc + / - Brain permeability in mice, as measured by vascular depletion of antibody levels in the brain 24 hours after peripheral injection. Antibody CD98hc.04.064 increased approximately 2-fold compared to control. Antibody CD98hc.04.063 increased approximately 1.5-fold compared to control. See Example 17.

[0064] Figure 5 The 2+1 anti-CD98hc bispecific antibody is shown in hCD98hc + / - Serum PK in mice. Brain-penetrating antibodies have the highest serum clearance. See Example 17.

[0065] Figure 6 The results show that 2+1 anti-CD98hc bispecific antibody can effectively inhibit the expression of hCD98hc in hCD98hc cells 24 hours after peripheral injection. + / - Brain / serum ratio in mice. See Example 17.

[0066] Figure 7A modest antibody-dependent cell-mediated cytotoxicity (ADCC) response of the 2+1 anti-CD98hc bispecific antibody against BBB cell lines was shown. See Example 20.

[0067] Figure 8 Antibody levels in vascular depleted brain fractions of huCD98hc knock-in mice following administration of 2_+1 anti-CD98hc bispecific antibodies and matched controls (having the same Fab and Fc domains but without a scFv that specifically binds to huCD98hc) are shown. Antibody levels are shown as fold change compared to matched controls. See Example 22.

[0068] Fig. 9 Shown are antibody levels in the serum of huCD98hc knock-in mice following administration of 2_+1 anti-CD98hc bispecific antibodies and matched controls (having the same Fab and Fc domains but without a scFv that specifically binds to huCD98hc). See Example 22.

[0069] Fig.10 The activity of the 2+1 anti-CD98hc bispecific antibody in an in vitro sTREM2 assay is shown. See Example 24.

[0070] Fig.11 Shown are absolute reticulocyte counts in non-human primates (NHPs) following administration of 2+1 anti-CD98hc bispecific antibodies. See Example 26.

[0071] Fig.12 Serum and CSF levels of 2+1 anti-CD98hc bispecific antibody after the first and second doses are shown. See Example 27.

[0072] Fig.13 Antibody concentrations in NHP brain fractions after administration of 2+1 anti-CD98hc bispecific antibodies are shown. See Example 28.

[0073] Fig.14 Soluble TREM2 levels in serum and CSF of NHPs after administration of 2+1 anti-CD98hc bispecific antibodies are shown. See Example 29.

[0074] Fig.15 Shown are the levels of CSF-1 in the CSF of NHPs after administration of 2+1 anti-CD98hc bispecific antibodies. See Example 29. DETAILED DESCRIPTION

[0075] The present disclosure relates to antigen-binding domains that specifically bind to human CD98 heavy chain (CD98hc), and antibodies and antigen-binding fragments thereof comprising such antigen-binding domains, methods for preparing and using such antigen-binding domains, antibodies and antigen-binding fragments thereof; pharmaceutical compositions comprising such antigen-binding domains, antibodies and antigen-binding fragments thereof; nucleic acids encoding such antigen-binding domains, antibodies and antigen-binding fragments thereof; and host cells comprising nucleic acids encoding such antigen-binding domains, antibodies and antigen-binding fragments thereof.

[0076] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are routinely employed using conventional methods, such as, for example, widely utilized methods such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel et al., eds., (2003); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000).

[0077] definition

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

[0079] The terms "blood-brain barrier" and "BBB" refer to a network of brain capillary endothelial cells that is tightly sealed by tight junctions and is characterized by low levels of nonspecific paracellular and transcellular transport.

[0080] "Central nervous system antigens" or "CNS antigens" are antigens expressed in the CNS (including the brain) that can be targeted by antibodies or small molecules. Examples of such antigens include, but are not limited to, beta-secretase 1 (BACE1), amyloid beta protein (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), Parkin, presenilin 1, presenilin 2, beta-glucocerebrosidase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR ), interleukin 6 receptor (IL6R), TNF receptor 1 (TNFR1), interleukin 1β (IL1(3)), caspase 6, sortilin (SORT), triggering receptor 2 on myeloid cells (TREM2), CD33 or sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), glycoprotein non-metastatic melanoma protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4 domain A4A (MS4A4A), transmembrane 4 domain A 6A (MS4A6A), ubiquitin protein ligase E3A (UBE3A) or transmembrane protein 106B (TMEM106b).

[0081] "Brain antigens" are CNS antigens expressed in the brain.

[0082] Unless otherwise indicated, the terms "CD98hc", "CD98hc polypeptide" and "CD98hc protein" used interchangeably herein 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). CD98hc is also known 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 aspects, the term encompasses wild-type sequences and naturally occurring variant sequences, such as splice variants or allelic variants. In some aspects, the term encompasses "full-length" unprocessed CD98hc and any form of CD98hc produced by intracellular processing. In some aspects, 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.

[0083] MELQPPEASIAVVSIPRQLPGSHSEAGVQGLSAGDDSELGSHCVAQTGLELLASGDPLPSASQNAEMIETGSDCVTQAGLQLLASSDPPALASKNAEVTGTMSQDTEVDMKEVELNELEPEKQPMNAASGAAMSLAGAEKNGLVKIKVAEDEAEAAAAAKFTGLSKEELLKVAGS PGWVRTRWALLLLFWLGWLGMLAGAVVIIVRAPRCRELPAQKWWHTGALYRIGDLQAFQGHGAGNLAGLKGRLDYLSSLKVKGLVLGPIHKNQKDDVAQTDLLQIDPNFGSKEDFDSLLQSAKKKSIRVILDLTPNYRGENSWFSTQVDTVATKVKDALEFWLQAGVDGFQVRDIENLKDASSFLAEWQNITKGFSEDRLLIAGTNSSDLQQILSLLESNKDLLLTSSYLSDSGSTGEHTKSLVTQYLNATGNRWCSWSLSQARLLTSFLPAQLLRLYQLMLFTLPGTPVFSYGDEIGLDAAALPGQPMEAPVMLWDESSFPDIPGAVSANMTVKGQSEDPGSLLSLFRRLSDQRSKERSLLHGDFHAFSAGPGLFSYIRHWDQNERFLVVLNFGDVGLSAGLQASDLPASASLPAKADLLLSTQPGREEGSPLELERLKLEPHEGLLLRFPYAA(SEQ ID NO:435)

[0084] As used herein, the terms "antibody" and "immunoglobulin" are used interchangeably and refer to antibody molecules (e.g., glycoproteins) that are recognized and specifically bound to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing) by at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term "antibody" encompasses monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific (e.g., bispecific) antibodies, and any other immunoglobulin molecules, as long as the antibody exhibits the desired biological activity. Antibodies may belong to any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), which are referred to as α, δ, ε, γ, and μ, respectively, based on the identity of their heavy chain constant regions. Different classes of antibodies have different and well-known subunit structures and three-dimensional configurations. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6.

[0085] The terms "anti-CD98hc antibody," "antibody that binds to CD98hc," and "antibody that specifically binds to CD98hc" refer to antibodies that are capable of binding to CD98hc with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting CD98hc. In one aspect, 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, e.g., as measured by radioimmunoassay (RIA). In certain aspects, an antibody that binds to CD98hc has a binding affinity of <10 μM, <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 M or smaller, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 In certain aspects, the anti-CD98hc antibody binds to an epitope of CD98hc that is conserved between CD98hc of different species.

[0086] The term "antibody fragment" refers to a part of an antibody. An "antigen-binding fragment" of an antibody refers to a part of an antibody that binds to an antigen. An antigen-binding fragment of an antibody may include an antigen-determining region (e.g., a complementary determining region (CDR)) of an antibody. Examples of antigen-binding fragments of an antibody include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies and single-chain antibodies. An antigen-binding fragment of an antibody may be monovalent or multivalent (e.g., bivalent). An antigen-binding fragment of an antibody may be monospecific or multispecific (e.g., bispecific). An antigen-binding fragment of an antibody may be derived from any animal species, such as a rodent (e.g., a mouse, a rat or a hamster) and a human, or may be artificially produced.

[0087] An "antigen binding domain" or "antigen binding region" refers to a monovalent portion of an antibody that binds to an antigen. An "antigen binding domain" may include an antigenic determining region (e.g., a complementarity determining region (CDR)) of an antibody. An antibody or antigen binding fragment thereof (including monospecific and multispecific (e.g., bispecific) antibodies or antigen binding fragments thereof) may comprise an antigen binding domain.

[0088] 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 for targeting CD98hc and / or as a diagnostic agent, therapeutic agent, or for transporting molecules or compounds across the BBB. In one aspect, the extent of binding of the 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, e.g., as measured by radioimmunoassay (RIA). In certain aspects, the antibody that binds to CD98hc has a binding affinity of <0.1 μM, <1 μM, <10 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 M or smaller, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 In certain aspects, the anti-CD98hc antigen binding domain binds to an epitope of CD98hc that is conserved between CD98hc of different species.

[0089] The terms "full-length antibody", "intact antibody" or "complete antibody" are used interchangeably to refer to an antibody in its substantially complete form, rather than an antibody fragment. Specifically, complete antibodies include those having heavy and light chains, including an Fc region. The constant region may be a native sequence constant region (e.g., a human native sequence constant region) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

[0090] "Native IgG antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light ("L") chains and two identical heavy ("H") chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V H ), followed by a number of constant domains. Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain.

[0091] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a name that reflects the ability to crystallize easily. The Fab fragment consists of an entire light chain and the variable region domain (V H ) and the first constant domain of a heavy chain (C H 1) Composition. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen binding site. Pepsin treatment of an antibody produces a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen binding activities and is still able to cross-link antigen. The Fab' fragment differs from the Fab fragment in that the C H The carboxyl terminus of the constant domain has a few additional residues, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) of the constant domains carry a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0092] The Fc fragment comprises the carboxyl terminal portions of the two heavy chains held together by disulfide bonds.The effector functions of antibodies are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcR) found on certain types of cells.

[0093] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain in a tight non-covalent association. From the folding of these two domains, six hypervariable loops (3 loops each from the H chain and L chain) that contribute to the amino acid residues for antigen binding and confer antigen binding specificity to the antibody are emanated. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although its affinity is lower than that of the complete binding site.

[0094] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising a VH antibody domain and a VL antibody domain linked into a single polypeptide chain. In some aspects, the scFv polypeptide also comprises a VH antibody domain and a VL antibody domain. H Domain and V L The polypeptide linker between the domains enables the sFv to form the desired structure for antigen binding.

[0095] The term "diabody" refers to small antibody fragments prepared by: constructing a V H Domain and V L sFv fragments (see previous paragraph) with short linkers (about 5-10 residues) between the domains, allowing interchain rather than intrachain pairing of the variable domains, thereby generating a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossed" sFv fragments, in which the V domains of the two antibodies are H Domain and V L Domains are present on different polypeptide chains.

[0096] As used herein, "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, wherein the antibody comprises 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. Figure 1A This format is illustrated.

[0097] As used herein, "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; wherein one antigen binding domain that binds to human CD98hc is linked to the C-terminus of one of the two antibody heavy chains, and the other antigen binding domain that binds 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. Figure 1B This format is shown. In some aspects, the two scFv antigen binding domains that bind to human CD98hc can comprise different amino acid sequences. Figure 1C This format is shown.

[0098] As used herein, the term "variable region" or "variable domain" is used interchangeably and is common in the art. A variable region generally refers to a part of an antibody, generally a part of a light chain or a heavy chain, generally about 110 to 120 amino acids or 110 to 125 amino acids of the amino terminal in a mature heavy chain, and about 90 to 115 amino acids of the amino terminal in a mature light chain, the sequence of the variable region is very different between antibodies, and is used for the binding and specificity of a particular antibody to its specific antigen. The variability of the sequence is concentrated in the region called the complementary determining region (CDR), and the highly conserved region in the variable domain is called the framework region (FR). It is not desirable to be bound by any particular mechanism or theory, and it is believed that the CDR of the light chain and the heavy chain is primarily responsible for the interaction and specificity of the antibody with the antigen. In some respects, the variable region is a human variable region. In some respects, the variable region includes rodent or mouse CDR and human framework region (FR). In some respects, the variable region is a primate (e.g., non-human primate) variable region. In some aspects, the variable region includes rodent or mouse CDR and primate (e.g., non-human primate) framework region (FR). The term "Kabat numbering" and similar terms are recognized in the art and refer to the numbering system of amino acid residues in the heavy chain and light chain variable regions of antibodies or their antigen-binding fragments. In some aspects, CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA and Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35 (which may optionally include one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDRH1), amino acid positions 50 to 65 (CDRH2), and amino acid positions 95 to 102 (CDRH3). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDRL1), amino acid positions 50 to 56 (CDRL2), and amino acid positions 89 to 97 (CDRL3). In some aspects, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme. Chothia referred instead to the positions of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).The ends of the Chothia CDRH1 loops, when numbered using the Kabat numbering convention, vary between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. In some aspects, the CDRs may be "contacting" CDRs. "Contacting" CDRs are based on analysis of available complex crystal structures. The residues from each of these CDRs are indicated below.

[0099]

[0100] The CDRs may include the following "extended CDRs": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these extended CDR definitions, the variable domain residues are numbered according to Kabat et al. (supra).

[0101] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0102] As used herein, the term "heavy chain" when used to refer to an antibody can refer to any different types based on the constant region amino acid sequence, for example, alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), which produce IgA, IgD, IgE, IgG and IgM class antibodies, respectively, including IgG subclasses, for example, IgG1, IgG2, IgG3 and IgG4. Heavy chain amino acid sequences are well known in the art. In some aspects, the heavy chain is a human heavy chain.

[0103] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0104] As used herein, the term "light chain" when used to refer to an antibody can refer to any different types of amino acid sequences based on the constant region, for example, kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.

[0105] As used herein, the term "constant region" is a region of an antibody that is not an antibody variable region, such as a carboxyl terminal portion of a light chain and / or a heavy chain, which is not directly involved in the binding of the antibody to an antigen but may exhibit various effector functions, such as interactions with Fc receptors. The constant region of an immunoglobulin molecule typically has an amino acid sequence that is more conservative relative to an immunoglobulin variable domain. In certain aspects, an antibody or antigen-binding fragment comprises a constant region or portion thereof sufficient to achieve antibody-dependent cell-mediated cytotoxicity (ADCC).

[0106] "Constant domain" refers to the domain within the constant region that is capable of forming an immunoglobulin fold. The constant domain includes CH1, CH2, CH3 and CL domains.

[0107] When referring to an antibody or its antigen-binding fragment, the term "monoclonal" refers to a homogeneous antibody or antigen-binding fragment group that participates in highly specific recognition and binding to a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies that typically contain different antibodies for different antigenic determinants. The term "monoclonal" antibody or its antigen-binding fragment encompasses complete and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising antibodies or antibody portions, and any other modified immunoglobulin molecules comprising antigen recognition sites. In addition, "monoclonal" antibodies or their antigen-binding fragments refer to such antibodies and their antigen-binding fragments prepared in any number of ways, including but not limited to by hybridomas, phage selection, recombinant expression, and transgenic animals.

[0108] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof in which the amino acid sequence is derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of an antibody or antigen-binding fragment thereof derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity and capacity, while the constant regions are homologous to sequences in an antibody or antigen-binding fragment thereof derived from another mammalian species (usually human) to avoid causing an immune response in that species.

[0109] The term "humanized" antibody or antigen-binding fragment thereof refers to a form of a non-human (e.g., murine) antibody or antigen-binding fragment that is a specific immunoglobulin chain, a chimeric immunoglobulin, or a fragment thereof that contains minimal non-human (e.g., murine) sequences. Typically, a humanized antibody or antigen-binding fragment thereof is a human immunoglobulin in which residues from a complementary determining region (CDR) are replaced ("CDR grafting") with residues from a CDR of a molecule of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and capacity (Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988)). Humanized antibodies or their antigen-binding fragments can be further modified by replacing additional residues in the Fv framework region and / or in the replaced non-human residues to improve and optimize the specificity, affinity and / or ability of the antibody or its antigen-binding fragment. In general, a humanized antibody or its antigen-binding fragment will comprise a VH and a VL, wherein the VH and VL comprise at least one and generally two or three substantially all of the CDR regions corresponding to non-human immunoglobulins, and all or substantially all of the FR regions are those of the human immunoglobulin consensus sequence. A humanized antibody or its antigen-binding fragment may also comprise at least a portion of an immunoglobulin constant region or an Fc region (generally a human immunoglobulin constant region or an Fc region). Examples of methods for producing humanized antibodies are described in U.S. Pat. No. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 91 (3): 969-973 (1994) and Roguska et al., Protein Eng. 9 (10): 895-904 (1996). In some aspects, a "humanized antibody" is a resurfaced antibody.

[0110] The term "human" antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin locus, wherein such antibody or antigen-binding fragment is prepared using any technique known in the art. This definition of human antibodies or antigen-binding fragments thereof includes complete or full-length antibodies and fragments thereof.

[0111] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as herein defined.

[0112] As used herein, an "acceptor human framework" is a V sequence comprising a human immunoglobulin framework or a human consensus framework. L or V HThe framework of the amino acid sequence of the framework. The acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may comprise pre-existing amino acid sequence changes. In some aspects, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less or 2 or less. When pre-existing amino acid changes are present in VH, in some aspects, those changes are present only at three, two or one of positions 71H, 73H and 78H; for example, the amino acid residues at those positions may be 71A, 73T and / or 78A. In some aspects, the VL acceptor human framework is identical in sequence to the V L The human immunoglobulin framework sequences or human consensus framework sequences are identical.

[0113] The "human consensus framework" is a representation of the human immunoglobulin V L or V H The framework of the most commonly occurring amino acid residues in a selection of framework sequences. L or V H The selection of sequences is from a subset of variable domain sequences. Typically, the subset of sequences is a subset as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include for V L , the subgroup may be subgroup κI, κII, κIII or κIV as in Kabat et al. (supra). In addition, for V H , the subgroup may be subgroup I, subgroup II or subgroup III as in Kabat et al. (supra).

[0114] For example, an "amino acid modification" at a specified position of an antibody of the present disclosure refers to a substitution or deletion of a specified residue, or the insertion of at least one amino acid residue adjacent to a specified residue. Insertion "adjacent" to a specified residue means insertion within one to two residues thereof. Insertion may be at the N-terminus or C-terminus of a specified residue. In some aspects, an amino acid modification is a substitution.

[0115] Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype.

[0116] The term "Fc region" or "fragment crystallizable region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) in the Fc region may be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Therefore, the composition of the complete antibody may include an antibody population from which all K447 residues have been removed, an antibody population from which the K447 residue has not been removed, and an antibody population having an antibody containing the K447 residue and an antibody population containing a mixture of antibodies without the K447 residue. The native sequence Fc region suitable for use in the antibodies of the present disclosure includes human IgG1, IgG2, IgG3, and IgG4.

[0117] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0118] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification, in some aspects one or more amino acid substitutions. In some aspects, the variant Fc region has at least one amino acid substitution in the native sequence Fc region or in the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions, and in some aspects, about one to about five amino acid substitutions, compared to the native sequence Fc region or the Fc region of a parent polypeptide. In some aspects, the variant Fc region has at least 80% homology with the native sequence Fc region and / or with the Fc region of a parent polypeptide, at least 90% homology therewith, or at least 95% homology therewith.

[0119] "Fc receptor" or "FcR" describes a receptor that is bound to the Fc region of an antibody. In some aspects, FcR is a native sequence human FcR. In some aspects, FcR is an FcR (gamma receptor) that binds to an IgG antibody, and includes receptors of FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors, and FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor") with similar amino acid sequences, the difference being mainly in its cytoplasmic domain. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif ("ITAM") in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif ("ITIM") in its cytoplasmic domain. Other FcRs, including those to be identified in the future, are covered by the term "FcR" herein. FcR can also extend the serum half-life of antibodies.

[0120] "Binding affinity" generally refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or antigen-binding fragment thereof and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (K D Affinity can be measured and / or expressed in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant (K D ) and equilibrium association constant (K A ). D By k off / k on The quotient of is calculated, and K A By k on / k off The quotient is calculated. on refers to the association rate constant of, for example, an antibody or its antigen-binding fragment with an antigen, while 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 It can be determined by techniques known to those of ordinary skill in the art, such as or KinExA.

[0121] With respect to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds to" or "specifically for" a particular polypeptide or an epitope on a particular polypeptide target means that the binding is measurably different from non-specific interactions. Specific binding can be measured, for example, by measuring the binding of a molecule and comparing it to the binding of a control molecule. For example, specific binding can be measured by competition with a control molecule similar to the target, such as an excess of unlabeled target. In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, then specific binding is indicated. As used herein, the term "specific binding" or "specifically binds to" or "specifically for" a particular polypeptide or an epitope on a particular polypeptide target can be measured, for example, by determining that the molecule has about 10 binding sites to the target. -4 M or lower, 10 -5 M or lower, 10 -6 M or lower, 10 -7 M or lower, 10 -8 M or lower, 10 -9 M or lower, 10 -10 M or lower, 10 -11 M or lower, 10 -12 M or lower or KD in 10 -4 M to 10 -6 M or 10 -6 M to 10 -10 M or 10 -7 M to 10 -9 Affinity is exhibited by a KD in the range of 100-200 M. As will be appreciated by those skilled in the art, affinity and KD values ​​are inversely related. High affinity for an antigen is measured by a low KD value. In some aspects, the term "specific binding" refers to binding in which a molecule binds to a specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0122] The term "joint" or "connected" refers to a covalent connection between two polypeptides or two heterologous molecules. In some aspects, the joint is a chemical joint. In some aspects, the joint comprises a peptide bond, and the two polypeptides or two heterologous molecules are directly or via one or more additional amino acids connected to each other. A glycine joint is a joint comprising one or more glycines but not other amino acids, such as GGGG (SEQ ID NO: 351). A glycine-rich joint is a joint comprising one or more glycines and may contain other amino acids, as long as glycine is the main species in the joint, such as GGGNGG, wherein N is any amino acid (SEQ ID NO: 352). A glycine-serine joint is a joint containing any proportion of glycine and serine, such as GGGS (SEQ ID NO: 353). Similarly, a proline joint is a joint comprising one or more prolines but not other amino acids. A proline-rich joint is a joint comprising one or more prolines, and may contain other amino acids, as long as proline is the main species in the joint.

[0123] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide or antibody sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art needed to achieve maximal alignment over the full length of the sequences being compared.

[0124] The term "epitope" includes any determinant that can be bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets the antigen, and when the antigen is a polypeptide, includes specific amino acids that directly contact the antibody. Most commonly, epitopes are present on polypeptides, but in some cases, may be present on other types of molecules such as nucleic acids. Epitope determinants may include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural features and / or specific charge characteristics. Typically, an antibody that is specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of polypeptides and / or macromolecules.

