CD98HC antigen-binding domains and uses therefor

Abstract

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

Description

CD98HC ANTIGEN-BINDING DOMAINS AND USES THEREFOR CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application Nos. 63 / 627,641, filed January 31, 2024; 63 / 660,916, filed June 17, 2024; 63 / 660,918, filed June 17, 2024; 63 / 681,496, filed August 9, 2024; 63 / 681,568, filed August 9, 2024; and 63 / 744,021, filed January 10, 2025, each of which is herein incorporated by reference in its entirety. REFERENCE TO SEQUENCE LISTING SUMBITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing (Name: 4503_028PC06_SequenceListing_ST26.xml; Size: 174,405 bytes; and Date of Creation: January 27, 2025) is herein incorporated by reference in its entirety. FIELD OF THE PRESENT DISCLOSURE

[0003] 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

[0004] Passive transfer of substances from blood to brain is restricted by the blood brain barrier (BBB). The BBB provides precise control of central nervous system (CNS) homeostasis allowing for proper neuronal function and also protecting neural tissue from toxins and pathogens. Alterations of the BBB are an important component of pathology and progression of different neurological diseases. However, the BBB poses a problem with regard to delivering therapeutics to the CNS. While recombinant proteins and antibody therapeutics have shown much success outside the CNS, such biologics do not cross the BBB efficiently. As a result, delivery of some therapeutics to the CNS has relied on injection of the therapeutic directly into the CNS. However, such injections are invasive procedures that have efficacy that is limited by the rapid export of cerebral spinal fluid (CSF) containing the therapeutic from the brain to the blood. Alternatively, atherapeutic intended for the CNS may be administered systemically at a high dose to allow for sufficient penetration of the BBB by the therapeutic. However, this approach may result in unintended effects due to the high dose in the periphery or increased manufacturing and formulation burdens to achieve the high dose. Accordingly, improved products and methods for delivering therapeutics across the BBB are needed. SUMMARY OF THE PRESENT DISCLOSURE

[0005] Provided herein are antigen-binding domains, complexes, fusion proteins, antibodies, and multi-specific proteins that specifically bind to human CD98 heavy chain (CD98hc), and methods of making and using the same.

[0006] Provided herein are antigen-binding domains that specifically bind 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 comprising the amino acid sequences of: SEQ ID NOs: 70, 71, 12, 16, 17 and 18, respectively; SEQ ID NOs: 70, 31, 12, 16, 17, and 18, respectively; SEQ ID NOs: 70, 33, 12, 16, 17, and 18, respectively; SEQ ID NOs: 70, 34, 12, 16, 17 and 18, respectively; or SEQ ID NOs:70, 35, 12, 16, 17 and 18, respectively.

[0007] Provided herein are antigen-binding domains that specifically bind 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 comprising the amino acid sequences of: SEQ ID NOs: 70, 7412, 16, 17, and 18, respectively; SEQ ID NOs: 70, 75, 12, 16, 17, and 18, respectively; SEQ ID NOs: 70, 7612, 16, 17, and 18, respectively; SEQ ID NOs: 70, 77, 12, 16, 17, and 18, respectively; SEQ ID NOs: 70, 78, 12, 16, 17, and 18, respectively; SEQ ID NOs:70, 79, 12, 16, 17, and 18, respectively; SEQ ID NOs:70, 80, 12, 16, 17, and 18, respectively;SEQ ID NOs:70, 31, 12, 91, 17, and 18, respectively; SEQ ID NOs:70, 31, 12, 92, 17, and 18, respectively; SEQ ID NOs:70, 31, 12, 93, 17, and 18, respectively; SEQ ID NOs:70, 31, 12, 94, 17, and 18, respectively; SEQ ID NOs:70, 31, 12, 95, 17, and 18, respectively; SEQ ID NOs:70, 31, 12, 96, 17, and 18, respectively; SEQ ID NOs:70, 31, 12, 97, 17, and 18, respectively; SEQ ID NOs:70, 76, 12, 93, 17, and 18, respectively; SEQ ID NOs:70, 90, 12, 16, 17, and 18, respectively; SEQ ID NOs:70, 31, 81, 16, 17, and 18, respectively; SEQ ID NOs:70, 31, 82, 16, 17, and 18, respectively; SEQ ID NOs:70, 31, 83, 16, 17, and 18, respectively; SEQ ID NOs:70, 31, 84, 16, 17, and 18, respectively SEQ ID NOs:70, 31, 85, 16, 17, and 18, respectively; SEQ ID NOs:70, 31, 86, 16, 17, and 18, respectively; SEQ ID NOs:70, 31, 87, 16, 17, and 18, respectively; SEQ ID NOs:70, 31, 88, 16, 17, and 18, respectively; SEQ ID NOs:70, 31, 89, 16, 17, and 18, respectively; SEQ ID NOs:70, 31, 12, 98, 17, and 18, respectively; SEQ ID NOs:70, 31, 12, 99, 17, and 18, respectively; SEQ ID NOs:70, 31, 12, 100, 17, and 18, respectively; SEQ ID NOs:70, 31, 12, 101, 17, and 18, respectively; SEQ ID NOs:70, 31, 12, 102, 17, and 18, respectively; SEQ ID NOs:70, 31, 12, 103, 17, and 18, respectively; SEQ ID NOs:70, 31, 12, 16, 17, and 104, respectively; SEQ ID NOs:70, 31, 12, 16, 17, and 105, respectively; SEQ ID NOs:70, 31, 12, 16, 17, and 106, respectively; SEQ ID NOs:70, 31, 12, 16, 17, and 107, respectively; SEQ ID NOs:70, 31, 12, 16, 17, and 108, respectively; SEQ ID NOs:70, 31, 12, 16, 17, and 109, respectively; SEQ ID NOs:70, 31, 12, 16, 17, and 110, respectively; SEQ ID NOs:70, 31, 12, 16, 17, and 111, respectively; SEQ ID NOs:70, 31, 12, 16, 17, and 112, respectively;SEQ ID NOs:70, 31, 12, 16, 17, and 113, respectively; SEQ ID NOs:73, 31, 12, 16, 17, and 18, respectively; or SEQ ID NOs:73, 31, 12, 16, 17, and 113, respectively.

[0008] In some aspects, the antigen-binding domain comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequences of: SEQ ID NOs: 5 and 6, respectively; SEQ ID NOs: 22 and 23, respectively; SEQ ID NOs: 24 and 23, respectively; SEQ ID NOs: 26 and 23, respectively; or SEQ ID NOs: 28 and 23, respectively.

[0009] In some aspects, the antigen-binding domain comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequences of: SEQ ID NOs: 114 and 23, respectively; SEQ ID NOs: 115 and 23, respectively; SEQ ID NOs: 116 and 23, respectively; SEQ ID NOs: 117 and 23, respectively; SEQ ID NOs: 118 and 23, respectively; SEQ ID NOs:119 and 23, respectively; SEQ ID NOs:120 and 23, respectively; SEQ ID NOs:22 and 121, respectively; SEQ ID NOs:22 and 122, respectively; SEQ ID NOs:22 and 123, respectively; SEQ ID NOs:22 and 124, respectively; SEQ ID NOs:22 and 125, respectively; SEQ ID NOs:22 and 126, respectively; SEQ ID NOs:22 and 127, respectively; SEQ ID NOs:116 and 123, respectively; SEQ ID NOs:128 and 23, respectively; SEQ ID NOs:129 and 23, respectively; SEQ ID NOs:130 and 23, respectively; SEQ ID NOs:131 and 23, respectively