[0125] 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. Peptide scanning mutagenesis or high-throughput alanine scanning mutagenesis can be used to determine the ability of an antibody to bind to the same epitope as a reference antibody. In the latter approach, a comprehensive mutant library of an antigen or portion thereof (e.g., an extracellular domain) can be generated by mutating each individual amino acid residue to alanine (or if the amino acid residue is alanine, to another residue, such as serine) and testing each mutant for binding to a target antibody or antigen-binding fragment thereof.

[0126] An antibody is said to "competitively inhibit" binding of a reference antibody to a given epitope if the antibody preferentially binds to a given epitope or overlapping epitope such that the antibody blocks binding of the reference antibody to the epitope to some extent. Competitive inhibition can be determined by any method known in the art, such as a competition ELISA assay. An antibody can be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0127] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition that is not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form in which they occur in nature. In some aspects, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0128] As used herein, "substantially pure" refers to a material that is at least 50% pure (ie, free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0129] The term "expression system" refers to one or more nucleic acid molecules comprising a coding sequence and control sequences in operable linkage, together with a host cell and / or other in vitro transcription and translation machinery, that enables production of one or more proteins encoded by the nucleic acid molecules.

[0130] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA to which an additional DNA segment can be connected. Another type of vector is a bacteriophage vector. Another type of vector is a viral vector, in which an additional DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in the host cells they are introduced into (e.g., bacterial vectors and free mammalian vectors with bacterial replication origins). Other vectors (e.g., non-free mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can guide the expression of the genes to which they are operatively connected. Such vectors are referred to herein as "recombinant expression vectors", or simply "expression vectors". In general, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms.

[0131] "Polynucleotide" or "nucleic acid" as used interchangeably herein refers to polymers of nucleotides of any length, and includes DNA and RNA. The nucleotides may 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 by a synthetic reaction.

[0132] "Host cell" includes a single cell or cell culture that can be or has been a recipient of one or more vectors for incorporating a polynucleotide insert. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with one or more polynucleotides of the invention.

[0133] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed.

[0134] As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural course of the individual being treated during the course of a clinical lesion. Desirable therapeutic effects include reducing the rate of progression of a particular disease, condition, or illness, improving or alleviating the pathological state of a particular disease, condition, or illness, and relieving a particular disease, condition, or illness or improving the prognosis of a particular disease, condition, or illness. For example, if one or more symptoms associated with a particular disease, condition, or illness are alleviated or eliminated, then the individual is successfully "treated."

[0135] As used herein, the terms "administer," "administering," "administration," and the like refer to methods that can be used to deliver a drug (eg, an anti-human antibody or antigen-binding fragment thereof) to a desired site of biological action.

[0136] "Effective amount" refers to at least the following amount: the amount is effective to achieve the desired treatment result at the necessary dose and for the necessary time period. The effective amount can be provided by one or more administrations. The effective amount is also the amount in which the beneficial effects of the treatment outweigh any toxic or harmful effects of the treatment. For therapeutic use, the beneficial or desired results include clinical results such as: alleviating one or more symptoms caused by the disease, increasing the quality of life of the person suffering from the disease, reducing the dose of other drug treatments required for the treatment of the disease, enhancing the effect of another drug treatment such as by targeting, delaying the progression of the disease, and / or prolonging the survival period. The effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve therapeutic treatment. As understood in clinical situations, the effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Therefore, an "effective amount" can be considered in the case of administering one or more therapeutic agents, and if combined with one or more other agents, a desirable result can be achieved or achieved, then a single agent can be considered to be given in an effective amount.

[0137] As used herein, the terms "subject" and "patient" are used interchangeably. The subject can be a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.). In some aspects, the subject is a cynomolgus monkey. In some aspects, the subject is a human.

[0138] As used herein, administration "in conjunction with" or "in combination with" another compound or composition includes simultaneous administration and / or administration at different times. Administration in conjunction also encompasses administration in a co-formulation or in a separate composition, including at different dosing frequencies or intervals, and using the same route of administration or different routes of administration. In some aspects, administration in conjunction is administration as part of the same treatment regimen.

[0139] As used herein, "neurological disorder" refers to a disease or disorder that affects the CNS and / or has its etiology in the CNS. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, eye diseases or disorders, viral or microbial infections, inflammation, ischemia, neurodegenerative diseases, epilepsy, behavioral disorders, and lysosomal storage diseases.

[0140] As used herein, "lysosomal storage disease" or (LSD) refers to an inherited metabolic disease characterized by excessive accumulation of substrates (such as undigested or partially digested macromolecules) in various cells of an organ, ultimately leading to cellular dysfunction and clinical abnormalities. LSD has been defined as a lysosomal functional defect, usually classified by the accumulated substrate, and includes sphingolipidosis, oligosaccharidosis, mucolipidosis, mucopolysaccharidosis, lipoprotein storage disease, neuronal ceroid lipofuscinosis, etc. LSD may also include other protein deficiencies or defects that cause macromolecular accumulation, such as proteins necessary for normal post-translational modification of lysosomal enzymes, or proteins that are important for proper lysosomal transport. LSD is a disease caused by a single gene defect. Nearly seventy percent of LSDs are caused by enzyme defects, and the rest are caused by defects in enzyme activators or related proteins.

[0141] "Protein replacement therapy" or "PRT" refers to medical treatments that supplement or replace specific proteins that are deficient or missing in a patient's body.

[0142] "Enzyme replacement therapy enzyme" or "ERT enzyme" refers to an enzyme that is deficient in lysosomal storage diseases. "ERT enzyme variant" refers to a functional variant of a wild-type ERT enzyme or a fragment thereof, including alleles and splice variants, wherein, for example, when assayed under the same conditions, the ERT enzyme variant has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the activity of the corresponding wild-type ERT enzyme or a fragment thereof. A "catalytically active fragment" of an ERT enzyme refers to a portion of a full-length ERT enzyme or a variant thereof, wherein, for example, when assayed under the same conditions, the catalytically active fragment has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the activity of the corresponding full-length ERT enzyme or a variant thereof.

[0143] 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 the stated value or range and as low as 10% below the stated value or range are still within the intended meaning of the stated value or range. It should be understood that whenever the wording "about" or "approximately" is used herein to describe an aspect, other similar aspects referring to the specific value or range are also provided.

[0144] 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" is a reference to one to many antibodies, such as molar amounts, and includes equivalents thereof known to those skilled in the art, and so on.

[0145] It should be understood that whenever the wording "comprising" is used to describe aspects herein, other similar aspects described as "consisting of" and / or "consisting essentially of" are also provided. In the present disclosure, "comprises", "comprising", "containing", "having" and the like may mean "includes", "including", and the like; "consisting essentially of" or "consists essentially of" is open-ended, allowing for more than the listed contents, as long as the basic or novel features of the listed contents are not changed by the presence of the listed contents, but does not include prior art aspects.

[0146] Anti-CD98hc antigen binding domain

[0147] Provided herein are antigen binding domains that specifically bind to human CD98hc.

[0148] Such antigen binding domains are capable of crossing the blood-brain barrier (BBB) ​​and are capable of transporting other agents (e.g., therapeutically active agents) associated with the antigen binding domain across the BBB. Thus, in some aspects, provided herein are antigen binding domains that specifically bind to human CD98hc that are capable of internalization in BBB epithelial cells, such as HCMEC / D3 cells.

[0149] In some aspects, the antigen binding domain that specifically binds to human CD98hc comprises six CDRs of an antibody listed in Tables 9 and 10 (i.e., three VH CDRs of an antibody listed in Table 9 and three VL CDRs of the same antibody listed in Table 10) or six CDRs of an antibody listed in Tables 14 and 15 (i.e., three VH CDRs of an antibody listed in Table 14 and three VL CDRs of the same antibody listed in Table 15), or six CDRs of an antibody listed in Tables 23 or 26.

[0150] In some aspects, the antigen binding domain that specifically binds to human CD98hc comprises six CDRs of an antibody listed in Tables 8, 13, 22 or 25. In some aspects, the CDRs of such an antigen binding domain can be identified according to the Chothia numbering scheme, which refers to the positions of immunoglobulin structural loops (see, e.g., Chothia C and Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226). Generally, when using the Kabat numbering convention, the Chothia CDR-H1 loop occurs at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop occurs at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop occurs at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop occurs at light chain amino acids 24 to 34, the Chothia CDR-L2 loop occurs at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop occurs at light chain amino acids 89 to 97. 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 because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34).

[0151] In some aspects, the antigen binding domain that specifically binds to human CD98hc comprises six Chothia CDRs of the antibodies listed in Tables 8, 13, 22 or 25. In some aspects, such as 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 aspects, provided herein is an antigen binding domain that specifically binds to human CD98hc and comprises a combination of Kabat CDRs and Chothia CDRs.

[0152] In some aspects, the CDRs of the antigen binding domain that specifically binds to human CD98hc can be determined according to MacCallumRM et al., (1996) J Mol Biol 262:732-745. See also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In some aspects, provided herein is an antigen binding domain that specifically binds to human CD98hc and comprises the VH CDRs and VL CDRs of the antibodies listed in Tables 8, 13, 22 or 25 as determined by the method of MacCallumRM et al.

[0153] In some aspects, the CDRs of the antigen binding domain that specifically binds to human CD98hc can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable region, which represents a compromise between the Kabat CDR and the Chothia structural loop, and is used by 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 and comprises a VH CDR and a VL CDR of an antibody listed in Table 8, 13, 22 or 25 as determined by the AbM numbering scheme.

[0154] In some aspects, the antigen binding domain that specifically binds to human CD98hc comprises six IMGT CDRs of an antibody listed in Tables 8, 13, 22 or 25 according to the IMGT numbering system 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 to 35, VH-CDR2 is located at positions 51 to 57, VH-CDR3 is located at positions 93 to 102, VL-CDR1 is located at positions 27 to 32, VL-CDR2 is located at positions 50 to 52, and VL-CDR3 is located at positions 89 to 97.

[0155] In some aspects, the antigen binding domains specifically binding to human CD98hc provided herein are described by their individual VL domains or their individual VH domains or by their individual 3 VL CDRs or their individual 3 VH CDRs. See, e.g., Rader C et al., (1998) PNAS 95:8910-8915, which is incorporated herein by reference in its entirety, describing the humanization of mouse anti-αvβ3 antibodies by identifying complementary light chains or heavy chains from human light chain or heavy chain libraries, respectively, resulting in humanized antibody variants having the same or higher affinity than the original antibody. See also Clackson T et al., (1991) Nature 352:624-628, which is incorporated herein by reference in its entirety, describing a method for producing antibodies that bind to a specific antigen by using a specific VL domain (or VH domain) and screening a library of complementary variable domains. The screening yielded 14 new partners for a particular VH domain and 13 new partners for a particular VL domain, all of which were strong binders as determined by ELISA. See also, Kim SJ and Hong HJ, (2007) J Microbiol 45:572-577, which is incorporated herein by reference in its entirety, describing a method for generating antibodies that bind to a particular antigen by using a particular VH domain and screening a library (e.g., a human VL library) of complementary VL domains; the selected VL domains in turn can be used to guide the selection of additional complementary (e.g., human) VH domains.

[0156] In some aspects, the antigen binding domain that specifically binds to human CD98hc comprises the VH of an antibody listed in Tables 8, 13, 22, or 25.

[0157] In some aspects, the antigen binding domain that specifically binds to human CD98hc comprises the VL of an antibody listed in Tables 8, 13, 22, or 25.

[0158] In some aspects, the 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.

[0159] In some aspects, the 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 the VH amino acid sequence of an antibody in Table 8, 13, 22 or 25, and (ii) a VL comprising an amino acid sequence that is at least 80% identical to the VL amino acid sequence of the same antibody in Table 8, 13, 22 or 25. In some aspects, the antigen binding domain that specifically binds to human CD98hc also includes the CDRs of an antibody in Table 8, 13, 22 or 25 (e.g., the amino acids that are not identical in VH and / or VL are located outside the CDRs).

[0160] In some aspects, the 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 the VH amino acid sequence of an antibody in Table 8, 13, 22 or 25, and (ii) a VL comprising an amino acid sequence that is at least 85% identical to the VL amino acid sequence of the same antibody in Table 8, 13, 22 or 25. In some aspects, the antigen binding domain that specifically binds to human CD98hc also includes the CDRs of an antibody in Table 8, 13, 22 or 25 (e.g., the amino acids that are not identical in VH and / or VL are located outside the CDRs).

[0161] In some aspects, the 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 the VH amino acid sequence of an antibody in Table 8, 13, 22 or 25, and (ii) a VL comprising an amino acid sequence that is at least 90% identical to the VL amino acid sequence of the same antibody in Table 8, 13, 22 or 25. In some aspects, the antigen binding domain that specifically binds to human CD98hc also includes the CDRs of an antibody in Table 8, 13, 22 or 25 (e.g., the amino acids that are not identical in VH and / or VL are located outside the CDRs).

[0162] In some aspects, the 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 the VH amino acid sequence of an antibody in Table 8, 13, 22 or 25, and (ii) a VL comprising an amino acid sequence that is at least 95% identical to the VL amino acid sequence of the same antibody in Table 8, 13, 22 or 25. In some aspects, the antigen binding domain that specifically binds to human CD98hc also includes the CDRs of an antibody in Table 8, 13, 22 or 25 (e.g., the amino acids that are not identical in VH and / or VL are located outside the CDRs).

[0163] In some aspects, the 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 the VH amino acid sequence of an antibody in Table 8, 13, 22 or 25, and (ii) a VL comprising an amino acid sequence that is at least 96% identical to the VL amino acid sequence of the same antibody in Table 8, 13, 22 or 25. In some aspects, the antigen binding domain that specifically binds to human CD98hc also includes the CDRs of an antibody in Table 8, 13, 22 or 25 (e.g., the amino acids that are not identical in VH and / or VL are located outside the CDRs).

[0164] In some aspects, the 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 the VH amino acid sequence of an antibody in Table 8, 13, 22 or 25, and (ii) a VL comprising an amino acid sequence that is at least 97% identical to the VL amino acid sequence of the same antibody in Table 8, 13, 22 or 25. In some aspects, the antigen binding domain that specifically binds to human CD98hc also includes the CDRs of an antibody in Table 8, 13, 22 or 25 (e.g., the amino acids that are not identical in VH and / or VL are located outside the CDRs).

[0165] In some aspects, the 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 the VH amino acid sequence of an antibody in Table 8, 13, 22 or 25, and (ii) a VL comprising an amino acid sequence that is at least 98% identical to the VL amino acid sequence of the same antibody in Table 8, 13, 22 or 25. In some aspects, the antigen binding domain that specifically binds to human CD98hc also includes the CDRs of an antibody in Table 8, 13, 22 or 25 (e.g., the amino acids that are not identical in VH and / or VL are located outside the CDRs).

[0166] In some aspects, the 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 the VH amino acid sequence of an antibody in Table 8, 13, 22 or 25, and (ii) a VL comprising an amino acid sequence that is at least 99% identical to the VL amino acid sequence of the same antibody in Table 8, 13, 22 or 25. In some aspects, the antigen binding domain that specifically binds to human CD98hc also includes the CDRs of an antibody in Table 8, 13, 22 or 25 (e.g., the amino acids that are not identical in VH and / or VL are located outside the CDRs).

[0167] In some aspects, provided herein is an antigen binding domain that binds to the same CD98hc epitope as an antibody comprising a VH amino acid sequence of an antibody in Table 8, 13, 22 or 25 and a VL amino acid sequence of the same antibody in Table 8, 13, 22 or 25.

[0168] In some aspects, provided herein is an antigen binding domain that competitively inhibits binding of CD98hc to an antibody comprising a VH amino acid sequence of an antibody in Table 8, 13, 22 or 25 and a VL amino acid sequence of the same antibody in Table 8, 13, 22 or 25.

[0169] In some aspects, the antigen binding domain that specifically binds to human CD98hc comprises VH and VL on a single polypeptide chain (e.g., VH and VL in Tables 8, 13, 22, or 25). In some aspects, the antigen binding domain comprises an scFv. The scFv may comprise a VH at the N-terminus of the VL or a VL at the N-terminus of the VH. The scFv may comprise, for example, a linker between the VH and the VL. Thus, the scFv may be in the orientation VH-linker-VL or VL-linker-VH. The length of such a linker may be about 5 to about 25 amino acids. The length of such a linker may be about 5 to about 20 amino acids. The length of such a linker may be about 10 to about 25 amino acids. The length of such a linker may be about 10 to about 20 amino acids. 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).

[0170] In some aspects, the antigen binding domain that specifically binds to human CD98hc comprises a VH on a first polypeptide and a VL on a second polypeptide (eg, Fab).

[0171] In some aspects, the antigen binding domain that specifically binds to human CD98hc comprises an antigen binding fragment of a heavy chain-only antibody (eg, a VHH or a nanobody).

[0172] In some aspects, the antigen binding domain that specifically binds to human CD98hc is a mouse antigen binding domain. In some aspects, the antigen binding domain that specifically binds to human CD98hc is a chimeric antigen binding domain. In some aspects, the antigen binding domain that specifically binds to human CD98hc is a humanized antigen binding domain. In some aspects, the antigen binding domain that specifically binds to human CD98hc is a human antigen binding domain.

[0173] In some aspects, an antigen binding domain provided herein that specifically binds to human CD98hc also binds to cynomolgus monkey CD98hc.

[0174] In some aspects, the antigen binding domains provided herein specifically bind to human CD98hc with an affinity of no more than 250 nM (e.g., 10 μM to 250 nM, 5 μM to 250 nM, 1 μM to 250 nM, 1 nM to 250 nM, or 3 nM to 250 nM), an affinity of no more than 200 nM (e.g., 10 μM to 200 nM, 5 μM to 200 nM, 1 μM to 200 nM, 1 nM to 200 nM, or 3 nM to 200 nM), or an affinity of no more than 150 nM (e.g., 10 μM to 150 nM, 5 μM to 150 nM, 1 μM to 150 nM, 1 nM to 150 nM, or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, for example, using a Carterra LSA platform. In some aspects, the antigen binding domains provided herein specifically bind to human 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.

[0175] In some aspects, the antigen binding domain that specifically binds to human CD98hc has an ELISA OD of at least 0.45. 450 Binds to human CD98hc. In some aspects, the antigen binding domain that specifically binds to human CD98hc has an ELISA OD of at least 0.45 450 Binds to cynomolgus monkey CD98hc. In some aspects, the antigen binding domain that specifically binds to human CD98hc has an ELISA OD of at least 0.45 450 Binds to human CD98hc and has an ELISA OD of at least 0.45 450 Binds to cynomolgus monkey CD98hc.

[0176] In some aspects, the antigen binding domains provided herein that specifically bind to human CD98hc have an affinity of no more than 250 nM (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), an affinity of no more than 200 nM (e.g., 10 μM to 200 nM, 5 μM to 200 nM, 1 μM to 20 0nM, 10μM to 250nM, 1nM to 200nM or 3nM to 200nM) or binds to cynomolgus monkey CD98hc with an affinity of no more than 150nM (e.g., 10μM to 150nM, 5μM to 150nM, 1μM to 150nM, 10μM to 250nM, 1nM to 150nM or 3nM to 150nM), optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, for example, using a Carterra LSA platform. In some aspects, the antigen binding domain provided herein that specifically binds to cynomolgus monkey CD98hc binds to 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.

[0177] In some aspects, an antigen binding domain provided herein that specifically binds to human CD98hc binds to each of human CD98hc and cynomolgus monkey CD98hc with an affinity of no more than 250 nM (e.g., 1 μM to 250 nM, 1 nM to 250 nM, or 3 nM to 250 nM), an affinity of no more than 200 nM (e.g., 1 μM to 200 nM, 1 nM to 200 nM, or 3 nM to 200 nM), or an affinity of no more than 150 nM (e.g., 1 μM to 150 nM, 1 nM to 150 nM, or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, for example, using a Carterra LSA platform. In some aspects, the antigen binding domain provided herein that specifically binds to human CD98hc binds to each of 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.

[0178] In some aspects, the antigen binding domain specifically bound to human CD98hc provided herein binds to human CD98hc with an affinity of 3.1nM to 210nM. In some aspects, the antigen binding domain specifically bound to human CD98hc provided herein binds to human CD98hc with an affinity of 18nM to 35nM. In some aspects, the antigen binding domain specifically bound to human CD98hc provided herein binds to human CD98hc with an affinity of 12nM to 34nM. In some aspects, in some aspects, the antigen binding domain specifically bound to human CD98hc provided herein binds to cynomolgus monkey CD98hc with an affinity of 3.2 to 145nm. In some aspects, the antigen binding domain specifically bound to human CD98hc provided herein binds to cynomolgus monkey CD98hc with an affinity of 340nM to 1.5μM. In some aspects, the antigen binding domains specifically binding to human CD98hc provided herein bind to cynomolgus monkey CD98hc with an affinity of 120nM to 880nM. In some aspects, the antigen binding domains specifically binding to human CD98hc provided herein bind to human CD98hc with an affinity of 3.1nM to 210nM and to cynomolgus monkey CD98hc with an affinity of 3.2nM to 145nM, optionally wherein affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, for example, using a Carterra LSA platform.

[0179] In some aspects, relative to CD98hc cell surface expression on HCMEC / D3 cells treated with an isotype control, the antigen binding domains provided herein that specifically bind to human CD98hc do not reduce CD98hc cell surface expression on HCMED / D3 cells by more than 20%. Cell surface expression can be measured, for example, using Western blot or FACS.

[0180] In some aspects, relative to the cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control, the antigen binding domains provided herein that specifically bind to human CD98hc do not increase the cell surface expression of CD98hc on HCMEC / D3 cells by more than 50%. Cell surface expression can be measured, for example, using Western blot or FACS.

[0181] In some aspects, the antigen binding domains provided herein that specifically bind to human CD98hc do not reduce the cell surface expression of CD98hc on HCMED / D3 cells by more than 20% relative to the cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control, and do not increase the cell surface expression of CD98hc on HCMED / D3 cells by more than 50% relative to the cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, for example, using Western blots or FACS.

[0182] In some aspects, the antigen binding domains provided herein that specifically bind to human CD98hc accumulate at least 1.5 times more than the isotype control in the brain of vascular-depleted human CD98hc knock-in mice after peripheral injection. In some aspects, the antigen binding domains provided herein that specifically bind to human CD98hc accumulate at least 1 times more than the isotype control in the brain of vascular-depleted mice.

[0183] In some aspects, 24 hours after administration to a mouse, the brain:serum concentration ratio of an antigen binding domain provided herein that specifically binds to human CD98hc is increased at least 5-fold compared to an isotype control.

[0184] Also provided herein are antigen binding domains that bind to the same CD98hc epitope as the CD98hc antigen binding domains provided herein. Also provided herein are antigen binding domains that competitively inhibit binding of the CD98hc antigen binding domains provided herein to CD98hc.

[0185] Agents comprising an anti-CD98hc antigen binding domain

[0186] Provided herein are agents (eg, fusion proteins, multispecific (eg, bispecific) proteins, antibodies, antigen-binding fragments thereof, etc.) comprising an antigen-binding domain that specifically binds to human CD98hc.

[0187] Fusion Protein

[0188] In some aspects, the fusion protein provided herein comprises an antigen binding domain and a heterologous protein or polypeptide that specifically binds to human CD98hc. In some aspects, the heterologous protein is a protein or polypeptide or a fragment thereof that can be used for protein replacement therapy (PRT). In some aspects, the heterologous polypeptide is an enzyme (e.g., an enzyme for enzyme replacement therapy (ERT)) or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide is an ERT enzyme or an ERT enzyme variant, 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 a granule protein precursor (PGRN), a sphingolipid activator protein progenitor (PSAP) or a survival motor neuron protein (SMN). In some aspects, the heterologous protein is an enzyme 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 β-galactosidase, glucosylceramidase, β-hexosaminidase A, β-hexosaminidase B, arylsulfatase A, β-galactosidase, acid ceramidase, α-glucosidase, lysosomal acid lipase, lysosomal protease, its synthetic enzyme substitutes such as larodinase, idursulphase, elosulphase alpha or galsuphase, or variants thereof, or catalytically active fragments thereof.In some aspects, the heterologous protein is a protein or enzyme selected from the group consisting of agglutinin (APOJ), reelin, tripeptidyl peptidase 1 (CLN2 / TPP1), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-α-glucosamine N-acetyltransferase (HGSNAT), and N-acetyl-α-glucosaminidase (NAGLU), α-L iduronidase (IDUA), iduronate-2-sulfatase (IDS), N-acetylgalactosamine-6-sulfatase (GALNS), N-sulfoglucosamine sulfohydrolase (S GSH), N-acetylgalactosamine-4-sulfatase (arylsulfatase B; ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramide β-galactosidase, glucoceramidase, β-hexosaminidase A, β-hexosaminidase B, arylsulfatase A, β-galactosidase, acid ceramidase, α-glucosidase, lysosomal acid lipase, lysosomal protease, its synthetic enzyme substitute, such as laronidase, idursulfase, elosulfase α or galactosidase, or its variant, or its catalytically active fragment. In some aspects, the heterologous protein in the fusion protein is located at the N-terminus of the antigen binding domain that specifically binds to human CD98hc. In some aspects, the heterologous protein or polypeptide in the fusion protein is located at the C-terminus of the antigen binding domain that specifically binds to human CD98hc. In some aspects, the heterologous protein or polypeptide and the antigen binding domain that specifically binds to human CD98hc are directly linked via a peptide bond. In some aspects, the heterologous fusion protein and the antigen binding domain that specifically binds to human CD98hc are linked via a joint (e.g., a peptide joint). In some aspects, the fusion protein comprises an antigen binding domain and a heterologous protein or polypeptide and an Fc part. In some aspects, the antigen binding domain and the heterologous protein or polypeptide are connected to the N-terminal of the Fc part of the fusion protein. In other aspects, the antigen binding domain is connected to the N-terminal of the Fc part of the fusion protein and the heterologous protein or polypeptide is connected to the C-terminal of the Fc part of the fusion protein. In other aspects, the antigen binding domain is connected to the C-terminal of the Fc part of the fusion protein and the heterologous protein or polypeptide is connected to the N-terminal of the Fc part of the fusion protein.

[0189] Bispecific and multispecific proteins

[0190] In some aspects, the antibodies or antigen binding fragments thereof provided herein comprise an antigen binding domain that specifically binds to human CD98hc. In some aspects, 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 a brain antigen. In some aspects, the CNS antigen or brain antigen is not CD98hc. Also provided herein are antibodies or antigen binding fragments that bind to the same CD98hc epitope as the CD98hc antigen binding domain provided herein. Also provided herein are antibodies or antigen binding fragments that competitively inhibit the binding of the CD98hc antigen binding domain provided herein to CD98hc.

[0191] In some aspects, the multispecific protein provided herein comprises a first antigen binding domain and a second antigen binding domain that binds to human CD98hc. 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 aspects, the CNS antigen or brain antigen is not CD98hc.

[0192] In some aspects, the multispecific proteins provided herein comprise an antigen binding domain that binds to human CD98hc attached to an antibody or antigen binding fragment thereof. The antibody or antigen binding fragment thereof can bind to a CNS antigen or a brain antigen. In some aspects, the CNS antigen or brain antigen is not CD98hc. In some aspects, such multispecific proteins can be in a 2+1 antibody format (e.g. Figure 1A ) or a 2+2 antibody format (as Figure 1B and 1C shown).

[0193] In some aspects, the multispecific proteins provided herein comprise a CD98hc antigen binding domain, which is a scFv connected to an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains. In some aspects, the scFv is connected to the C-terminus of one of the two antibody heavy chains, for example via a protein linker.

[0194] In some aspects, the multispecific protein comprises 1) an antigen binding domain that binds CD98hc, 2) a second antigen binding domain that binds to a different CNS or brain antigen, and 3) an Fc region, wherein the CD98hc antigen binding domain and the second antigen binding domain are linked or connected to the Fc region of the multispecific protein. In other aspects, the multispecific protein comprises 1) an antigen binding domain that comprises a heavy chain variable region and binds to CD98hc, 2) a second antigen binding domain that comprises a heavy chain variable region and binds to a different CNS or brain antigen, and 3) an Fc region, wherein the CD98hc antigen binding domain and the second antigen binding domain are linked or connected to the Fc region of the multispecific protein. In some aspects, the multispecific protein includes an antigen binding domain that binds CD98hc, a second antigen binding domain that binds to a different CNS or brain antigen, and an Fc region. In some aspects, the CD98hc antigen binding domain and the second antigen binding domain are linked or connected to the N-terminus of the Fc portion of the multispecific protein. In other aspects, the CD98hc antigen binding domain is linked or attached 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 aspects, the CD98hc antigen binding domain is linked or attached 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.

[0195] In some aspects, the multispecific proteins provided herein comprise two copies of a CD98hc antigen binding domain that is a scFv and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, wherein one of the two copies of the antigen binding domain is connected to the C-terminus of one antibody heavy chain, and wherein the other copy of the antigen binding domain is connected to the C-terminus of another antibody heavy chain. In some aspects, the scFv is connected to the heavy chain via a protein linker.

[0196] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof or multispecific proteins provided herein can be multispecific, such as bispecific. Many different formats and uses of bispecific binding molecules are known in the art (reviewed in, for example, Kontermann; Drug Discov Today, July 2015; 20(7):838-47; MAbs, March to April 2012; 4(2):182-97). The bispecific proteins according to the present invention are not limited to any particular bispecific format or method of producing it. Therefore, the bispecific proteins of the present disclosure may include various configurations having a first antigen binding domain that binds to human CD98hc and a second antigen binding domain that binds to, for example, a CNS antigen or a brain antigen.

[0197] Examples of bispecific molecules that can be used in the present disclosure include, for example, (i) a single antibody with two arms comprising different antigen binding domains; (ii) a single chain antibody specific for two different epitopes, for example via two scFvs linked in series via an additional peptide linker; (iii) a dual variable domain antibody (DVD-Ig), in which each light and heavy chain contains two variable domains linked in series via a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig.TM.) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iv) a chemically linked bispecific (Fab')2 fragment; (v) a Tandab, which is a fusion of two single chain diabodies, thereby generating a tetravalent bispecific antibody with two binding sites for each target antigen; (vi) a flexible antibody (flexibody), which is a combination of an scFv and a diabody, thereby generating a multivalent molecule; (vii) a so-called "lock and key" (dock and key). (viii) so-called scorpion molecules, which contain, for example, two scFvs fused to both ends of a human Fab arm; and (ix) diabodies. Other examples of antibody structures are described in WO2019 / 246288, which is incorporated by reference.

[0198] In some aspects, the fusion protein, antibody or antigen binding fragment thereof or multispecific protein provided herein is multivalent (e.g., bivalent). In some aspects, the fusion protein, antibody or antigen binding fragment thereof or multispecific protein provided herein is trivalent (e.g., in a 2+1 antibody format). In some aspects, the trivalent format comprises a single CD98hc antigen binding domain provided herein and two antigen binding domains that bind to CNS antigens or brain antigens. The two antigen binding domains that bind to CNS antigens or brain antigens may comprise the same amino acid sequence, or may comprise different amino acid sequences. In some aspects, the CD98hc antigen binding domain is a scFv. In some aspects, the CD98hc antigen binding domain is a VHH.

[0199] In some aspects, the fusion protein, antibody or antigen-binding fragment thereof or multispecific protein provided herein is tetravalent (e.g., in a 2+2 antibody format). In some aspects, the tetravalent format comprises two CD98hc antigen-binding domains provided herein and two antigen-binding domains that bind to CNS antigens or brain antigens. The two CD98hc antigen-binding domains may comprise the same amino acid sequence, or may comprise different amino acid sequences. In some aspects, the two CD98hc antigen-binding domains comprise the same amino acid sequence. In some aspects, one or both of the CD98hc antigen-binding domains are scFv. The two antigen-binding domains that bind to CNS antigens or brain antigens may comprise the same amino acid sequence, or may comprise different amino acid sequences.

[0200] The fusion proteins, antibodies or antigen binding fragments thereof or multispecific proteins provided herein may include, for example, a linker, an antibody or antigen binding fragment thereof or other antigen binding domain that connects the CD98hc antigen binding domain to a heterologous protein. The linker may be, for example, a glycine linker, a glycine-rich linker or a glycine-serine linker. The linker may include the amino acid sequence (GGGGS) x3 (SEQ ID NO: 183). The linker may include the amino acid sequence (GGSGG) x3 (SEQ ID NO: 338). The linker may include the amino acid sequence GGSGG (no repeats) (SEQ ID NO: 354). The length of the linker may be 1 to 20 amino acids.

[0201] Fusion proteins, antibodies or their antigen-binding fragments or multispecific proteins provided herein may include constant regions. In some aspects, the CD98hc antigen-binding domains provided herein are connected to constant regions (e.g., the C-terminal end of constant regions). In some aspects, the constant domains are human constant domains. In some aspects, the constant domains are mouse, rat, rabbit or monkey (e.g., cynomolgus monkey) constant domains. 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.

[0202] In some aspects, the fusion protein, antibody or its antigen-binding fragment or multi-specific protein provided herein comprises a heavy chain and a light chain. For the heavy chain, in some aspects, the heavy chain of the antigen-binding protein described herein can be an alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In some aspects, the heavy chain may include human alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In some aspects, the heavy chain includes a human gamma (γ) heavy chain constant region. In some aspects, the heavy chain includes an amino acid sequence of an IgG1 heavy chain constant region. In some aspects, the heavy chain includes an amino acid sequence of an IgG2 (e.g., IgG2a or IgG2b) heavy chain constant region. In some aspects, the heavy chain includes an amino acid sequence of an IgG4 heavy chain constant region. For the light chain, in some aspects, the light chain is a kappa light chain. In some aspects, 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.

[0203] In some aspects, fusion protein, antibody or its antigen-binding fragment or multi-specific protein provided herein include constant region, and the constant region includes the amino acid sequence of the constant region of IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule or human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule. In some aspects, fusion protein, antibody or its antigen-binding fragment or multi-specific protein provided herein include constant region, and the constant region includes the amino acid sequence of the constant region of IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass (e.g., IgG2a and IgG2b) immunoglobulin molecule. In some aspects, the constant region comprises the amino acid sequence of a constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, an immunoglobulin molecule of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b).

[0204] Non-limiting examples of human constant region sequences are described in, e.g., U.S. Pat. No. 5,693,780 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

[0205] In some aspects, the constant regions provided herein comprise a knob mutation. In some aspects, the constant regions provided herein comprise a hole mutation. Thus, in some aspects, the fusion proteins, antibodies, or antigen-binding fragments thereof, or multispecific proteins provided herein may comprise a constant region comprising a knob mutation and a constant region comprising a hole mutation.

[0206] FC domain

[0207] The fusion protein, antibody or its antigen-binding fragment or multispecific protein provided herein may include an Fc domain or a fragment thereof. In some aspects, the Fc domain belongs to the IgG category, the IgM category or the IgA category. In some aspects, the Fc domain or its fragment is an IgG Fc domain or its fragment. In some aspects, the Fc domain or its fragment is a human IgG Fc domain or its fragment. In some aspects, the Fc domain or its fragment is a human IgG1 Fc domain or its fragment. In some aspects, the Fc domain or its fragment is a human IgG2 Fc domain or its fragment. In some aspects, the Fc domain or its fragment is a human IgG4 Fc domain or its fragment.

[0208] In some aspects provided herein, the fusion protein, antibody or antigen-binding fragment thereof or multispecific protein provided comprises a modified Fc domain or fragment thereof. In some aspects, the modified Fc domain or fragment thereof is a modified IgG1 Fc comprising one or more modifications. For example, in some aspects, the IgG1 modified Fc comprises one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some aspects, the one or more amino acid substitutions are selected from N297A (Bolt S et al. (1993) Eur J Immunol 23:403-411), D265A (Shields et al. (2001) RJ Biol. Chem. 276, 6591-6604), L234A, L235A (Hutchins et al. (1995) Proc Natl Acad Sci USA, 92: 11980-11984; Alegre et al., (1994) Transplantation 57: 1537-1543.31; Xu et al., (2000) Cell Immunol, 200: 16-26), G237A (Alegre et al. (1994) Transplantation 57: 1537-1543.31; Xu et al. (2000) Cell Immunol, 200: 16-26). 256E, wherein the amino acid positions are according to the EU numbering convention. In some aspects, the bispecific antibody comprises the amino acid substitutions L234A, L235A and P331S (LALAPS) according to EU numbering. In some aspects of any of the modified IgG1 Fc, the Fc comprises N325S and L328F mutations according to EU numbering. In some aspects of any of the modified IgG1 Fc, the Fc comprises P329G or P329S according to EU numbering.

[0209] In some aspects provided herein, the fusion protein, antibody or its antigen-binding fragment or multi-specific protein provided herein is a bispecific fusion protein, antibody or its antigen-binding fragment or multi-specific protein. Bispecific molecules include, for example, κ-λ bodies, dual affinity redirection molecules (DARTs), knob-in-hole antibodies, strand exchange engineered domain bodies (strand-exchange engineered domain body) (SEEDbody) and bispecific bodies (DuoBody). In some aspects, the bispecific fusion protein, antibody or its antigen-binding fragment or multi-specific protein provided herein comprises a knob mutation and a mortar mutation. In some aspects, the knob mutation includes an amino acid substitution T366W according to EU numbering. In some aspects, the mortar mutation includes an amino acid substitution T366S, L368A and Y407V according to EU numbering.

[0210] In some aspects provided herein, the fusion protein, antibody or antigen-binding fragment thereof or multispecific protein provided herein comprises a mutation that promotes heterodimerization of the Fc region. In some aspects, the bispecific dimerization Fc region provided herein is formed by an Fc region containing amino acid mutations, substitutions, additions or deletions to promote heterodimerization, wherein different polypeptides containing different Fc regions can dimerize to produce a heterodimer configuration. In some aspects, the bispecific antibody of the present disclosure comprises a first Fc sequence containing a first CH3 region, and a second Fc sequence containing a second CH3 region, wherein the sequences of the first and second CH3 regions are different, and the heterodimer interaction between the first and second CH3 regions is made stronger than each homodimer interaction of the first and second CH3 regions.

[0211] Methods for promoting heterodimerization of the Fc region include making amino acid deletions, additions or substitutions to the amino acid sequence of the Fc region, such as by including a set of "knobs and holes" deletions, additions or substitutions, or including amino acid deletions, additions or substitutions to affect the electrostatic steering of the Fc, thereby favoring attractive interactions between different polypeptide chains. Methods for promoting heterodimerization of complementary Fc polypeptides have been previously described, for example, in the following literature: Ridgway et al., 1996, Protein Eng, 9:617-621; Merchant et al., 1998, Nature Biotechnol, 16:677-681; Moore et al., 2011, MAbs, 3:546-557; Von Kreudenstein et al., 2013, 5:646-654; Gunasekaran et al., 2010, J Biol Chem, 285:19637-19464; Leaver-Fay et al., 2016, Structure, 24:641-651; Ha et al., 2016, Frontiers in Immunology, 7:1; Davis et al., 2010, Protein Eng Des Sel, 23: 195-202; WO1996 / 027011; WO1998 / 050431; WO2006 / 028936; WO 2009 / 089004; WO2011 / 143545; WO2014 / 067011; WO2012 / 058768; WO2018 / 027025; US2014 / 0363426; US2015 / 0307628; US2018 / 0016354; US2015 / 0239991; US2017 / 0058054; USPN5731168; USPN7183076; USPN9701759; USPN9605084; USPN9650446; USPN8216805; USPN8765412; and USPN8258268.

[0212] In some aspects, the complementary Fc polypeptides of the Fc heterodimers include mutations to change the charge polarity on the Fc dimer interface, such that co-expression of electrostatically matched Fc regions supports favorable attractive interactions, thereby promoting the formation of desired Fc heterodimers; whereas unfavorable repulsive charge interactions inhibit the formation of unwanted Fc homodimers (Guneskaran et al., 2010, J Biol Chem, 285: 19637-19646). When co-expressed in cells, association between polypeptide chains is possible, but due to charge repulsion, the chains will not substantially self-associate.

[0213] In addition, the complementary Fc polypeptides of the Fc heterodimers include a "knob-and-hole" configuration to promote heterodimerization of the two Fc polypeptides. The "knob-and-hole" technology is described in, for example, U.S. Patent Nos. 5,731,168; 7,695,936; 8,216,805; 8,765,412; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method includes introducing a protrusion ("knob") at the interface of a first polypeptide, and introducing a corresponding cavity ("hole") at the interface of a second polypeptide, so that the protrusion can be located in the cavity, thereby promoting the formation of heterodimers and hindering the formation of homodimers. The protrusion is constructed by replacing the small amino acid side chains on the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine), a compensatory cavity with the same or similar size as the protrusion is generated in the interface of the second polypeptide. Protrusions and cavities can be prepared by changing the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis, or by peptide synthesis. In some aspects, the knob is modified to include the amino acid substitution T366W in one of the two subunits of the Fc domain, while the hole is modified to include the amino acid substitution T366S, L368A and Y407V in the other subunit of the two subunits of the Fc domain. In some aspects, the subunit of the Fc domain modified by the knob further includes the amino acid substitution S354C, and the subunit of the Fc domain modified by the hole further includes the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of a disulfide bond between the two subunits of the Fc domain, thus further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). Thus, in such a configuration, the first Fc polypeptide comprises amino acid modifications to form a "knob" and the second Fc polypeptide comprises amino acid modifications to form a "hole", thereby forming an Fc heterodimer comprising complementary Fc polypeptides.

[0214] The following table lists exemplary paired amino acid modifications (EU numbering) of complementary Fc polypeptides in the Fc heterodimer configuration.

[0215] Table 1: Exemplary paired Fc modifications of heterodimeric Fc domains

[0216] First Fc polypeptide Second Fc polypeptide T366Y Y407T T366W T366S / L368W / Y407V T366W T366S / L368A / Y407V T366W / S354C T366S / L368A / Y407V / Y349C T350V / L351Y / F405A / Y407V T350V / T366L / K392L / T394W K360D / D399M / Y407A E345R / Q347R / T366V / K409V K409D / K392D D399K / E356K K360E / K409W Q347R / D399V / F405T L360E / K409W / Y349C Q347R / D399V / F405T / S354C K370E / K409W E357N / D399V / F405T

[0217] Some agents provided herein include antigen-binding fragments of antibodies. Antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv and scFv fragments and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, WO 93 / 16185; and U.S. Patent Nos. 5571894 and 5587458. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5869046.

[0218] Diabodies are antibody fragments that may be bivalent and / or bispecific and have two antigen binding sites. See, e.g., EP404097; WO 1993 / 01161; Hudson et al. Nat. Med. 9:129-134 (2003). Mini trifunctional antibodies and mini tetrafunctional antibodies are also described in Hudson et al. Nat. Med. 9:129-134 (2003). Single domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable domains or all or a portion of the light chain variable domains of an antibody. In some aspects, single domain antibodies are human single domain antibodies (see, e.g., U.S. Pat. No. 6,248,516).

[0219] Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein.

[0220] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof or multispecific proteins provided herein can be chimeric. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4816567. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit or non-human primate such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class switching" antibody, in which the class or subclass has been changed from the class or subclass of the parent antibody.

[0221] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof or multispecific proteins provided herein can be humanized. Generally, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibodies. In some aspects, humanized antibodies are substantially non-immunogenic in people. In some aspects, humanized antibodies have substantially the same affinity for targets as antibodies from another species from which the humanized antibodies are derived. See, e.g., U.S. Patent Nos. 5530101, 5693761; 5693762; and 5585089. In some aspects, amino acids that can be modified without weakening the natural affinity of the antigen-binding domain while reducing its immunogenicity are identified for antibody variable domains. See, e.g., U.S. Patent Nos. 5766886 and 5869619. Generally, humanized antibodies comprise one or more variable domains in which CDRs (or portions thereof) are derived from non-human antibodies, and framework regions (FRs) (or portions thereof) are derived from human antibody sequences. Humanized antibodies may comprise at least a portion of a human constant region. In some aspects, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (eg, the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0222] Humanized antibodies and methods for making them are reviewed in, e.g., Almagro et al. Front. Biosci. 13: 1619-1633 (2008), and further described in, e.g., 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 "fittest match" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies having a particular subgroup of light chain variable regions or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al., J. Immunol. 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson et al. Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al. J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al. J. Biol. Chem. 271: 22611-22618 (1996)).

[0223] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof or multispecific proteins provided herein can be human. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk et al. Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg Curr. Opin. Immunol. 20: 450-459 (2008).

[0224] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce a complete human antibody or a complete antibody with a human variable region in response to an antigenic challenge. Mouse strains that are defective in mouse antibody production can be engineered with large fragments of human Ig loci, with the expectation that such mice will produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can retain a wide range of variable gene diversity and appropriate regulation of antibody production and expression. By utilizing mouse mechanisms for antibody diversification and selection and lack of immune tolerance to human proteins, the human antibody library reproduced in these mouse strains can produce high-affinity fully human antibodies for any target antigen including human antigens. Using hybridoma technology, antigen-specific human MAbs with desired specificity can be produced and selected. Some exemplary methods are described in U.S. Patent No. 5545807, EP 546073 and EP 546073. See also, for example, description of XENOMOUSE TM US Patent Nos. 6075181 and 6150584 for the technology; description US Patent No. 5770429 for technology; description KM US Patent No. 7041870 and description of the technology Human variable regions from intact antibodies produced by such animals can be further modified, for example by combining with different human constant regions.

[0225] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol. 133: 3001 (1984) and Boerner et al. J. Immunol. 147: 86 (1991)). Human antibodies produced by human B cell hybridoma technology are also described in Li et al. Proc. Natl. Acad. Sci. USA, 1 03: 3557-3562 (2006). Additional methods include those described in, for example, U.S. Patent No. 7189826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines). Human hybridoma technology (trioma technology) is also described in Vollmers et al. Histology and Histopathology 20 (3): 927-937 (2005) and Vollmers et al. Methods and Findings in Experimental and Clinical Pharmacology 27 (3): 185-91 (2005). Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. The technology for selecting human antibodies from antibody libraries is described below.

[0226] In some aspects provided herein, antibodies are human antibodies isolated by in vitro methods and / or screening combinatorial libraries for antibodies having one or more desired activities. Suitable examples include, but are not limited to, phage display (CAT, Morphosys, Dyax, Biosite / Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosome display (CAT), yeast display (Adimab), etc. In certain phage display methods, pools of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phages, as described in Winter et al. Ann. Rev. Immunol. 12: 433-455 (1994). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having 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. J. Immunol. Methods 284(-2):1 19-132 (2004). Phages typically display antibody fragments in the form of single-chain Fv (scFv) fragments or in the form of Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the initial library can be cloned (e.g., cloned from a human) to provide antibodies from a single source for a wide range of non-self antigens and self antigens without any immunization, as described by Griffiths et al. EMBO J. 12: 725-734 (1993). Finally, the initial library can also be prepared synthetically by cloning unrearranged V gene segments from stem cells, and using PCR primers containing random sequences to encode hypervariable CDR3 regions and achieve in vitro rearrangement, as described by Hoogenboom et al. J. Mol. Biol., 227: 381-388, 1992. Patents describing human antibody phage libraries are published, for example: U.S. Patent No. 5750373 and U.S. Patent Publication No. 2007 / 0292936 and 2009 / 0002360. Antibodies separated from human antibody libraries are considered to be human antibodies or human antibody fragments herein.

[0227] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein can comprise an antigen binding domain that binds to a CNS antigen or a brain antigen. In some aspects, the CNS antigen or brain antigen can be beta-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor for myeloid cells 2 (TREM2), CD33, or sialic acid-binding Ig-like lectin 3 (Siglec3), transmembrane 4-domain A4A (MS4A4A), or transmembrane 4-domain A4A. 6A (MS4A6A) or transmembrane protein 106B (TMEM106b). In some aspects, the CNS antigen or brain antigen can be β-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin protein, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), Parkin, presenilin 1, presenilin 2, β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), ubiquitin protein ligase E3A (UBE3A), γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor 2 on myeloid cells (TREM2), CD33, sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), sialic acid-binding Ig-like lectin 11 (Siglec11), glycoprotein non-metastatic melanoma protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4 domain A4A (MS4A4A), transmembrane 4 domain A 6A (MS4A6A), MSA4A4E, transmembrane protein 106B (TMEM106b), CR1, ABCA1, ABCA7, HLA-DR1, HLA-DR5, IL1RAP, TREML2, IL-34, SORL1 and ADAM1.

[0228] In some aspects, the CNS or brain antigen is located on a cancer cell within the central nervous system. In some aspects, the CNS or brain antigen is a cell surface target on a blood cancer cell selected from B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b. In some aspects, the CNS or brain antigen is a tumor cell target selected from the group consisting of siglec-3 or CD33, siglec-5, siglec-7, siglec-9, siglec 14, PILRA, IL18-BP, MerTK, ACKR1, ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (Cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), folate receptor alpha, GAL-3BP (galectin binding protein), protein), GD2, GD3, GloboH (globohexasylceramide), gp100, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine-rich repeat-containing LRRC15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon cancer kinase), RON, ROR1, TF (tissue factor), and TROP2.

[0229] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof or multispecific proteins provided herein may include antigen-binding domains that bind to CNS antigens or brain antigens. As provided herein, the multispecific proteins provided herein may include antigen-binding domains that bind to CNS antigens or brain antigens. The antigen-binding domains that bind to CNS antigens or brain antigens may include VH and VL. Exemplary CNS antigen binding VH and VL sequences are provided below. Additional VH and VL and antigen-binding domain sequences are found in US2017 / 0224702, US2018 / 0002433, US2021 / 0236634 and US2021 / 0238265, each of which is incorporated herein by reference in its entirety.

[0230] Table 2: Exemplary CNS Antigens

[0231]

[0232] As provided herein, the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins provided herein are capable of crossing the BBB because the anti-CD98 antigen-binding domain in the fusion proteins, antibodies or antigen-binding fragments thereof, or multispecific proteins are capable of crossing the BBB.

[0233] In some aspects, the internalization degree of the fusion protein, antibody or antigen-binding fragment thereof or multispecific protein provided herein in the blood-brain barrier epithelial cell is more than 10 times higher than the internalization degree of the isotype control. The blood-brain barrier endothelial cell can be, for example, a HCMEC / D3 cell.

[0234] In some aspects, relative to the cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control, the fusion proteins, antibodies, or antigen-binding fragments thereof, or multispecific proteins provided herein do not reduce the cell surface expression of CD98hc on HCMEC / D3 cells by more than 20%. Cell surface expression can be measured, for example, using Western blot or FACS.

[0235] In some aspects, relative to the cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control, the fusion proteins, antibodies, or antigen-binding fragments thereof, or multispecific proteins provided herein do not increase the cell surface expression of CD98hc on HCMEC / D3 cells by more than 50%. Cell surface expression can be measured, for example, using Western blot or FACS.

[0236] In some aspects, the fusion proteins, antibodies, or antigen-binding fragments thereof, or multispecific proteins provided herein do not reduce the cell surface expression of CD98hc on HCMEC / D3 cells by more than 20% relative to the cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control, and do not increase the cell surface expression of CD98hc on HCMEC / D3 cells by more than 50% relative to the cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, for example, using Western blotting or FACS.

[0237] In some aspects, the fusion proteins, antibodies, or antigen-binding fragments thereof, or multispecific proteins provided herein accumulate at least 1.5 times more in the brain of vascular-depleted mice than the isotype control. In some aspects, the fusion proteins, antibodies, or antigen-binding fragments thereof, or multispecific proteins provided herein accumulate at least 2 times more in the brain of vascular-depleted mice than the isotype control.

[0238] In some aspects, 24 hours after administration to a mouse, the brain:serum concentration ratio of a fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein is increased at least 5-fold compared to an isotype control.

[0239] In some aspects, the fusion proteins, antibodies, or antigen-binding fragments thereof, or multispecific proteins provided herein have an equilibrium dissociation constant (K) of about 3 nM to about 225 nM. D ) binds to human CD98hc. In some aspects, the fusion protein, antibody, or antigen-binding fragment thereof, or multispecific protein provided herein has a K of about 3 nM to about 225 nM. D Binds to cynomolgus monkey CD98hc. In some aspects, the fusion proteins, antibodies, or antigen-binding fragments thereof, or multispecific proteins provided herein bind to cynomolgus monkey CD98hc with an equilibrium dissociation constant (K) of about 3 nM to about 225 nM. D ) binds to human CD98hc and has a K of about 3 nM to about 225 nM D Binds to cynomolgus monkey CD98hc.

[0240] Polynucleotides and methods for preparing anti-CD98hc antigen binding domains and agents containing the same

[0241] In some aspects, provided herein are polynucleotides comprising nucleotide sequences encoding an antigen-binding domain, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein described herein that specifically binds to human CD98hc, or a domain thereof described herein, and vectors, such as vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells).

[0242] In some aspects, the polynucleotides provided herein include nucleic acid molecules encoding the heavy chain of the antigen binding domain specifically bound to human CD98hc provided herein. In some aspects, the polynucleotides provided herein include nucleic acid molecules encoding the light chain of the antigen binding domain specifically bound to human CD98hc provided herein. In some aspects, the polynucleotides provided herein include nucleic acid molecules encoding the heavy chain of the antigen binding domain specifically bound to human CD98hc provided herein and nucleic acid molecules encoding the light chain of the antigen binding domain specifically bound to human CD98hc provided herein.

[0243] In some aspects, provided herein are combinations or compositions of polynucleotides. In some aspects, 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, for example, wherein the first polynucleotide encodes a first heavy chain, the second polynucleotide encodes a second heavy chain and an antigen binding domain specifically bound to human CD98hc provided herein, and the third polynucleotide encodes a light chain. In some aspects, the antigen binding domain bound to human CD98hc is a scFv. In some aspects, the first heavy chain comprises a knob mutation and the second heavy chain comprises a mortar mutation. In some aspects, the first heavy chain comprises a mortar mutation and the second heavy chain comprises a knob mutation.

[0244] In some aspects, 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 / or second antigen binding domains that bind to human CD98hc are scFv. In some aspects, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation. In some aspects, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0245] In some aspects, the combination or composition comprises 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 provided herein that specifically binds to human CD98hc, and wherein the second polynucleotide encodes a light chain.

[0246] Also provided herein are polynucleotides comprising a nucleotide sequence encoding an antigen binding domain, fusion protein, antibody, antigen binding fragment thereof or multispecific protein specifically binding to human CD98hc as described herein, or a domain thereof as described herein, the polynucleotides, for example, are optimized by codon / RNA optimization, substitution with a heterologous signal sequence and / or elimination of mRNA instability elements. Methods for generating optimized nucleic acids for recombinant expression by introducing codon changes (e.g., codon changes encoding the same amino acid due to the degeneracy of the genetic code) and / or eliminating inhibitory regions in mRNA can therefore be performed by adjusting the optimization methods described in, for example, U.S. Pat. Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132 and 6,794,498.

[0247] A polynucleotide comprising a nucleotide sequence encoding an antigen binding domain, fusion protein, antibody, antigen binding fragment thereof, or multi-specific protein or a domain thereof described herein that specifically binds to human CD98hc as described herein can be generated from a nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from a hybridoma cell that produces the target antibody. Such PCR amplification methods can be used to obtain nucleic acids that contain, for example, sequences encoding an antigen binding domain, a light chain and / or a heavy chain of an antibody or an antigen binding fragment thereof. The amplified nucleic acid can be cloned into a vector for expression in a host cell and further cloned, for example, to produce an antigen binding domain, fusion protein, antibody, antigen binding fragment thereof, or multi-specific protein or a domain thereof described herein that specifically binds to human CD98hc as described herein.

[0248] The polynucleotides provided herein can be, for example, in RNA form or DNA form. DNA includes cDNA, genomic DNA and synthetic DNA, and the DNA can be double-stranded or single-stranded. If the DNA is single-stranded, it can be a coding strand or a non-coding (antisense) strand. In some aspects, the polynucleotides are cDNA or lack DNA of one or more endogenous introns. In some aspects, the polynucleotides are non-naturally occurring polynucleotides. In some aspects, the polynucleotides are recombinantly produced. In some aspects, the polynucleotides are separated. In some aspects, the polynucleotides are substantially pure.

[0249] 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, at least one nucleoside modification is selected from the group consisting of: pyridine-4-ketone ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propargyl-uridine, 1-propargyl-pseudouridine, 5-taurine methyl uridine, 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine, 1-taurine methyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4- Thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-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, pyrrolo-cytidine, pyrrolo 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylaminoformyladenosine, N6-threonylaminoformyladenosine, 2-methylthio-N6-threonylaminoformyladenosine, N6,N6-dimethyladenosine, 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-guanosine, 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.

[0250] In some aspects, provided herein are polynucleotides encoding fusion proteins, the fusion proteins comprising antigen binding proteins and heterologous polypeptides provided herein. In some aspects, the heterologous polypeptide comprises an antigen binding domain that binds to the following: β-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), Parkin, presenilin 1, presenilin 2, β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), γ-secretase, death receptor 6 (DR6) , amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), caspase 6, sorting protein (SORT), myeloid cell triggering 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), glycoprotein non-metastatic melanoma protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4 domain A4A (MS4A4A), transmembrane 4 domain A 6A (MS4A6A) or transmembrane protein 106B (TMEM106b) or a portion thereof. In some aspects, the heterologous polypeptide comprises an antigen binding domain that binds to ubiquitin protein ligase E3A (UBE3A). In some aspects, provided herein are polynucleotides encoding fusion proteins comprising an antigen binding domain provided herein and a heterologous polypeptide.In some aspects, the heterologous polypeptide comprises the following amino acid sequence: beta-secretase 1 (BACE1), Aβ, 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 neurotrophic factor Receptor (p75NTR), caspase 6, sorting protein (SORT), myeloid cell triggering 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), glycoprotein non-metastatic melanoma protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4 domain A4A (MS4A4A), transmembrane 4 domain A6A (MS4A6A) or transmembrane protein 106B (TMEM106b) or part thereof. In some aspects, the multispecific protein comprises an antigen binding domain that binds to ubiquitin protein ligase E3A (UBE3A). In some embodiments, the polynucleotide is mRNA (e.g., synthetic mRNA).

[0251] In some embodiments, disclosed herein are polynucleotides encoding fusion proteins disclosed herein that include heterologous polypeptides. In some aspects, the heterologous polypeptide is an ERT enzyme or an ERT enzyme variant, or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide includes β-glucocerebrosidase (GBA), progranulin (PGRN), prosphingolipid activator protein (PSAP) or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide in the fusion protein provided herein is a growth factor. In some aspects, the heterologous polypeptide in the fusion protein provided herein is a decoy receptor. In some aspects, the heterologous polypeptide in the fusion protein provided herein is progranulin (PGRN), prosphingolipid activator protein (PSAP) or survival motor neuron protein (SMN). In some aspects, the heterologous protein is an enzyme selected from the group consisting of alpha-L iduronidase (IDUA), iduronate-2-sulfatase (IDS), N-acetylgalactosamine-6-sulfatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulfatase (arylsulfatase B; ARSB), acid sphingomyelinase (ASM), beta-glucocerebrosidase (GCase or GBA), galactosylceramide beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta-hexosaminidase B, arylsulfatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme surrogate thereof, such as laronidase, iduronic acid enzyme, ilosulfase alpha or galactosidase, or a variant thereof, or a catalytically active fragment thereof.In some aspects, the heterologous protein is an enzyme selected from the group consisting of: apoJ, nebulin, ubiquitin protein ligase E3A (UBE3A), tripeptidyl peptidase 1 (CLN2 / TPP1), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-α-glucosamine N-acetyltransferase (HGSNAT) and N-acetyl-α-glucosaminidase (NAGLU), α-L iduronidase (IDUA), iduronate-2-sulfate enzyme (IDS), N-acetylgalactosamine-6-sulfatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulfatase (arylsulfatase B; ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramide β-galactosidase, glucosylceramidase, β-hexosaminidase A, β-hexosaminidase B, arylsulfatase A, β-galactosidase, acid ceramidase, α-glucosidase, lysosomal acid lipase, lysosomal protease, their synthetase substitutes, such as laronidase, iduronidase, iluronidase α or galactosidase or variants or 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 β-galactosidase, glucosylceramidase, β-hexosaminidase A, β-hexosaminidase B, arylsulfatase A, β-galactosidase, acid ceramidase, α-glucosidase, lysosomal acid lipase, lysosomal protease, its synthetic enzyme substitute, such as laronidase, idursulfase, elosulfase α or galactosidase or its variant or catalytically active fragment thereof. In some embodiments, the polynucleotide is mRNA (e.g., synthetic mRNA).

[0252] In certain aspects, provided herein are vectors (e.g., expression vectors) comprising polynucleotides encoding nucleotide sequences of antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multi-specific proteins or domains thereof described herein that specifically bind to human CD98hc, which are described herein, and which are used for recombinant expression in host cells (e.g., mammalian cells). Vectors for producing antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multi-specific proteins that specifically bind to human CD98hc, or domains thereof described herein, can be produced, for example, by recombinant DNA technology using techniques well known in the art. These methods include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination. Also provided are replicable vectors comprising nucleotide sequences encoding antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multi-specific proteins that specifically bind to human CD98hc, or domains thereof described herein, which vectors are operably linked to promoters. Such vectors may, for example, include nucleotide sequences encoding constant regions of antigen binding domains, antibodies, or antigen binding fragments thereof (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Pat. No. 5,122,464), and variable domains of antigen binding domains, antibodies, or antigen binding fragments thereof may be cloned into such vectors to express the entire heavy chain, the entire light chain, or the entire heavy and light chains. In some embodiments, the vector is a gene therapy vector (e.g., AAV or lentiviral vector).

[0253] In certain aspects, provided herein is an expression system comprising a polynucleotide comprising a nucleotide sequence encoding an antigen binding domain, a fusion protein, an antibody, an antigen binding fragment thereof, or a multispecific protein or a domain thereof as described herein that specifically binds to human CD98hc. The expression system may be included on a vector. The expression system may also be introduced into a host cell chromosome. In some aspects, the expression system is a cell-free expression system. In some aspects, the expression system comprises a host cell comprising a polynucleotide and / or a vector as provided herein.

[0254] Therefore, cells, such as host cells, are also provided herein, which contain polynucleotides and / or vectors for recombinantly expressing antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multispecific proteins or domains thereof described herein that specifically bind to human CD98hc. In some aspects, in order to express double-chain antigen binding proteins, vectors encoding heavy chains and light chains, respectively, can be co-expressed in host cells to express the entire immunoglobulin. In some aspects, the host cell contains two different vectors, a first vector comprising a polynucleotide encoding the heavy chain of the antigen binding protein described herein and a second vector comprising a polynucleotide encoding the light chain of the antigen binding protein. In some aspects, the first host cell comprises a first vector comprising a polynucleotide encoding a heavy chain, and the second host cell comprises a second vector comprising a polynucleotide encoding a light chain. In some aspects, a host cell population comprising such a first host cell and such a second host cell is provided herein.

[0255] In some aspects, provided herein are methods for producing antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multi-specific proteins or domains thereof described herein that specifically bind to human CD98hc as described herein. In some aspects, provided herein are methods for producing single-chain antigen binding domains, Fc domains, and heterologous proteins or polypeptides that specifically bind to human CD98hc as described herein in host cells. In some aspects, provided herein are methods for producing single-chain antigen binding domains, Fc domains, and second antigen binding domains that specifically bind to human CD98hc as described herein in host cells. The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multi-specific proteins or domains thereof that specifically bind to human CD98hc as described herein.

[0256] 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 described herein that specifically bind to human CD98hc, or the antigen-binding domains of domains thereof described herein (see, e.g., U.S. Pat. No. 5,807,715). Such host-expression systems represent vehicles by which the target coding sequence can be produced and subsequently purified, and also represent cells that, when transformed or transfected with appropriate nucleotide coding sequences, can express in situ the antigen-binding domains, fusion proteins, antibodies, antigen-binding fragments thereof, or multispecific proteins described herein that specifically bind to human CD98hc, or the domains thereof described herein. These include, but are not limited to, microorganisms, such as bacteria (e.g., E. coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Pichia pastoris) transformed with recombinant yeast expression vectors containing antibody coding sequences; Pichia); an insect cell system infected with a recombinant viral expression vector (e.g., baculovirus) containing the antibody coding sequence; a plant cell system (e.g., green algae, such as Chlamydomonas reinhardtii) infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) containing the coding sequence or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing the coding sequence; or a mammalian cell system (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7030, HsS78Bst, HeLa and NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20 and BMT10 cells). In some aspects, the cell used to express the antigen binding domain, fusion protein, antibody, antigen binding fragment or multispecific protein described herein that specifically binds to human CD98hc or the domain thereof described herein is a CHO cell, such as a CHO GS System TM (Lonza) CHO cells. In some aspects, the cells used for the antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multispecific proteins described herein that specifically bind to human CD98hc or domains thereof described herein are human cells, such as human cell lines. In some aspects, the mammalian expression vector is pOptiVEC TMOr 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 or domains thereof described herein that specifically bind to human CD98hc. For example, mammalian cells (such as Chinese hamster ovary (CHO) cells) combined with vectors (such as major intermediate early gene promoter elements from human cytomegalovirus) are effective expression systems for antibodies (Foecking MK and Hofstetter H (1986) Gene 45: 101-105; and Cockett MI et al., (1990) Biotechnology 8: 662-667). In some aspects, antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multispecific proteins or domains thereof described herein that specifically bind to human CD98hc described herein are produced by CHO cells or NS0 cells.

[0257] In addition, a host cell strain may be selected to modulate the expression of the inserted sequence, or to modify and process the gene product in a desired specific manner. 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 having cellular machinery for appropriate 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, BT20 and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells.

[0258] Once the antigen binding domain, fusion protein, antibody, antigen binding fragment or multispecific protein specifically binding to human CD98hc described herein or domain thereof described herein is produced by recombinant expression, it can be purified by any purification method known in the art, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography, especially affinity chromatography for specific antigens after protein A and sizing column chromatography), centrifugation, differential solubility or by any other standard technique for purifying proteins. In addition, the antigen binding domain, fusion protein, antibody, antigen binding fragment or multispecific protein specifically binding to human CD98hc described herein or domain thereof described herein can be fused to a heterologous polypeptide sequence for purification.

[0259] In some aspects, once the antigen binding domain, fusion protein, antibody, antigen binding fragment or multi-specific protein described herein that specifically binds to human CD98hc is isolated or purified. Typically, the isolated antigen binding domain, fusion protein, antibody, antigen binding fragment or multi-specific protein described herein that specifically binds to human CD98hc is substantially free of other proteins. For example, in some aspects, the preparation of the antigen binding domain, fusion protein, antibody, antigen binding fragment or multi-specific protein described herein that specifically binds to human CD98hc is substantially free of cellular material and / or chemical precursors.

[0260] Pharmaceutical composition

[0261] Provided herein are compositions comprising antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multi-specific proteins specifically binding to human CD98hc as described herein. In some aspects, antigen binding domains, fusion proteins, antibodies, antigen binding fragments thereof, or multi-specific proteins specifically binding to human CD98hc with the desired purity are present in a formulation, the formulation including, for example, a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the dose and concentration used. Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may include antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, the suspensions may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives.

[0262] In some aspects, the pharmaceutical composition comprises an antigen binding domain, fusion protein, antibody, antigen binding fragment thereof or multispecific protein described herein that specifically binds to human CD98hc and a pharmaceutically acceptable carrier (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th edition (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd edition, Pharmaceutical Press (2000)). In some aspects, the pharmaceutical composition described herein is used as a medicament. The composition to be used for in vivo administration can be sterile. This is easily achieved by filtering through, for example, a sterile filtration membrane.

[0263] Also provided herein are pharmaceutical compositions comprising polynucleotides encoding antigen binding domains, fusion proteins, antibodies or antigen binding fragments thereof or multi-specific proteins specifically binding to human CD98hc as described herein. In some embodiments, the polynucleotides are RNA. In some embodiments, the polynucleotides are synthetic mRNA. In some embodiments, the pharmaceutical compositions comprising polynucleotides further comprise lipid-based transfection reagents.

[0264] The pharmaceutical compositions described herein can be used to exert a biological effect in vivo or in vitro. For example, the pharmaceutical compositions described herein can be used to cross the blood-brain barrier, for example, in a subject.

[0265] In some aspects, provided herein is a pharmaceutical composition for treating diseases or illnesses such as: neuropathy, neurodegenerative diseases, cancer, eye diseases, epilepsy, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders and CNS inflammation. In some aspects, provided herein is a pharmaceutical composition for treating diseases or illnesses such as: Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer and traumatic brain injury. In some aspects, provided herein is a pharmaceutical composition for treating frontotemporal dementia.

[0266] 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.

[0267] Methods of using anti-CD98hc antigen binding domains and agents comprising the same

[0268] Antigen binding domains that specifically bind to human CD98hc, fusion proteins comprising such antigen binding domains, antibodies, antigen binding fragments thereof, and multispecific proteins as provided herein can be advantageously transported across the blood-brain barrier. Therefore, provided herein is a method of administering 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, an antigen binding fragment thereof, or a multispecific protein, or transporting it across the blood-brain barrier of a subject, 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, an antigen binding fragment thereof, or a multispecific protein.

[0269] Given that antigen binding domains that specifically bind to human CD98hc, fusion proteins comprising such antigen binding domains, antibodies, antigen binding fragments thereof, and multispecific proteins as provided herein can be transported across the blood-brain barrier, they can be used to treat nervous system diseases or disorders. In some aspects, the method of treating a nervous system disease or disorder in a subject comprises administering to the subject an antigen binding protein that specifically binds to human CD98hc, a fusion protein comprising an antigen binding protein that specifically binds to human CD98hc, an antibody, an antigen binding fragment thereof, or a multispecific protein. Nervous system diseases or disorders can be, for example, neuropathy disorders, neurodegenerative diseases, cancer, eye disease disorders, epilepsy, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, or CNS inflammation. The nervous system disease or disorder can be, for example, a neurodegenerative disease (such as Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, Gaucher disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), a tauopathy (such as Alzheimer's disease and supranuclear palsy), a prion disease (such as bovine spongiform encephalopathy, scrapie, Creutz-feldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker syndrome), or a prion disease (such as bovine spongiform encephalopathy, scrapie, Creutz-feldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker syndrome, ... disease, chronic wasting disease and fatal familial insomnia), bulbar palsy, motor neuron disease, heterogeneous degenerative diseases of the nervous system (such as Canavan disease, Huntington disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, Lafora disease, Rett syndrome,In some aspects, the neurological disease or disorder is a dementia. In some aspects, the neurological disease or disorder is a frontotemporal dementia. In some aspects, the neurological disease or disorder is a frontotemporal dementia. In some aspects, the neurological disease or disorder is a frontotemporal dementia. In some aspects, the neurological disease or disorder is a frontotemporal dementia. In some aspects, the neurological disease or disorder is Parkinson's disease. In some aspects, the neurological disease or disorder is frontotemporal epilepsy. In some embodiments, the neurological disease or disorder is autism. In some aspects, the neurological disease or disorder is anencephaly.

[0270] In some aspects, provided herein are methods of treating lysosomal storage diseases using the fusion proteins disclosed herein. In some aspects, the lysosomal storage disease is selected from Gaucher disease, ceroid lipofuscinosis (Patton disease), mucopolysaccharidosis (MPS) type I, MPS type II, and MPS type III.

[0271] Antigen binding domains that specifically bind to human CD98hc, fusion proteins comprising such antigen binding domains, antibodies, antigen binding fragments thereof, and multispecific proteins as provided herein can be used to detect antigens (e.g., CNS antigens or brain antigens). Antigen binding domains that specifically bind to human CD98hc and fusion proteins, antibodies, antigen binding fragments thereof, and multispecific proteins comprising such antigen binding domains for such purposes can be labeled. Exemplary labels include, for example, radioisotopes (e.g., 64CU) and fluorescent labeling. Therefore, a method for detecting an antigen using an antigen binding protein specifically binding to human CD98hc as described herein or a fusion protein, antibody, antigen binding fragment thereof, or multi-specific protein comprising an antigen binding protein specifically binding to human CD98hc is provided. In some aspects, the method for detecting an antigen in the CNS (e.g., brain) of a subject comprises administering an antigen binding protein specifically binding to human CD98hc or a fusion protein, antibody, antigen binding fragment thereof, or multi-specific protein comprising an antigen binding protein specifically binding to human CD98hc to an antigen in the CNS (e.g., brain). Such methods may also include, for example, positron emission tomography (PET) imaging of the subject. In some aspects, disclosed herein is a method for detecting CNS antigens in vitro, comprising contacting an in vitro sample with a fusion protein, antibody, or antigen binding fragment thereof, or multi-specific protein disclosed herein, and positioning an imaging agent in the sample.

[0272] Antigen binding domains that specifically bind to human CD98hc as provided herein and fusion proteins, antibodies, antigen binding fragments thereof, and multispecific proteins comprising such antigen binding domains can be used for prognosis, diagnosis, monitoring, and / or screening applications, including in vivo applications that are well known and standard to those skilled in the art and based on the present description. 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 as diagnostic agents. In some aspects, 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.

[0273] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0274] By reference to the following examples, the present disclosure will be more fully understood. However, they should not be construed as limiting the scope of the present disclosure. All citations throughout the present disclosure are hereby expressly incorporated herein by reference.

[0275] Example

[0276] Example 1: Generation of Avi-His tagged variants of CD98 heavy chain (CD98hc)

[0277] In order to produce the Avi-His tag variants of the extracellular domain (ECD) of people, cynomolgus monkey (cyno) and mouse CD98 heavy chain (CD98hc), the following method was used.By cloning the synthetic gene based on cDNA into a mammalian expression vector, then transiently transfected and expressed in Expi293 cells, mammalian expression of people, cynomolgus monkey and mouse BBB receptor antigen variants (SEQ ID NO:1-3) was carried out.Construct includes a heterologous signal peptide and a C-terminal Avi-His tag, to allow purification and biotinylation.In short, according to the manufacturer's instructions, the plasmid encoding antigen was transfected using Expifectamine 293 transfection kit (ThermoFisherA14524).Five days after transfection, culture supernatant was harvested, clarified by centrifugation, and purified in a drop column format using HisPur Ni-NTA resin (Thermo Scientific 88223). Use 0.2 μm filter unit to filter 200mL culture supernatant, and the resin slurry in 3mL PBS is added in the supernatant filtered.Sample is incubated overnight at 4 ℃ of vibrations, and on the second day, beads are loaded in 20mL drop column, washed with 10mL His-Select washing buffer (Millipore Sigma H5288), and eluted with 5mL His-Pur elution buffer (Millipore SigmaH5413).Amicon Ultra-15 centrifugal filter unit (Millipore UFC9010) is used to exchange the eluent buffer into PBS.By using Nanodrop 8000 (ThermoFisher) to measure the absorbance at 280nm, determine the quantification of antibody concentration.Determine the purity of antigen by SDS-PAGE.Use size exclusion chromatography (SEC) to analyze the aggregation of antigen.According to the explanation of manufacturer, use BirA biotin protein ligase kit (AVIDITY) to carry out biotinylation to some antigens.

[0278] Table 3. Avi-His tag variants of CD98hc ECD

[0279]

[0280]

[0281] Example 2: Production of CHO cell lines overexpressing CD98hc

[0282] As described herein, several cell lines were generated for screening binding of anti-CD98hc antibodies. Briefly, pLenti-EF1a constructs expressing full-length human CD98hc and mouse CD98hc (SEQ ID NOs: 4-5, respectively) were used to generate CHO cells stably expressing human CD98hc and mouse CD98hc. Lentivirus constructs (Genecopoeia) were used to express human CD98hc with puromycin selection and mouse CD98hc with GFP as a reporter gene. Lentivirus was generated using ViraSafe TM The lentiviral packaging system (CellBiolabs VPK-206) was generated from the transfection of 293T cells. Subsequently, Chinese hamster ovary (CHO) cells were transduced using the supernatant containing the lentivirus. Two days after transduction, puromycin was added to the culture medium as a selection pressure for human CD98hc. Mouse CD98hc was FACS sorted on a FACS Aria system (BD Biosciences) to obtain GFP-positive cells. The cell surface expression of the obtained CHO cells stably expressing human CD98hc and mouse CD98hc was analyzed by flow cytometry.

[0283] Table 4. Full-length CD98hc sequence

[0284]

[0285]

[0286] Example 3: Generation of CD98hc humanized mice

[0287] A humanized mouse line expressing the extracellular domain of human CD98hc was generated (Taconic Biosciences GmbH (Germany)). The mouse ECD was replaced with the human version using CRISPR while retaining the mouse intracellular and transmembrane portions under the control of the mouse promoter. Brain sections from these mice were evaluated by IHC and / or Western blotting to confirm the expression and localization of the human ECD in vivo (data not shown).

[0288] Example 4: Production of anti-CD98hc hybridoma antibodies

[0289] To obtain antibodies against CD98hc, the following steps were used to generate hybridomas. BALB / c mice or Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) were immunized twice a week by subcutaneous or intraperitoneal injection of purified human, cynomolgus monkey and / or mouse CD98hc extracellular domain polypeptides (with or without adjuvant) obtained as described in Example 1 above. Lymph nodes for the generation of hybridoma cell lines were harvested from mice three days after a total of 6-8 injections and the last booster injection.

[0290] Serum from animals was analyzed for reactivity to CD98hc by FACS against CHO cells overexpressing human or mouse CD98hc and by ELISA against human, cynomolgus monkey and mouse CD98hc Avi-His polypeptides (described below). Lymphocytes were isolated from animals whose sera showed strong binding to CHO cells overexpressing human or mouse CD98hc and fused with a myeloma fusion partner via electrofusion (Hybrimune, BTX, Holliston, MA) to generate mouse myeloma cells and incubated overnight at 37°C, 5% CO2 in Clonacell-HY medium C (Stemcell Technologies, Vancouver, BC, Canada, catalog number 03803).

[0291] The next day, the fused cells were centrifuged and resuspended in Clonacell-HY medium E (Stemcell Technologies, catalog number 03805) with HAT (Sigma Aldrich, catalog number H0262). The cells were inoculated into T225 flasks and grown for 6 days at 37°C, 5% CO2, and then the hybridoma library was cryopreserved. After thawing the mouse hybridoma library, the cells were resuspended in 10 ml of ClonaCell-HY medium C (Jackson ImmunoResearch, West Grove, PA) with anti-mouse IgG Fc-FITC, and then gently mixed with 90 ml of methylcellulose-based ClonaCell-HY medium D (Stemcell Technologies, catalog number 03804) containing HAT components. The cells were plated in Nunc OmniTrays (Thermo Fisher Scientific, Rochester, NY) and grown for 11 days at 37°C, 5% CO2. The Clonepix 2 (Molecular Devices, Sunnyvale, CA) system was used to select IgG positive colonies and transfer them to 96-well plates with high glucose DMEM medium containing 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 number 03805). This approach has been performed in two hybridoma campaigns (No. 3 and No. 4).

[0292] Alternatively, fused hybridomas were recovered overnight, resuspended in ClonaCell-HY Medium C containing anti-mouse IgG Fc-FITC, and then mixed into Clonacell-HY Medium D. Cells were plated, grown, and selected using the Clonepix 2 system described above. This approach has been performed in two additional hybridoma campaigns (No. 1 and No. 2).

[0293] A total of four hybridoma campaigns were performed and a total of 898 IgG-positive hybridoma clones were isolated.After six days in culture, tissue culture supernatants of the hybridomas were screened by FACS on CHO cells overexpressing human or mouse CD98hc (as described below).

[0294] Example 5: Screening of anti-CD98hc antibody hybridoma supernatants by FACS

[0295] IgG-positive hybridoma supernatants (898 in total) were initially screened by FACS to determine their ability to differentially bind to CHO cells overexpressing human or mouse CD98hc cells compared to CHO parental cells. Overexpressing cells were harvested, washed, and labeled with different concentrations and combinations of CellTrace cell proliferation dyes CFSE and Violet (ThermoFisher, catalog numbers C34554 and 34557, respectively) to create cell populations with unique barcodes. Barcoded cells (5x10 per cell population) were then plated and labeled with 10% IgG. 4 ) were dispensed into 96-well U-bottom plates and incubated on ice for 30 minutes with 50 μl of hybridoma cell culture supernatant or 1 μg / ml of commercially purified mouse anti-human CD98hc monoclonal antibody (Sigma Aldrich, catalog number SAB 4700503) and rat anti-mouse CD98hc antibody (Bio-Rad Laboratories, Hercules, CA, catalog number MCA2684). After this primary incubation, the supernatant was removed via centrifugation, the cells were washed twice with 175 μl of ice-cold FACS buffer (PBS + 1% FBS + 2mM EDTA), and then the cells were further incubated on ice with anti-mouse IgG Fc-allophycocyanin (APC) or anti-rat IgG Fc-APC (Jackson Labs, catalog numbers 115-136-071 and 112-136-071, respectively), 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 propidium iodide (BD Biosciences, catalog number 556463). The binding intensity of the cells was analyzed using the FACS Canto system (BD Biosciences), and a sorting gate was drawn to exclude dead (i.e., propidium iodide-positive) cells. For each barcoded cell population, the ratio of the APC mean fluorescence intensity (MFI) of each tested anti-CD98hc hybridoma supernatant divided by the secondary antibody MFI was calculated and reported in Table 5.

[0296] From four hybridoma campaigns, a total of 121 hybridoma clones showed a 2-fold increase in the MFI ratio of the CHO cells that stably overexpressed human CD98hc. 103 of these clones combined with the HCMEC / D3 endothelial cells of endogenous expression human CD98hc had a 5-fold increase in the MFI ratio. No hybridoma clones were shown to be combined with the CHO cells that stably overexpressed mouse CD98hc. Therefore, 121 of the 898 clones were determined to be "FACS positive", and 104 of these FACS positive clones had unique CD98hc sequences. Table 3 shows the data of the FACS positive clone subset.

[0297] Table 5. FACS MFI values ​​of unique anti-CD98hc antibodies identified from hybridoma campaigns

[0298]

[0299] Example 6: Screening of anti-CD98hc antibody hybridoma supernatants by recombinant CD98hc protein binding assay

[0300] Hybridoma culture supernatants from 121 hybridomas (obtained from Example 5) were screened to determine their binding ability to Avi-His-tagged human, cynomolgus monkey and mouse CD98hc (prepared as described in Example 1), and compared with the binding to Avi-His-tagged human TfR (control protein). Briefly, 96-well polystyrene plates were coated with 5 μg / ml streptavidin (Thermo Fisher, catalog number PI21125) in coating buffer (0.05M carbonate buffer, pH 9.6, Sigma, catalog number C3041) overnight at 4°C. The coated plates were then blocked with ELISA diluent (PBS + 0.5% BSA + 0.05% Tween 20) for one hour. The blocking buffer was removed, and human, cynomolgus monkey or mouse Avi-His-tagged CD98hc and Avi-His-tagged human TfR polypeptides were added to the ELISA diluent at 1 μg / ml and captured at room temperature for 1 hour. After washing three times with 300 μl PBST (PBS + 0.05% Tween 20, Thermo28352), hybridoma cell culture supernatant or 1 μg / ml commercially purified mouse anti-human CD98hc monoclonal antibody (Sigma Aldrich, catalog number SAB4700503) and rat anti-mouse CD98hc antibody (Bio-Rad Laboratories, Hercules, CA, catalog number MCA2684) (50 μl / well) were added to each well. After incubation at room temperature for 30 minutes, the plate was washed three times with 300 μl of PBST. The anti-mouse IgG Fc-HRP or anti-rat IgG Fc-HRP (Jackson Immunoresearch, catalog numbers 115-035-071 and 112-036-071, respectively) secondary antibody was diluted 1: 5000 in ELISA diluent, added to each well at 50 μl / well, and incubated at room temperature for 30 minutes with shaking. After the last set of washes (3x300 μl in PBST), 50 μl / well BioFx TMB substrate (Surmodics, Eden Praire, MN, catalog number TMBW-1000-01) was added to the wells. The reaction was then quenched with 50 μl / well of 2N sulfuric acid after 5-10 minutes. The absorbance of the plate at 450 nm was read on SpectraMax M5 (Molecular Devices, Sunnyvale, CA) using SoftMax Pro software. The ELISA data for the selected antibodies are shown in Table 4 below.

[0301] From the hybridoma supernatant screening, 120 of 121 anti-CD98hc hybridoma clones had OD values ​​of 1.1 for human and cynomolgus monkey CD98hc. 450The value was greater than 0.45. One hybridoma clone, CD98HC.02.020, bound to mouse CD98hc.

[0302] Table 6. ELISA OD of unique anti-CD98hc antibodies identified from hybridoma campaigns 3 and 4 450 value.

[0303]

[0304]

[0305] Example 7: In vitro internalization of anti-CD98hc antibodies into blood-brain barrier endothelial cell lines

[0306] The supernatant from 121 anti-CD98hc hybridoma clones was purified on Hamilton STAR platform (Hamilton Company, Reno, NV) using ProPlus Phytip columns (Biotage, Uppsala, Sweden, catalog number PTH 91-20-07). In brief, the antibody from the supernatant was captured by the protein A coupled to the resin-packed tip, washed twice with PBS, eluted with Pierce IgG elution buffer (ThermoFisher, catalog number 21004), and neutralized to final pH of 6.0 with 1M Tris-HCl pH8. The absorbance at 280nm was measured using Nanodrop 8000 (ThermoFisher) to determine the concentration of purified antibodies. The ability of hybridoma purified antibodies to internalize into endothelial cells was then tested.

[0307] Internalization into the endothelial cells of the blood-brain barrier is the first stage of transcytosis that crosses the BBB and enters the brain. In order to identify anti-CD98hc antibodies with internalization ability, HCMEC / D3 cells were inoculated in black-walled transparent bottom 96-well plates (No. 3904, Corning) at 2.5*10^4 cells / well. The next day, cells were treated with 6 μg / ml anti-CD98hc antibodies, which were pre-conjugated with an equal concentration of pHrodo-Red labeling reagent (Z25612, Invitrogen) in 100 μl culture medium (EBM2, Lonza). Human IgG isotypes and anti-CD98hc reference antibodies with known internalization ability were included in the assay as negative and positive controls, respectively.

[0308] The plate was then placed in an IncuCyte machine (Live Cell Analysis System) and images were captured every 2 hours over the course of 24 hours. Images were then processed and analyzed using the IncuCyte software. Internalization data (pHrodoRed positive area μm) at the 24 hour time point 2 / images), shown in Table 5 as relative fold change relative to isotype hIgG1.

[0309] Table 7. Internalization fold changes of purified unique anti-CD98hc antibodies identified from hybridoma campaigns relative to isotype hIgG1

[0310]

[0311]

[0312] Example 8: Molecular cloning of anti-CD98hc antibodies

[0313] Anti-CD98hc antibodies from the above 121 hybridomas were cloned as follows: Anti-CD98hc antibodies selected based on FACS positive data (as described in Example 5 above). 1-2x10 5 Hybridoma cells were washed with PBS and resuspended in 200 μl RNAlater (Invitrogen, catalog number AM7021). Samples were stored at -80 ° C and sent to Abterra Biosciences (San Diego, CA) for sequencing. In short, RNA was extracted and cDNA synthesis was performed. The variable regions of IgG / IgM, IgK and IgL were amplified using proprietary primers and 5'RACE strategy. Hybridoma variable region amplicons were sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA). The reads of hybridomas were processed by Abterra's Reptor analysis process. Table 8 below provides exemplary unique amino acid sequences of variable heavy chains and variable light chains of anti-CD98hc antibodies. Tables 9 and 10 provide CDR sequences (Kabat) for each unique VH and VL sequence in Table 8.

[0314] Table 8. Unique VH / VL sequences derived from hybridoma campaigns

[0315]

[0316]

[0317]

[0318]

[0319] Table 9. CDR heavy chain sequences derived from hybridoma campaigns

[0320]

[0321]

[0322]

[0323] Table 10. CDR light chain sequences derived from hybridoma campaigns

[0324]

[0325]

[0326] Example 9: Reformatting hybridoma antibodies into 2+1 bispecific antibodies

[0327] Antibodies were selected to be formatted into a 2+1 bispecific format based on various criteria: 1) the antibodies covered a wide range of affinities based on ELISA and FACS binding assays, 2) the antibodies covered a wide range of internalization activities, 3) no prominent high-risk modification sites were identified in the CDRs, and 4) the antibodies were phylogenetically distinct from each other within the obtained hybridoma sequences. Cross-reactivity of antibodies between human and cynomolgus CD98hc was also used as a criterion for selection. From 121 anti-CD98hc antibodies, a panel of 24 anti-CD98hc hybridoma antibodies were selected based on these criteria to be reformatted into scFvs in a VH-linker-VL format.

[0328] An IgG isotype antibody without target specificity ("inert isotype control antibody"), with a knob-hole mutation in the heavy chain constant region, was formatted as a 2+1 bispecific antibody. The anti-CD98hc scFv (VH and VL) was attached to the IgG isotype antibody via a linker with a "hole" mutation (the "hole side" of the IgG isotype antibody) located at the C-terminus of the constant region. The IgG isotype antibody attached to the scFv is called a 2+1 bispecific antibody, as Figure 1A As shown. The 20 amino acid linker linking the VH and VL domains within the scFv is GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 182) (Bird et al., Science 1988 October 21; 242(4877): 423-6), and the linker linking the C-terminus of the Fc'hole' domain to the scFv is (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).

[0329] Using the framework in SEQ ID NO: 184, the scFv (VH and VL) sequences were formatted into IgG heavy chain expression constructs with "hole" mutations ("heavy chain-hole" constructs). 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 includes a set of mutations (H435R, Y436F) that minimize the binding of protein A (Tustian et al., MAbs. 2016 May-June; 8(4): 828-38) (SEQ ID NO: 185). In some cases, the heavy chain-hole and heavy chain-knob also include mutations that reduce effector function, such as L234A / L235A / P331S (LALAPS) or N325S / L328F (NSLF) (SEQ ID NOs: 188-193).

[0330] The example structure of a 2+1 bispecific antibody comprises the following components: 1) an isotype control hIgG1 wild-type antibody with a knob (T366W) mutation and a hole mutation (T366S_L368A_Y407V) in the constant region, 2) a (G4S)x3 linker between the "hole side" of the hIgG1 antibody and the scFv, 3) the VH sequence of the scFv, 4) a 20 amino acid linker sequence between the VH and VL of the scFv, and 5) the VL sequence of the scFv. Table 11 shows examples of heavy chains with various knob or hole mutations and light chain sequences.

[0331] Table 11. 2+1 bispecific antibody sequences

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339] To produce 2+1 bispecific antibodies, Expi293 cells (Invitrogen) were transiently transfected according to the manufacturer's instructions. For 20 mL culture, 20 ug of total DNA consisting of 3 expression plasmids (heavy chain-hole-scFv sequence, heavy chain-knob sequence and light chain sequence) was used. The molar ratio of heavy chain-knob, heavy chain-hole-scFv and light chain plasmids was optimized to 1:3:6 to achieve high purity 2+1 bispecific antibodies. Cell culture supernatant was harvested 5 days after transfection. The clarified supernatant was purified using a drop column with MabSelect Sure resin (Cytiva), washed with PBS, and eluted with an elution buffer of pH 3.5, and neutralized with Tris-HCl to a final pH of 5.5-6.0. The neutralized eluate containing the antibody was dialyzed into PBS. By using Nanodrop8000 (ThermoFisher) or Lunatic (Unchained Labs) to measure the absorbance at 280nm, determine the quantification of antibody concentration. The purity of 2+1 bispecific antibodies was determined by SDS-PAGE. Size exclusion chromatography (SEC) was used to analyze the aggregation of 2+1 bispecific antibodies. Next, 22 of the 24 antibodies were produced and purified into 2+1 bispecific antibodies. 22 of the 2+1 bispecific antibodies were analyzed via ELISA, FACS and internalization to confirm that binding was retained in the 2+1 format (data not shown).

[0340] Example 10: Production and screening of anti-CD98hc antibodies using single B cell clones

[0341] Anti-CD98hc antibodies were also produced via B cell cloning technology. BALB / c mice (Charles River Laboratories, Wilmington, MA) were immunized twice a week by subcutaneous or intraperitoneal injection of purified human, cynomolgus monkey and / or mouse CD98hc extracellular domain polypeptides (with or without adjuvant) (as described in Example 1 above). After a total of 14 injections and three days after the last booster injection, lymph nodes and spleens were harvested from mice, and FACS showed strong binding of mouse serum to CHO cells overexpressing human or mouse CD98hc.

[0342] Tissue is processed into single cell suspension, and negative selection magnetic bead kit (Stemcell Technologies, Vancouver, BC, Canada, catalog number 19844) is used to enrich B cells. Anti-mouse CD16 / CD32 (2.4G2, catalog number 553142, BD Biosciences, San Jose, CA) and unlabeled irrelevant Avi-His labeled protein block cells. Then use Lightening link test kit (Novus Biologics, catalog number ab236553, 336-0010, Littletown, CO), anti-mouse IgG BV605 (Poly4053, Biolegend, catalog number 405327, San Diego, CA) and anti-mouse IgM BV786 (II / 41, BD Bioscience, catalog number 743328) AF488 and AF647 labeled huCD98hc Avi-His protein pair cells are dyed. Cells are washed and subsequently stained with anti-mouse CD19 BV421 (6D5, Biolegend, catalog number 115537), anti-mouse CD138 BV421 (281-2, Biolegend, catalog number 142508), anti-human CD3 PE (UCHT1, Biolegend, catalog number 100206). Finally, viability dye 7-AAD (BD Biosciences, catalog number 559925) is added before sorting. FACS ARIA II cell sorter (BD Biosciences) is used to sort the CD19 positive and / or CD138 positive plasmablasts and memory B cell populations of IgM negative, IgG positive and human CD98hc positive.

[0343] The sorted cells are processed to sequence the single cell B cell receptor using Chromium single cell 5′ gel beads v2, which are then loaded into 3 holes of the K chip to form emulsion gel beads (Gel Beads-in-emulsion) (GEM) in the Chromium controller (10X Genomics, Pleasanton, CA, catalog numbers 1000266 and 1000287). After GEM cleaning, the GEM-RT product is purified, and the cDNA of the antibody heavy chain and light chain is amplified using the Chromium single cell mouse BCR amplification kit (10X Genomics, catalog number 1000255). GEX and VDJ libraries are prepared according to the manufacturer's instructions, and the Agilent BioAnalyzer high sensitivity DNA kit is used for evaluation, and quantification is performed by qPCR (KAPA library quantitative kit, catalog number 07960140001, Roche Sequencing, Wilmington, MA). Libraries were dual-indexed sequenced using the Illumina 150 cycle kit on an Illumina MiSeq or NovaSeq (Illumina, San Diego, CA). Sequencing data were processed using the 10x Genomics Cell Ranger mkfastq, count, and vdj pipelines. The resulting VDJ sequences were post-processed to identify unique IgG pairs.

[0344] 450 unique sequences were identified, from which a set of 94 sequences with different CDRH3 sequences were selected, synthesized and expressed as chimeras in a 2+1 bispecific antibody format.

[0345] In order to produce 2+1 bispecific antibodies, Expi293 cells (Invitrogen) were transiently transfected in 96-well deep-well plates according to the manufacturer's instructions. For the 0.8 mL culture in each well of the 96 deep-well plates, 0.8 ug of total DNA consisting of three expression plasmids (heavy chain-hole-scFv, heavy chain-knob and light chain) was used. The molar ratio of three types of plasmids (heavy chain-knob, heavy chain-hole-scFv and light chain plasmid) was optimized to 1:3:6 to achieve high-purity 2+1 bispecific antibodies. Cell culture supernatants were harvested 5 days after transfection. As described in Example 7, the clarified supernatant was purified using the ProPlusPhytip column on the Hamilton STAR platform. The concentration of the purified antibody was determined by measuring the absorbance at 280 nm using Nanodrop 8000 (ThermoFisher).

[0346] As in Example 6, the purified 2+1 anti-CD98hc bispecific antibody was screened for binding to human and cynomolgus monkey CD98hc by ELISA. The purified 2+1 bispecific antibody was screened in a concentration range of 1-30ug / mL. The ratio of the binding of ELISA to human and cynomolgus monkey CD98 and control cynomolgus monkey TfR AVI-His labeled protein to the second antibody alone was calculated, and the values ​​are listed in Table 10A. The ability of the purified 2+1 bispecific antibody to bind to CHO cells overexpressing human or mouse CD98hc or cells endogenously expressing human CD98hc was also screened by FACS. The antibody was incubated with transfected CHO, parental CHO or HCMEC / D3, washed, and then stained with goat anti-human IgG Fc (Jackson Immunoresearch, 109-136-098) and labeled with fixable activity eFluor 450 (eBioscience, catalog 65-0863-14). The data were acquired on a FACS Canto (BD Biosciences). Table 12B indicates the mean fluorescence intensity ratio of the transfected cell lines to the parental CHO binding. The binding ratio of endogenously expressed human brain endothelial cells to the second only is also listed in Table 12B. Of the 94 2+1 anti-CD98hc bispecific antibodies screened, 47 were positive by ELISA, with a signal at least 5 times higher than the second alone, and 16 were ELISA and FACS positive compared to unexpressed cells, with FACS binding increased by 3 times or more. Table 13 shows the sequences of 2+1 anti-CD98hc anti-CD98hc bispecific antibodies combined by ELISA and FACS. The CDR sequences (Kabat) are shown in Tables 14 and 15.

[0347] Table 12A: ELISA characteristics of 2+1 anti-CD98hc bispecific antibodies derived from a single B cell clone

[0348]

[0349] Table 12B: FACS characteristics of anti-CD98hc 2+1 bispecific antibodies derived from a single B cell clone

[0350]

[0351]

[0352] Table 13: Antibody sequences of anti-CD98hc 2+1 bispecific antibodies from a single B cell clone

[0353]

[0354]

[0355]

[0356] Table 14. Heavy chain CDR sequences of anti-CD98hc 2+1 bispecific antibodies from a single B cell clone

[0357]

[0358]

[0359] Table 15. Light chain CDR sequences of anti-CD98hc 2+1 bispecific antibodies from a single B cell clone

[0360]

[0361]

[0362] Example 11: ELISA binding of 2+1 anti-CD98hc bispecific antibody

[0363] To determine the binding retention of the reformatted 2+1 anti-CD98hc bispecific antibody by ELISA, a high binding ELISA plate (Thermo Fisher) was coated with streptavidin (1 μg / ml, Thermo Fisher) overnight at 4°C. The plate was then washed three times with phosphate-buffered saline containing 0.05% Tween-20 (PBST) and incubated with blocking buffer (PBS+1% BSA, 1 h, room temperature). The plate was washed three times again with PBST and incubated with biotinylated human, cynomolgus monkey and mouse CD98hc and an irrelevant antigen, human transferrin receptor (1 μg / ml, 1 h, room temperature). The plate was washed three times again with PBST and incubated with anti-CD98hc antibody (1 μg / ml, 1 h, room temperature). The plate was washed three times again with PBST and incubated with the second detection antibody, anti-human horseradish peroxidase conjugated antibody (1:5000 dilution, 30 min, room temperature). The plates were washed three times for the final time and incubated with TMB-ELISA substrate solution (Thermo Fisher) and then quenched with H2SO4. The SpectraMax M5 (Molecular Devices) was then used to quantify the 450 nm (OD 450) were analyzed for anti-CD98hc binding by optical density at . The binding properties of various 2+1 anti-CD98hc bispecific antibodies to CD98hc (human, cynomolgus monkey, mouse) and control human TfR are summarized in Table 16 below, shown as fold change relative to isotype hIgG. All 2+1 anti-CD98hc antibodies showed binding specificity to human CD98hc compared to mouse CD98hc and controls, and cross-reacted with cynomolgus monkey CD98hc to varying degrees.

[0364] Table 16. ELISA OD of reformatted 2+1 anti-CD98hc bispecific antibodies 450 value

[0365]

[0366]

[0367] Example 12: FACS binding of 2+1 anti-CD98hc bispecific antibody

[0368] In order to determine the binding retention of 2+1 anti-CD98hc bispecific antibodies by FACS, the binding of antibodies to HCMEC / D3 cells (cell lines derived from human brain endothelial cells), overexpressing human and mouse CD98hc CHO cell lines, and non-specific binding to parental CHO cells were evaluated. The cells were inoculated in tissue culture plates (50,000 cells / well) and washed and resuspended in FACS buffer (PBS+2% BSA+1mM EDTA). The 2+1 anti-CD98hc bispecific antibodies were then incubated with cells (5 μg / ml, 1 hour, on ice) and washed twice with FACS buffer. The cells were then incubated with an anti-mouse second antibody (Jackson Immunoresearch) conjugated to allophycocyanin (1: 1000, 30min, on ice) and then washed twice with FACS buffer. Next, the cells were resuspended in FACS buffer supplemented with 0.5% propidium iodide (Thermo Fisher). Anti-CD98hc binding was then analyzed on an iQue flow cytometer (IntelliCyt) by measuring mean fluorescence intensity (MFI). The FACS data are summarized in Table 17 below, showing the MFI fold change relative to the isotype hIgG1 control. All 2+1 anti-CD98hc antibodies showed binding specificity to hCMEC / D3 cells and to a lesser extent to the CHO-huCD98hc cell line.

[0369] Table 17. FACS MFI fold changes of reformatted 2+1 anti-CD98hc bispecific antibodies

[0370]

[0371]

[0372] Example 13: In vitro internalization of 2+1 anti-CD98hc bispecific antibody into blood-brain barrier endothelial cell line

[0373] As described in Example 7 above, internalization of 2+1 anti-CD98hc bispecific antibodies into hCMEC / D3 cells was determined. Anti-TfR hIgG1 antibodies were included as positive controls and isotype hIgG1 antibodies were included as negative controls. The fold change of the total integrated intensity of pHrodo-red signals in each well relative to isotype hIgG1 was calculated and summarized in Table 18 below. Table 168 shows that 22 of the 2+1 anti-CD98hc bispecific antibodies that retained target binding also showed internalization. All 2+1 anti-CD98hc antibodies were internalized into hCMEC / D3 cells to varying degrees.

[0374] Table 18. Internalization values ​​of 2+1 anti-CD98hc bispecific antibodies

[0375]

[0376]

[0377] Example 14: Epitope binning of 2+1 anti-CD98hc bispecific antibodies

[0378] Epitope binning analysis of 2+1 anti-CD98hc bispecific antibodies was performed by using a Carterra LSA instrument (Carterra, Salt Lake City, UT) for a classic sandwich experiment. Briefly, kinetic evaluation was performed using the HC200M and then tested in a binning assay, where the ability of the immobilized antibodies to form sandwich pairs with recombinant huCD98hc Avi-His and injected antibodies was tested. For each cycle, 200 nM huCD98hc Avi-His was injected onto the chip for five minutes, followed by injection of the test antibody (diluted to 40 μg / ml in running buffer) for five minutes, followed by two 30-second injections of Pierce TMIgG elution buffer (ThermoScientific) was used for regeneration. Data were processed and analyzed using epitope high-throughput binning analysis software (Carterra). Antibodies that can bind to antigens captured by immobilized antibodies are designated as "sandwich" or "pairing" antibodies, and these antibodies are assigned to epitope bins different from those of immobilized antibodies. The combined results of these experiments constructed a matrix of paired and unpaired antibodies, which allowed the generation of an epitope bin landscape for 2+1 anti-CD98hc bispecific antibodies. Some antibodies have overlapping binning features, indicating that they can recognize adjacent but not completely overlapping epitopes. Slight heterogeneity within the bin is indicated by adding letters such as a, b, c, and d. For antibodies that appear to overlap with two epitope bins, numbers are underlined to indicate overlapping bins. The epitope bins are summarized in Table 19. "Unique" means that the 2+1 anti-CD98hc bispecific antibody does not share an epitope with any other antibody (tested in the group).

[0379] Table 19: Epitope bins of 2+1 anti-CD98hc bispecific antibodies

[0380] box Sample ID 1a CD98hc.04.009, -.010, -.013, -.018, .064 1b CD98hc.04.046,-.022 1c CD98hc.04.054 1d CD98hc.04.062 1e CD98hc.03.010 1f CD98hc.04.047,-.065 1_2 CD98hc.04.011 1_2d CD98hc.04.063 2a CD98hc.04.003 2b CD98hc.04.045, CD98hc.03.002 2c CD98hc.04.012 2d CD98hc.03.031, CD98hc.04.058 unique CD98hc.03.004, CD98hc.04.006

[0381] Example 15: Binding kinetics of 2+1 anti-CD98hc bispecific antibody

[0382] The binding kinetics of 2+1 anti-CD98hc bispecific antibodies (from hybridoma campaigns) to human and cynomolgus CD98hc Avi-His were evaluated using a Carterra LSA instrument (Carterra, Salt Lake City, UT). The 2+1 anti-CD98hc bispecific antibodies were diluted 50 times in 10mM acetate, pH 5.0 (Carterra) to obtain a final concentration of 20μg / mL, prepared in duplicate. The HC200M sensor chip (Carterra) was activated using a single-channel flow cell, and a mixture of 100mM MES pH 5.5, 100mM sulfo-NHS, and 400mMEDC (all reconstituted in MES pH 5.5; before running the assay, each was immediately mixed in a vial with 100μl) was injected for 7 minutes. After switching to a multichannel array flow cell, the antibodies were injected into the activated chips in four 96-point arrays, and each chip was injected for 15 minutes. The remaining unconjugated active groups on the chip were then blocked by injecting 1 M ethanolamine pH 8.5 (Carterra) for 7 minutes using a single channel flow cell. The resulting sensor chip contained eight spots for each antibody with four different densities. Two independent experiments were performed as described below, resulting in N between one and eight determinations for each antibody. Spots that produced less than 20 RU of analyte binding were excluded from further analysis.

[0383] After initiation using running buffer HBS-EP+ (Teknova) with 0.5mg / ml BSA (MP Biomedicals), the ability of immobilized 2+1 anti-CD98hc bispecific antibodies to bind to several forms of CD98hc Avi-His (including the above-mentioned human and cynomolgus monkey orthologs) was tested. Each analyte was injected into the entire antibody array using a single-channel flow cell to generate an affinity estimate. Using running buffer, a series of six three-fold serial dilutions were performed for CD98hc Avi-His analytes for human and cynomolgus monkey CD98hc Avi-His starting from 600nM. The analyte was injected for 5 minutes and then dissociated for 10 minutes. After each analyte injection, the antibody was regenerated with Pierce IgG elution buffer (ThermoScientific). NextGenKIT high-throughput kinetic analysis software (Carterra) was used to process and analyze the data.

[0384] The equilibrium dissociation constant (KD) was calculated from the fitted association and dissociation rate constants (k-on and k-off) of the 2+1 anti-CD98hc bispecific antibodies of the present disclosure. These values ​​were combined, the mean and standard deviation were calculated, and a graph was prepared using GraphPadPrism. The KD values ​​are summarized in Table 20 below. As indicated in the table, some antibodies showed heterogeneous binding properties that did not fit the 1:1 binding model, so their rate constants could not be determined.

[0385] Table 20: Equilibrium dissociation constants (K) of 2+1 anti-CD98hc bispecific antibodies D )

[0386]

[0387]

[0388] N = number of determinations; NT = not tested; NA = not applicable; LB = low binding (below the limit of measurement); NB = no binding detected.

[0389] These results show that the 2+1 anti-CD98hc bispecific antibody exhibits an affinity range of about 3nM to 200nM for CD98hc Avi-His binding. Specifically, the 2+1 anti-CD98hc bispecific antibody binds to human CD98hc Avi-His with an affinity range of 3.1nM to 210nM; the anti-CD98hc antibody of the present disclosure binds to cynomolgus monkey CD98hc Avi-His with an affinity range of 3.2nM to 145nM.

[0390] Example 16: Ability of 2+1 anti-CD98hc bispecific antibodies to affect CD98hc levels or localization

[0391] Binding of 2+1 anti-CD98hc bispecific antibodies to their receptors on target cells can dysregulate their cell surface expression or recycling. To evaluate 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 cultured at 7.5*10^4 cells / cm 2 The cells were seeded in tissue culture plates in 100uL of culture medium (EBM2, Lonza). After 24 hours, the cells were treated with 20μg / ml 2+1 anti-CD98hc bispecific antibody for 24 hours and then analyzed by flow cytometry (FACS) or western blot. Quantitative FACS data revealed that there was no reduction in cell surface expression of CD98hc after 2+1 bispecific antibody treatment ( Figure 2 Similarly, using quantitative Western blot analysis, the total amount of CD98hc protein in antibody-treated cells did not vary by more than 50% compared to isotype controls ( Figure 3 ). Apparently, after treatment with some of these 2+1 anti-CD98hc bispecific antibodies, the cell surface expression of CD98hc increased, which could be due to a feedback effect induced by the cells in response to amino acid deprivation. It has been reported that cells treated with JPH203 and BCH exhibit this phenotype, which can inhibit the activity of the LAT1-CD98hc complex (Maimaiti M. et al., Scientific Reports. (2020) 10: 1292; et al., Journal of Experimental & Clinical Cancer Research (2018) 37: 234). Therefore, 2+1 anti-CD98hc bispecific antibodies that significantly increased CD98hc cell surface levels (> 2-fold) were excluded from further development (except CD98hc.04.003, which had other desirable properties (provided in other examples)).

[0392] Example 17: In vivo PK study of 2+1 anti-CD98hc bispecific antibody in huCD98hc ECD mice

[0393] Antibody production method: According to the manufacturer's instructions, antibodies for in vivo PK studies were produced via transient transfection of ExpiCHO cells (Invitrogen). For 400 mL of culture, 320 ug of total DNA consisting of 3 expression plasmids (heavy chain-hole-scFv sequence, heavy chain-knob sequence, and light chain sequence) was used. The molar ratio of the 3 plasmids was optimized to achieve a highly pure 2+1 anti-CD98hc bispecific antibody. Cell culture supernatant was harvested 10 days after transfection. Antibodies were purified using protein A affinity chromatography, followed by ion exchange chromatography on AKTA Avant 25 (Cytiva) to remove product-related impurities. The purified antibodies were dialyzed into PBS. Analytical characterization was performed by 280 nm absorbance, CE-SDS, size exclusion chromatography, and endotoxin measurement.

[0394] Study design: To establish the concept of brain penetration of the in vivo validation 2+1 anti-CD98hc bispecific antibody, knock-in (KI) mice were generated in which the extracellular domain region of mouse CD98hc was replaced by the extracellular domain region of human CD98hc in one of the two alleles. This was done because the 2+1 anti-CD98hc bispecific antibody specifically binds to primate CD98hc but not to mouse CD98hc. A double-dose strategy was used to determine the brain uptake of the injected antibody and the peripheral clearance over time. Briefly, 10 mg / kg of isotype IgG or 2+1 anti-CD98hc bispecific antibody was administered to heterozygous (huCD98hc+ / -KI) mouse groups (N=3) on days 1 and 15. Blood samples were then collected from antibody-treated animals at different time points, placed in serum separation tubes, and coagulated at room temperature (RT) and then centrifuged. The resulting supernatant representing serum was subsequently transferred to a new tube and stored at -80°C until analysis.

[0395] Brain sample preparation: Brain tissue was collected from antibody-treated mice on the 15th day (i.e. 24 hours after the second antibody injection). Prior to this, mice were anesthetized and subjected to cardiac perfusion with 15ml PBS to clear blood vessels. The brain tissue was then minced using a manual grinder and homogenized in 1ml HBSS buffer (MilliporeSigma No. 55037C) containing 10mM HEPES (No. 15630130, Gibco). Subsequently, the sample was centrifuged at a speed of 1000g for 5 minutes to precipitate the container portion. The supernatant representing the substance was then transferred to a new tube and mixed with 10x RIPA buffer (final concentration 1x), which included a mixture of protease and phosphatase inhibitors (cOmplete TM, Mini Protease Inhibitor Cocktail, Roche, catalog number 11836153001). The samples were incubated on a rotary mixer (nutator) for 20 minutes at 4°C, and then the samples were frozen on dry ice and stored at 80°C until analysis. The vascular fraction in the myelin fragments was cleared by centrifugation via % 18 dextran (70kDa, No. 31390, Sigma-Aldrich), followed by washing 2 times in HBSS buffer. Subsequently, the samples were dissolved in RIPA buffer (R0278, Sigma-Aldrich) and stored at -80°C until analysis. The validity of this method was confirmed using WB analysis, where brain endothelial cell markers (e.g., CD31 and Claudin-5) were not present in the parenchymal part (data not shown).

[0396] Pharmacokinetic (PK) analysis: Antibody concentrations in serum and brain samples were measured using MSD (Meso Scale Discovery) methods. In brief, 50 μl / well goat anti-human IgG1Ab (Jackson ImmunoResearch, No. 109-005-097) was added to MSD plates at 1 μg / ml in PBS, and incubated overnight at 4 ° C. The plates were then washed 3x with wash buffer (0.05% Tween-20 in 1xPBS), followed by incubation of 1 hour with blocking buffer (wash buffer containing 3% BSA) at RT. Subsequently, serum (1:10000 dilution in PBS) and brain (1:2 dilution in PBS) samples were added to the plates in duplicate, and incubated on a shaker at 500RPM for 1 hour. Human IgG1 isotype antibodies of known concentrations included in the assay were used to create a standard curve. Next, the plate was washed 3x with wash buffer, followed by addition of 0.2 μg / ml of Sulfo-Tag goat anti-human antibody (R32AJ-5, Meso Scale Discovery) in PBS at 30 μl / well and incubated on a shaker at 500 RPM for 1 hour. Finally, after washing 3x with wash buffer, 150 μl / well 1x read buffer (R92PC-2, Meso Scale Discovery) was added to the plate, which was then read using a Sector Imager S600 instrument.

[0397] By measuring antibody concentrations in vascular depleted brains, we determined that the levels of 2+1 anti-CD98hc bispecific antibodies were 2-fold higher than isotype IgG in HET huCD98hc KI mice ( Figure 4 Peripheral PK was determined by measuring antibody concentrations in plasma samples from antibody-treated mice ( Figure 5All tested 2+1 anti-CD98hc bispecific antibodies showed higher plasma clearance in HET huCD98hc KI mice compared to isotype IgG, consistent with CD98hc-mediated peripheral distribution. The three 2+1 anti-CD98hc bispecific antibodies tested (CD98hc.04.062, CD98hc.04.063, CD98hc.04.064) showed significant (5-17) fold changes in brain / serum ratios at the 24-hour time point tested ( Figure 6 ).

[0398] Example 18: Humanization of 2+1 anti-CD98hc bispecific antibody

[0399] Based on the 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 had the best brain penetration 24 hours after injection. One of the most commonly used methods for humanization of non-human antibodies is to transplant the CDR of a non-human antibody onto a human antibody acceptor framework. Typically, such CDR transplantation results in a weakening or complete loss of its affinity due to perturbations in the humanized antibody framework. Therefore, it may be necessary to retain certain residues from the mouse framework to replace the human residues at the corresponding positions (back mutations) to restore the weakened or lost affinity. Therefore, it is crucial to accurately predict the residues to be retained in the context of the selected human antibody germline acceptor framework (which can retain the function and paratope of the humanized antibody). In addition, for good manufacturability and downstream development, retaining or improving thermal stability and solubility is desired.

[0400] Structure-based antibody modeling was applied in the process of humanizing anti-CD98hc mouse monoclonal antibody (mAb) using the BioMOE module of MOE (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada). In short, the VH and VL sequences of the mouse mAb sequence to be humanized were compared with the functional germline sequences of people VL, VH, LJ, HJ taken from IMGT (http: / / www.imgt.org / ). Pseudogenes and ORFs were excluded. For each mouse mAb (query), the five most similar VL and five most similar VH germline sequences were selected and combined with the most similar VJ and HJ genes to generate 25 humanized sequences. The CDRs to be transplanted into the human framework were defined according to the AbM definition (http: / / www.bioinf.org.uk / abs / #cdrdef).

[0401] The frequency of VH and VL framework of two kinds of humanized sequences in the human library has selected these two kinds of humanized sequences.Query and humanized sequence are used to create Fv homology model.Utilize BioMOE module or antibody modeler module of MOE (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada) to create Fv homology model.AMBER10:EHT force field is used to carry out energy minimization in the whole antibody homology modeling process.The scoring criteria provided by MOE is calculated, analyzed and sorted based on Fv homology model, molecular descriptor (such as interaction energy between VL and VH, isoelectric point (3D pI) based on coordinates, hydrophobic patch and charged surface area).Utilize these molecular descriptors to prioritize the humanized mAb for downstream experimental program, and described molecular descriptor includes protein expression, purification and binding affinity test and functional determination.

[0402] The BioMOE module of MOE provides a tool, Mutation Site Properties, to visualize and classify residues that may undergo back mutations. Back mutations are defined as amino acid substitutions that revert to the original query sequence replacing the humanized sequence. Using this tool, the original query (reference) is compared to the primary amino acid sequence of the selected humanized variant and the 3D structure of the 3D Fv homology model, respectively.

[0403] Changes between the reference and humanized variants were categorized based on differences in amino acid type, potential for interaction with CDR residues, potential for impact on VL / VH pairing, and potential changes in hydrophobic and charged surface areas in and near the CDRs.

[0404] Mutations near the CDRs or VL / VH interface, with significant charge differences, or containing strong H-bond interactions were evaluated separately, and significantly disruptive mutations were restored to the original query residues. Thus, the humanized sequence may contain up to five back mutations. The query mouse mAb (mouse CD98hc.04.064) and the humanized mAbs are listed as scFvs in Table 21 below.

[0405] Table 21: Humanized anti-CD98hc scFv sequences

[0406]

[0407]

[0408] Table 22: Humanized anti-CD98hc sequences (VH and VL of scFv sequences in Table 21)

[0409]

[0410]

[0411] Table 23: Humanized anti-CD98hc CDR sequences (Kabat)

[0412]

[0413]

[0414]

[0415] Example 19: Affinity Optimization and Liability Elimination of 2+1 Anti-CD98hc Antibodies

[0416] Based on the characteristics of the humanized variants, CD98hc.04.064.4a was selected for further engineering. The affinity optimization of the CD98hc.04.04.064.4a variant is designed by removing the responsible sequence (such as high-risk isomerization sites and oxidation sites) and replacing the responsible sequence with residues with high frequency at this position in the immune repertoire. In addition, residues in the CDR regions predicted to have strong antigen binding interactions are selected for mutation analysis and evaluation for further affinity maturation. The variants selected for generation and further screening are shown in Table 24 below. The selected clones are produced as 2+1 anti-CD98hc bispecific antibodies in the above format (Example 9).

[0417] Table 24: Affinity-optimized 2+1 bispecific antibody variant sequences

[0418]

[0419]

[0420]

[0421] Table 25: Affinity optimized 2+1 bispecific antibody variant sequences (VH and VL of the sequences in Table 24)

[0422]

[0423]

[0424]

[0425]

[0426] Table 26: Affinity optimized 2+1 bispecific antibody CDR sequences (Kabat)

[0427]

[0428]

[0429] Example 20: Evaluation of in vitro effector function (CDC and ADCC)

[0430] To investigate the mechanism of reticulocyte depletion occurring in the 2+1 anti-CD98hc bispecific antibodies, the ability of these same antibodies to elicit in vitro effector responses, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), was evaluated.

[0431] CDC Methods and Results: The ability of 2+1 anti-CD98hc bispecific antibodies to drive complement deposition was measured on hCMEC / D3 cell lines expressing high levels of both receptors. Target cells were isolated, washed 1x in PBS, and diluted to 2x10^6 cells / mL in RPMI 1640 medium. 50 μL of target cells were dispensed into each well of a round-bottom 96-well plate (Falcon No. 351177) (1x10^5 cells / well). 25 μL of 4x antibodies diluted in the same medium were added to these cells. Cells and antibodies were incubated at 37°C for 15 min, and then 25 μL of pooled complement human serum (Innovative Research, IPLA-CSER) was added to each well as a complement source, and the plate was incubated at 37°C for another 2 h. Afterwards, the cells were washed 2x with FACS buffer (PBS + 2% FBS + 1mM EDTA) and resuspended in 50 μL FACS buffer + 0.25 μL / well propidium iodide (FischerScientific, BD 556463), and then analyzed on an iQue flow cytometer (IntelliCyt). CDC was analyzed as the ratio of PI+ single cells in each well. In this assay, none of the 2+1 anti-CD98hc bispecific antibodies induced a significant CDC response (data not shown).

[0432] ADCC Method and Results: The ability of 2+1 bispecific antibodies to cause antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated using the ADCC Reporter Bioassay system (No. G7010) from Promega. This system relies on an engineered Jurkat T cell line that stably expresses the FcgRIIIa receptor (V158 variant) and an NFAT response element that drives the expression of firefly luciferase. The target cells were diluted in assay buffer (RPMI+4% low IgG serum) at a concentration of 1.2x10^6 / mL, and 25 μL cells (30,000 per well) were dispensed into appropriate wells of a 96-well white assay plate. 25 μL 3x antibodies, also diluted in assay buffer, were added to the wells containing the cells. After adding the antibody to the target cells, the provided effector cells (frozen at 2x10^7 / mL) were thawed at 37°C and 630 μL was added to 3.6 mL of heated (37°C) assay buffer, gently mixed, and 25 μL of effector cells (75,000 per well, E:T ratio of 2.5) was immediately added to the well containing the target cells and antibody. The plate was then incubated at 37°C and 5% CO2 for 6 hours to allow activation of the receptor cells and expression of luciferase. After 6 hours, the plate was equilibrated to room temperature (15 minutes) while the 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 on a Bio Tek plate reader.

[0433] In wild-type human IgG1 format, certain 2+1 bispecific antibodies targeting CD98hc (CD98hc.04.064 and CD98hc.04.003) were able to drive significant ADCC signals ( Figure 7 ), indicating that utilizing an inactive Fc has advantages.

[0434] Example 21: Affinity data of 04.064 humanized group

[0435] The binding kinetics of humanized anti-CD98hc antibodies were evaluated using 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 ability of the captured antibodies to bind to human and cynomolgus CD98hc was tested as follows.

[0436] The recombinant human and cynomolgus CD98hc analytes described in the above method were diluted to a concentration of 1000 nM in running buffer (HBS-EP+, Teknova, No. 8022, with 0.5 mg / mL BSA, MP Biomedicals LLC, No. 820451), and then diluted 3 times to 250, 62.5 and 15.6 nM. Each sample was injected for 2 minutes to allow association, followed by dissociation in buffer alone for 10 minutes. After each sample injection, three 30-second injections of 10 mM glycine pH 1.7 were used for chip regeneration. Fresh antibodies were captured at the beginning of each cycle.

[0437] The data were analyzed using Biacore evaluation software to generate kinetic constants. The equilibrium dissociation constant (KD) was calculated from the fitted association and dissociation rate constants (k-on and k-off) for each anti-CD98hc antibody. These values ​​are summarized in Table 27 below.

[0438] Table 27: Equilibrium dissociation constants (K) of humanized 2+1 anti-CD98hc bispecific antibodies D )

[0439]

[0440] This data indicates that the 4.064 humanized panel binds to human CD98hc in the range of about 18 nM to 35 nM and to cynomolgus CD98hc in the range of about 340 nM to 1.5 μM.

[0441] Example 22: Serum and brain PK data of CD98hc.04.064 humanized group after intravenous injection into hCD98hc+ / +KI mice

[0442] Antibody Production Methods: 2+1 antibodies and matched control antibodies (same Fab and Fc domains, but lacking scFv targeting CD98hc) for in vivo PK studies were produced via transient transfection of ExpiCHO cells (Invitrogen) according to the manufacturer's instructions and purified as described in Example 17 above.

[0443] Study Design: For this study, homozygous knock-in (KI) mice (described in Example 17) were utilized (mice in which the extracellular domain region of mouse CD98hc was replaced by the extracellular domain region of human CD98hc in both alleles (huCD98hc+ / +)). A double-dose study was performed to determine brain uptake of injected antibodies and peripheral clearance over time. Briefly, groups of homozygous (huCD98hc+ / +KI) mice (N=3) were intravenously administered 10 mg / kg of isotype IgG or 2+1 anti-CD98hc bispecific antibodies on days 1 and 15. Blood samples were then collected from antibody-treated animals at different time points, placed in serum separator tubes, and coagulated at room temperature (RT) and then centrifuged. The resulting supernatant, representing serum, was then transferred to a fresh tube and stored at -80°C until analysis.

[0444] Samples from vascular depleted brain and serum were processed and analyzed as described in Example 17.

[0445] Figure 8 The antibody levels in the vascular depleted brain fraction of huCD98hcKI+ / + mice 24 hours after the second dose of 10 mg / kg of 2+1 anti-CD98hc and control antibodies are shown. Antibody levels are shown as fold change of CD98hc relative to matched controls with the same Fab and Fc but without the scFv binding domain. Figure 8 As described in, levels of the 2+1 anti-CD98hc bispecific CD98hc.04.064 humanized lead antibody were determined to be 3- to 4.5-fold higher than TD1 IgG in vascular-depleted brain fractions of huCD98hc+ / +KI mice. Peripheral PK was determined by measuring antibody concentrations in plasma samples of antibody-treated mice.

[0446] Fig. 9 The antibody levels in the serum of huCD98hcKI+ / + mice 24 hours after the second dose of 10 mg / kg of 2+1 anti-CD98hc and control antibodies are shown. The antibody levels in the vascular depleted brain fraction of huCD98hc KI+ / + mice 24 hours after the second dose of 10 mg / kg of 2+1 anti-CD98hc and control antibodies are shown. Fig. 9 As shown in Figure 2, all tested 2+1 anti-CD98hc bispecific 04.064 humanized antibodies showed similar clearance compared to control IgG in huCD98hc+ / +KI mice. CD98hc.04.064.1e was further engineered.

[0447] Example 23: Affinity data of engineered variants of CD98hc.04.064

[0448] The binding kinetics of 2+1 anti-CD98hc bispecific antibodies to recombinant human and cynomolgus CD98hc ECD Avi-His were evaluated using a Biacore T200 instrument (Global Life Sciences Solutions USA LLC, Marlborough, MA). The 2+1 anti-CD98hc bispecific antibodies were prepared by diluting to 50 μg / mL in a running buffer HBS-EP+ (Teknova) with 0.5 mg / mL BSA (MP Biomedicals), and captured by a goat anti-human κ polyclonal antibody (Southern Biotech) surface, which was prepared according to the recommendations of the instrument manufacturer. Two independent experiments were performed as described below, resulting in N of each antibody being determined between one and three times. Affinity was estimated by injecting each analyte into the capture antibody on flow cell 2, 3 or 4. The CD98hc ECD Avi-His analyte was diluted with running buffer, starting from 600 nM, and a series of five three-fold serial dilutions were performed. Analytes were injected without regeneration, from the lowest concentration to the highest concentration, each concentration lasting 5 minutes, and then dissociated for 15 minutes. After each analyte was injected continuously, the antibody was regenerated with 10 mM glycine pH 2.0 buffer (Cytiva). Data were processed and analyzed using Biacore T200 BiaEvaluation software (Cytiva).

[0449] The equilibrium dissociation constant (K D ) are calculated from the fitted association and dissociation rate constants (k-on and k-off) of the 2+1 anti-CD98hc bispecific antibodies of the present disclosure that bind to human and cynomolgus CD98hc ECD Avi-His. For measurements with N>1, the mean and standard deviation were calculated, and the measurements for all samples were combined and graphed using GraphPad Prism. K D The values ​​are summarized in Table 28 below.

[0450] Table 28: Equilibrium dissociation constants (K) of 2+1 humanized variants of CD98hc.04.064 in 2+1 format D ).

[0451]

[0452]

[0453] N = number of determinations; NA = not applicable;

[0454] *Inconsistent results obtained in additional equilibrium dissociation constant experiments.

[0455] These results indicate that the 2+1 anti-CD98hc bispecific antibodies bind to human and cynomolgus CD98hc ECD Avi-His with an affinity ranging from approximately 13 nM to 880 nM. Specifically, the 2+1 anti-CD98hc bispecific antibodies bind to human CD98hc ECD Avi-His with an affinity ranging from 12 nM to 34 nM, and to cynomolgus CD98hc ECD Avi-His with an affinity ranging from 120 nM to 880 nM.

[0456] Example 24: TD1-CD98hc bispecific antibody in an in vitro sTREM2 assay

[0457] In order to test the function of TD1-CD98hc antibody (used as a PK / PD model for delivering CD98hc-mediated antibodies to the brain), sTREM2 determination was performed. According to the manufacturer's protocol, human monocytes were isolated from whole blood using RosetteSep human monocyte enrichment mixture (Stemcell technologies) and Ficoll centrifugation. After lysing red blood cells with ACK lysis buffer, monocytes were resuspended in complete medium (RPMI, 10% FBS, Pen / Strep, L-glutamine, HEPES, non-essential amino acids and sodium pyruvate). In order to obtain macrophages from these isolated monocytes, 50ng / ml human M-CSF and 8% volume / volume human serum were added to the cells for 5-7 days.

[0458] Macrophages were differentiated from 4 human donors in 2 independent experiments. 5 Macrophages / well were stimulated with 1 ug / mL test antibody (huIgG1, TD1 or TD1-CD98hc) for 48 hours. Duplicate wells were considered as technical replicates. Supernatants were tested for sTREM2 by the MSD platform.

[0459] Technical replicates for each donor were averaged, and fold changes compared to the NSLF isotype control were then calculated and plotted. Fig.10 Each point in the graphs in represents a single donor. Statistical significance was calculated using matched one-way ANOVA.

[0460] like Fig.10 As shown, TD1-CD98hc was found to be active in an in vitro sTREM2 assay compared to hIgG1. TD1-CD98hc also promoted sTREM2, or better than TD1.

[0461] Example 25: TD1-CD98hc bispecific antibody and method for intravenous administration to non-human primates

[0462] A bispecific antibody was generated in a 2+1 (hole) format (referred to as "TD1-CD98hc") using: (i) CD98hc.04.064.1e scFV; and (ii) a specific antibody against human MSA4A (TD1). The complete amino acid sequence of TD1-CD98hc is as follows:

[0463] Variable light chain region:

[0464] DVVMTQSPLSLPVTLGQPASISCKSSRSLLYSAGKTYLSWFQ QRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDV GVYYCWQGIDFHQTFGGGTKVEIK (SEQ ID NO: 405).

[0465] Constant light chain region:

[0466] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 406).

[0467] Variable heavy chain region:

[0468] QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAP GQGLEWMGWINTYSGVPTYAQGFTGRFVFSLDTSVSTAYLQISSL KAEDTAVYYCARTMADYWGQGTLVTVSS (SEQ ID NO: 407).

[0469] CH+Fc No. 1 (pestle, T366W):

[0470] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:408).

[0471] CH+Fc number 2 (socket, T366S, L368A, Y407V):

[0472] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK(SEQ ID NO:409).

[0473] Linker targeting domain:

[0474] GGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGIIDPSDSETHYAQKFQGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARASYGKGYFDYWGQGTLVTVSSGGSEGKSSGSGSESKSTGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLNNINQKNYLAWYQQKPGQPPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSSPFTFGGGTKVEIK(SEQ ID NO:410).

[0475] The complete amino acid sequence of the antibody (TD1) specific for the individual MSA4A is as follows:

[0476] TD1 heavy chain hIgG1 NSLF (N325S, L328F)

[0477] QVQLVQSGSELKKPGASVKVSCKASGYAFTSYGLSWVRQAPGQGLEWMGWINTYSGVPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARTMADYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:411).

[0478] TD1 light chain

[0479] DVVMTQSPLSLPVTLGQPASISCKSSRSLLYSAGKTYLSWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGIDFHQTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:412).

[0480] A PK / PD study was conducted in juvenile male cynomolgus monkeys (n=9) following intravenous (iv) administration of 20 mg / kg each of the three antibodies (n=3) on days 1 and 29 (2 administrations in total).

[0481] Table 29: In vivo administration of anti-TD1-CD98hc bispecific antibodies

[0482]

[0483] The intravenous dose was administered as a slow push via the saphenous vein. The target dose level (mg / kg), target dose concentration (mg / mL), and target dose volume (mL / kg) were consistent between Groups 1-3.

[0484] Example 26: Hematological parameters after intravenous administration of TD1-CD98hc bispecific antibody to non-human primates

[0485] Blood samples (approximately 2 mL) were collected from the femoral vein into tubes containing potassium EDTA before and after dosing for hematology. The following parameters in Table 30 were tested:

[0486] Table 30: Hematological parameters tested after intravenous administration of anti-TD1-CD98hc antibodies

[0487]

[0488] The normal range of the reticulocyte count by the testing device is 25.7 -122.3 10 3 / μl.

[0489] Fig.11 Absolute reticulocyte counts during the study period are described.

[0490] Overall, no significant changes were observed in hematological parameters, and no persistent decrease in reticulocytes was observed during the study.

[0491] Example 27: CSF Penetration Following IV Administration of TD1-CD98hc Bispecific Antibody in Non-Human Primates

[0492] Cerebrospinal fluid (CSF) and serum were collected from NHPs at various times after the first and second administrations as described in Example 25. Serum and CSF were tested as described below.

[0493] Sandwich ELISA: 100 μL / well goat anti-human IgG, Monkey adsorbed-BIOTIN (Southern Biotech; catalog number 2049-08) working solution (0.5 μg / mL) was added to the SuperBlock TM 96-well Pierce TMThe 100 μ L / well calibration standards, QC and MRD diluted serum or CSF QC and test samples were incubated on a plate oscillator at 350 rpm for 1 hour ± 10 minutes. Serum or CSF quality control (QC) and test samples were diluted to the minimum required dilution (MRD) in assay buffer (1x TBS, 0.05% Tween-20 and 0.1% BSA) and then loaded onto the assay plate, while the standard calibration curve and assay buffer QC (AB-QC) were added directly to the assay plate. After incubation, the plate was washed 3 times with a plate washer with wash buffer (1XPBS and 0.05% Tween-20), and then 100 μ L / well calibration standards, QC and MRD diluted serum or CSF QC and test samples were added to the plate, and incubated at room temperature for 2 hours ± 15 minutes on a plate oscillator at 350RPM. The plate is then washed, and 100 μL / well goat anti-human IgG, monkey adsorption HRP (Southern Biotech; Catalog No. 2049-05) working solution (0.08 μg / mL) is added to the plate, and incubated at room temperature for 1 hour ± 10 minutes on a plate shaker at 350RPM. After HRP incubation, the plate is washed and 100 μL / well tetramethylbenzidine (TMB) substrate solution (Surmodics product No. TMBS-1000-01) is added, and incubated at room temperature (RT) for 15 ± 10 minutes, covered with aluminum foil or placed in a drawer to avoid light. After the incubation is complete, 100 μL / well stop solution (Surmodics product No. NSTP-1000-01) is added to the plate to stop the reaction. The plate is then placed on a plate shaker, and at room temperature, it is shaken at 350RPM for at least 2 minutes. The plate was then read on a Molecular Devices SpectraMax M5 instrument and raw optical density (OD450-540) signals were generated from each well. The concentration of the analyte in each unknown sample was determined by interpolating the concentration using the calibration standard curve. Standard regression was performed using SoftMax Pro (Molecular Devices / Version 7.1) with a weighting factor of 1 / Y 2 The 4-parameter logistic (4-PL) model was used.

[0494] Fig.12 This indicates that TD1-CD98hc exhibited increased serum clearance in NHPs compared to TD1 alone and isotype controls. Fig.12It was shown that CSF Cmax was increased in NHPs after administration of TD1-CD98hc compared to TD1 alone, with increases of approximately 13-fold at the 2-hour time point, approximately 10-fold at the 6-hour time point, and approximately 4-fold at the 24-hour time point following an initial 10 mg / kg dose, but attenuated after the 96-hour time point.

[0495] Example 28: Effects of the CD98hc Binding Arm on Brain Uptake and Brain Pharmacodynamics in Non-Human Primates

[0496] Brain sample preparation: Brain tissue was collected from antibody-treated cynomolgus monkeys at day 30 (i.e. 48 hours after the second antibody was administered) and subsequently frozen. Prior to this, the animals were anesthetized and subjected to cardiac perfusion with PBS to clear blood vessels. The brain tissue was then thawed, minced and homogenized in HBSS buffer (MilliporeSigma No. 55037C) containing 10mM HEPES (No. 15630130, Gibco) using a manual grinder. Subsequently, the sample was centrifuged at a speed of 1000g for 5 minutes to precipitate the container portion. The supernatant representing the substance was then transferred to a new tube and mixed with 10x RIPA buffer (final concentration 1x), which included a mixture of protease and phosphatase inhibitors (cOmplete TM , Mini Protease Inhibitor Cocktail, Roche, catalog number 11836153001). The samples were incubated on a rotary mixer at 4°C for 45 minutes, then frozen on dry ice and stored at 80°C until analysis. The vascular fraction in the myelin fragments was cleared by centrifugation with 18% dextran (70 kDa, No. 31390, Sigma-Aldrich), followed by washing 2 times in HBSS buffer. Subsequently, the samples were dissolved in RIPA buffer (R0278, Sigma-Aldrich) and stored at -80°C until analysis. We have previously confirmed the effectiveness of this method using Western blot analysis, in which brain endothelial cell markers (e.g., CD31 and Claudin-5) were absent in the parenchymal fraction (data not shown).

[0497] Pharmacokinetic (PK) analysis: Antibody concentration in brain samples was measured using MSD (Meso Scale Discovery) method. In brief, 50 μl / well goat anti-human IgG1 antibody was added to MSD plates with 1 μg / ml in PBS, and incubated overnight on a shaker at 500RPM at 4°C. The plate was then washed 3x with wash buffer (0.05% Tween 20 in 1xPBS), then incubated with blocking buffer (wash buffer containing 3% BSA) for 1 hour at room temperature. Subsequently, brain (diluted in PBS) samples were added to culture dishes in duplicate, and incubated 2 hours at room temperature, on a shaker at 500RPM. The plate was then washed 3x with wash buffer, then 0.5 μg / ml of Sulfo-Tag goat anti-human antibody (R32AJ-5, Meso Scale Discovery) in PBS was added with 40 μl / well, and incubated for 1 hour on a shaker at 500RPM. Finally, after washing 3x with washing buffer, 150 μl / well 1x reading buffer (R92PC-2, Meso Scale Discovery) was added to the plate, which was then read using a Sector Imager S600 instrument. Eleven 2-fold serial dilutions of each antibody (from 100-0.019 ng / ml) were used to generate a standard curve for each treatment. A four-parameter logistic regression fit curve was used to calculate the antibody concentration in the sample.

[0498] Fig.13 Results: Compared with TD1 alone, the CD98hc BBB-targeting arm increased brain uptake of TD1-CD98hc by up to 3-fold in the frontal cortex and up to 4-fold in the entorhinal cortex, whereas no significant increase was seen in the hippocampus.

[0499] Example 29: Pharmacodynamic Analysis of TD1-CD98hc Bispecific Antibody in NHP Samples

[0500] As described in Example 27, CSF from NHP test subjects was further tested for pharmacodynamic purposes, as described below.

[0501] CSF1R Method Summary (Brain Lysate)

[0502] Colony stimulating factor 1 receptor (CSF1R) concentrations in cynomolgus monkey brain lysates were measured using an enzyme-linked immunosorbent assay (ELISA) kit from R&D Systems (Cat. No. DY329). Mouse anti-human M-CSF R capture antibody (R&D Systems, Part No. 841246) was diluted to a working concentration of 4 μg / mL in 1x PBS (Corning; REF 21-040-CM) and coated on 96-well microplates (Nunc-Immuno Maxisorp; ThermoScientific Catalog No. 446612) at 100 μL / well and incubated overnight at room temperature without shaking. The next day, the plates were washed 3x 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 at the minimum dilution (MRD) in assay buffer (1x PBS, 1% BSA and 0.05% Tween-20). The plates were then washed and 100 μL / well of human M-CSF R standards, diluted QCs, and diluted samples in assay buffer were added to the appropriate wells and incubated for 2 hours at room temperature. The plates were washed and 100 μL / well of 100 ng / mL biotinylated goat anti-human M-CSF R detection antibody (R&D Systems, part number 841247) working solution in reagent diluent was added and incubated for 2 hours at room temperature. After the incubation was complete, the plates were washed and then Streptavidin-HRP (R&D Systems, part number 890803) diluted 1:200 in reagent diluent was added and incubated for 20 minutes at room temperature. The plate was finally washed sequentially and 100 μL / well substrate solution (R&D Systems, part number 895000 and 895001) was added and incubated at room temperature for 20 minutes. After the incubation was complete, 50 μL / well stop solution (R&D Systems, part number 895032) was added to the plate and mixed thoroughly. The plate was then read on a Molecular Devices SpectraMax M5 instrument and the raw optical density (OD) was generated from each well. 450-540 ) signal. The concentration of CSF1R in each unknown sample was determined by interpolation of the calibration standard curve. Standard regression was performed by SoftMax Pro (Molecular Devices, version 7.1) using a weighting factor of 1 / Y 2 The 4-parameter logistic (4-PL) model was used.

[0503] CSF1 Method Summary (CSF)

[0504] 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 number K151XRK-4). 96-well MSD GOLD small spot streptavidin plates (MSD, catalog number L45SA-1) were coated with 25 μL / well of biotinylated anti-human CSF1 antibody (MSD, catalog number C21XR-3) diluted 1:17.5 in diluent 100 (MSD, catalog number R50AA-4) and incubated at room temperature for 1 hour on a plate shaker set at 700 revolutions per minute (RPM).

[0505] Standards were prepared in Diluent 43 (MSD, Cat. No. R50AG-2) and further diluted 1:1 in assay buffer before loading onto the plate. QCs were prepared in Diluent 43 and diluted to the MRD in assay buffer. The diluted QCs were further diluted 1:1 with Diluent 43 and then loaded onto the plate. Test samples were diluted to the MRD in assay buffer. The diluted samples were further diluted 1:1 in Diluent 43 and then loaded onto the plate.

[0506] After washing, 50 μL of standards, QCs, 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 to 700 RPM. The plates were then washed and 50 μL of detection antibody (SULFO-TAG anti-human CSF1 antibody; MSD, catalog number D21XR-3) was diluted 1:100 in diluent 3 (MSD, catalog number R50AP-2), added to each well, and incubated for 1 hour at room temperature on a plate shaker set to 700 RPM. After the incubation was complete, the plates were washed and 150 μL / well MSD GOLD Read Buffer B (MSD, catalog number R60AM-2) was then added and the plates were read on an MSD Sector 600 imager. In the presence of read buffer, ruthenium produces a chemiluminescent signal when a voltage is applied. The signal intensity is proportional to the concentration of CSF1 present in the sample. The standard curve was measured using a weight of 1 / y. 2 CSF1 levels were quantified using a four-parameter logistic (4-PL) curve fitting equation. Data were analyzed using Microsoft Excel and GraphPad Prism 9.0.

[0507] sTREM2 Method Summary (Serum, CSF, and Brain Lysates)

[0508] Electrochemiluminescence (ECL) assays on the MSD platform were used to measure the concentration of myeloid cell triggering receptor 2 (TREM2) in cynomolgus monkey serum, cerebrospinal fluid (CSF) and brain lysates. 96-well standard MSD plates (catalog number L15XA) were coated with 50 μL / well capture antibody T2-8F11 (produced by Alector) and placed in 1X PBS (Corning; REF 21-040-CM) and left overnight on a plate oscillator set at 500 revolutions per minute (RPM) at 2-8°C. 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 advanced BSA in PBS). The plates were then washed and 50 μL / well of cynomolgus monkey TREM2-Fc standards (generated by Alector), QCs, and diluted samples in assay buffer (1x 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 binds to the immobilized T2-8F11 capture antibody. After washing away any unbound material, 50 μL / well of biotinylated goat anti-human TREM2 polyclonal antibody (R&D Systems, catalog number BAF1828) diluted to 100 ng / mL in assay buffer was added to the wells of the plate and incubated for 1 hour at RT on a shaker set at 500 RPM. After washing the plate, 50 μL of sulfonated tag streptavidin (MSD, catalog number R32AD-1) solution (0.2 μg / mL) in detection buffer was added and incubated at RT for 30 minutes on an oscillator set to 500 RPM. After the incubation was complete, the plate was washed and 150 μL / well 1x read buffer (prepared by diluting 4X read buffer T with surfactant (MSD, catalog number R92TC-1) with deionized water) was added, and the plate was read on an MSD Sector 600 imager. In the presence of read buffer, ruthenium produces a chemiluminescent signal when voltage is applied. The signal intensity is proportional to the concentration of TREM2 present in the sample. Standard regression was performed using a weighting factor of 1 / Y by SoftMax Pro (Molecular Devices, Version 7.1) 2 The 4-parameter logistic (4-PL) model was used.

[0509] Fig.14 It was shown 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. Fig.14Further, soluble TREM2 was not elevated in the CSF of NHPs treated with CD98hc compared with TD1 alone, suggesting that TD1-98hc is cleared from the CSF more rapidly than TD1 alone. Fig.15 Figure 2 illustrates CSF-1 levels in the CSF of NHPs treated with TD1-CD98hc or TD-1 alone. There was no sustained increase in CSF-1 levels in any of the groups.

Claims

1. An antigen binding domain that specifically binds to human CD98 heavy chain (CD98hc), wherein the antigen binding domain comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2 and CDR3 sequences, and the sequences comprise the following amino acid sequence: (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: 53-55 and 119-121, 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: 74-76 and 140-142, 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: 83-85 and 149-151, 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: 104-106 and 170-172, 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, 427 and 176-178, respectively; (1x) SEQ ID NOs: 421, 422, 112, 427 and 176-178, respectively; or (lxi) SEQ ID NOs: 421, 426, 112, 434, 177 and 178, respectively.

2. The antigen binding domain of claim 1, wherein the antigen binding domain comprises a VH and a VL, wherein the VH and VL comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of: (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:215 and 216, respectively. NO:216 and 217; (xxxvi) SEQ ID NO:218 and 219, respectively; (xxxvii) SEQ ID NO:220 and 221, respectively; (xxxviii) SEQ ID NO:222 and 223, respectively; (xxxix) SEQ ID NO:224 and 225, respectively; (xl) SEQ ID NO: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, wherein the antigen binding domain comprises VH and VL, wherein the VH comprises the following amino acid sequence: SEQ ID NO: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, 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, wherein the antigen binding domain comprises VH and VL, wherein the VL comprises the following amino acid sequence: SEQ ID NO:364,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,356,358,360,362,366,368,370,372,374,376,378,380,382,384,386,388,400,402 or 404.

5. The antigen binding domain of any one of claims 1 to 4, wherein the antigen binding domain comprises VH and VL, and the VH and VL comprise 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:215 and 216, respectively. NO:216 and 217; (xxxvi) SEQ ID NO:218 and 219, respectively; (xxxvii) SEQ ID NO:220 and 221, respectively; (xxxviii) SEQ ID NO:222 and 223, respectively; (xxxix) SEQ ID NO:224 and 225, respectively; (xl) SEQ ID NO: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, wherein the antigen binding domain is capable of crossing the blood-brain barrier (BBB).

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

8. The antigen binding domain of any one of claims 1 to 7, wherein the antigen binding domain binds to human CD98hc with an affinity between 50 nM and 500 nM.

9. The antigen binding domain of any one of claims 1 to 8, wherein the antigen binding domain binds to human CD98hc with an affinity between 1 nM and 50 nM.

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

11. The antigen binding domain of any one of claims 1 to 10, wherein the antigen binding domain has an ELISA OD of at least 0.

45. 450 Binds to human CD98hc and / or has an ELISA OD of at least 0.45 450 Binds to cynomolgus monkey CD98hc.

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, optionally wherein the blood-brain barrier epithelial cells are HCMEC / D3 cells.

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

14. The antigen binding domain of any one of claims 1 to 13, wherein the antigen binding domain binds to cynomolgus monkey CD98hc with an affinity of 3.2 nM to 1.5 μM.

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, wherein the antigen binding domain does not reduce the cell surface expression of CD98hc on HCMEC / D3 cells by more than 20% relative to the 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, wherein the antigen binding domain does not increase the cell surface expression of CD98hc on HCMEC / D3 cells by more than 50% relative to the 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, wherein the antigen binding domain accumulates at least 1.5-fold or at least 2-fold more in the brain of vascular-depleted mice than an isotype control.

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

20. An antigen binding domain as described in any one of claims 1 to 19, wherein the antigen binding domain comprises a VH and a VL, and the VH and VL comprise the following amino acid sequences: 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. 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, wherein the antigen binding domain comprises VH and VL, and the VH and VL comprise the amino acid sequences of SEQ ID NOs: 14 and 15, respectively.

22. The antigen binding domain of any one of claims 1 to 21, wherein the antigen binding domain comprises VH and VL on a single polypeptide chain.

23. The antigen binding domain of any one of claims 1 to 22, wherein the antigen binding domain comprises a single chain variable fragment (scFv).

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

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

26. The antigen binding domain of claim 24 or 25, wherein 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.

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 GGGGSGGGGSGGGGSGGGGS (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: 318-336.

29. The antigen binding domain of any one of claims 1 to 21, wherein the antigen binding domain comprises a VH on a first polypeptide and a VL on a second polypeptide.

30. The antigen binding domain of any one of claims 1 to 29, wherein the antigen binding domain is a murine, chimeric, humanized or human antigen binding domain, optionally wherein the antigen binding domain is a humanized antigen binding domain.

31. An antigen binding domain that specifically binds to human CD98hc, wherein the antigen binding domain is a VHH, the VHH comprising (i) the VH CDR1, VHCDR2 and VH CDR3 of the antigen binding domain of any one of claims 1 to 19 and 27 or (ii) the VH of the antigen binding domain of any one of claims 1 to 19 and 27, optionally wherein the VHH is 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 fusion protein of claim 32, wherein the heterologous protein or peptide comprises the following amino acid sequence: β-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, glucocerebrosidase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), glycoprotein non-metastatic melanoma protein B (GPNMB), gamma-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), caspase 6, sortin (SORT), triggering receptor 2 on myeloid cells (TREM2), CD33 or sialic acid-binding Ig-like lectin 3 (Siglec3), sialic acid-binding Ig-like lectin 5 (Siglec5), sialic acid-binding Ig-like lectin 7 (Siglec7), sialic acid-binding Ig-like lectin 9 (Siglec9), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4 domain A4A (MS4A4A), transmembrane 4 domain A 6A (MS4A6A) or transmembrane protein 106B (TMEM106b), clusterin (APOJ), reelin, ubiquitin protein ligase E3A (UBE3A), tripeptidyl peptidase 1 (CLN2 / TPP1), α-L-iduronidase (IDUA), iduronate 2-sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-α-glucosamine N-acetyltransferase (HGSNAT) and N-acetyl-α-glucosaminidase (NAGLU), N-sulfoglucosamine sulfohydrolase (SGSH) or parts thereof.

34. An antibody comprising the antigen binding domain of any one of claims 1 to 31.

35. An antibody or antigen-binding fragment thereof that binds to the same epitope of human CD98hc 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. A multispecific protein comprising a first antigen binding domain, which is the antigen binding domain of any one of claims 1 to 31 connected to a second antigen binding domain, optionally wherein the second antigen binding domain specifically binds to a CNS antigen.

38. A multispecific protein comprising an antigen binding domain as described in 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. The multispecific protein of claim 38, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region.

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

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

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

43. The multispecific protein of claim 42, wherein the multispecific protein is bivalent.

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

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

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

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

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

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

51. The multispecific protein of any one of claims 46 to 50, wherein the amino acid linker is a glycine-serine linker, optionally wherein the glycine-serine linker comprises the amino acid sequence (GGGGS) x 3 (SEQ ID NO: 183).

52. The multispecific protein of any one of claims 46 to 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. The multispecific protein of any one of claims 38 to 54, wherein the antibody or antigen-binding fragment thereof comprises a mutation that reduces effector function, optionally wherein the mutation that reduces effector function comprises (i) L234A, L235A and / or P331S, and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S.

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, 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.

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 a hole mutation and a mutation that reduces effector function, optionally wherein the mutation that reduces effector function comprises (i) L234A, L235A and / or P331S, and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S.

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. 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. The multispecific protein of any one of claims 37 to 59, wherein the multispecific protein has an equilibrium dissociation constant (K) of about 3 nM to about 225 nM. D ) binds to human CD98hc and / or with a K of about 3 nM to about 225 nM D Binds to cynomolgus monkey CD98hc.

61. The multispecific protein of any one of claims 37 to 60, wherein internalization of the multispecific protein in blood-brain barrier epithelial cells is greater than 10-fold compared to internalization of an isotype control, optionally wherein the blood-brain barrier epithelial cells are HCMEC / D3 cells.

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

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

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

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

66. The multispecific protein of claims 37 to 65, wherein the CNS antigen is β-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), prosaposin (PSAP), γ-secretase, death receptor 6 (DR6), amyloid precursor protein (A PP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor 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), sialic acid-binding Ig-like lectin 11 (Siglec11), glycoprotein non-metastatic melanoma protein B (GPNMB), paired immunoglobulin-like type 2 receptor alpha (PILRA), transmembrane 4 domain A4A (MS4A4A), transmembrane 4 domain A 6A (MS4A6A), MS4A4E, transmembrane protein 106B (TMEM106b), ubiquitin-protein ligase E3A (UBE3A), CR1, ABCA1, ABCA7, HLA-DR1, HLA-DR5, IL1RAP, TREML2, IL-34, SORL1, or ADAM1.

67. The multispecific protein of claim 66, wherein the CNS antigen is MS4A4A, optionally wherein (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 amino acid 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. The multispecific protein of claim 67, wherein the multispecific protein comprises the amino acid sequence of SEQ ID NOs: 405-410.

69. The fusion protein of claim 32 or 33, the antibody or antigen-binding fragment thereof 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. The fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein of any one of claims 32 to 69, wherein the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein is linked to an imaging agent.

71. A composition comprising a first polynucleotide, a second polynucleotide and a third polynucleotide, wherein the first polynucleotide, the second polynucleotide and the third polynucleotide encode a multi-specific protein as described in any one of claims 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. A composition comprising a first polynucleotide, a second polynucleotide and a third polynucleotide, wherein the first polynucleotide, the second polynucleotide and the third polynucleotide encode a multispecific protein as described in 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, optionally wherein the first antigen-binding domain and the second antigen-binding domain that bind to human CD98hc comprise the same amino acid sequence.

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

6.

75. 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. A composition comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide encode a multi-specific protein as described in any one of claims 37 to 44 and 47 to 70, wherein the first polynucleotide encodes a heavy chain and the antigen binding domain that specifically binds to human CD98hc, and wherein the 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 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 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. 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 the 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. A host cell comprising the polynucleotide of claim 78 or 79 or the vector of claim 80 or 81.

83. The host cell of claim 77 or 82, wherein the host cell is selected from the group consisting of: Escherichia coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NSO, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture.

84. A method for producing an antigen binding domain or a multi-specific protein, comprising culturing a host cell as described in any one of claims 77, 82 and 83, thereby producing the antigen binding domain or the multi-specific protein, optionally wherein the method also comprises separating the antigen binding domain or the multi-specific protein from the culture.

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

86. A pharmaceutical composition comprising (i) an antigen binding domain, fusion protein, antibody or antigen binding fragment thereof, or multispecific protein as described in any one of claims 1 to 70, and (ii) a pharmaceutically acceptable carrier.

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

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

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

91. The method of claim 89 or 90, wherein administration increases delivery of the fusion protein, antibody or antigen-binding fragment thereof, multispecific protein, or pharmaceutical composition to the frontal cortex, entorhinal cortex, and / or hippocampus.

92. The method of any one of claims 89 to 91, wherein the nervous system disease or disorder is selected from a neuropathy disorder, a neurodegenerative disease, cancer, an eye disease disorder, epilepsy, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation.

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's syndrome, Liddle's syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, encephalitis, traumatic brain injury, and marginal dominant age-associated TDP-43 encephalopathy (LATE).

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

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

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

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

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

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

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

103. A method of transporting a fusion protein, an antibody or its antigen-binding fragment, or a multi-specific protein across a subject's BBB, comprising administering to the subject a fusion protein, an antibody or its antigen-binding fragment, a multi-specific protein, or a pharmaceutical composition as described in any one of claims 32 to 70, 86 and 87.

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 following administration compared to an isotype control.

105. The method of claim 103 or 104, wherein the concentration of the fusion protein, antibody or antigen-binding fragment thereof, multi-specific 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. 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 a greater than 10% decrease in reticulocyte count in the subject compared to administration of an isotype control.

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. 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 as described in 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. A method of 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 localizing the imaging agent in the subject.

110. An in vitro method for detecting a CNS antigen, comprising contacting an in vitro sample with the fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein of claim 70, and localizing the imaging agent within the sample.

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

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

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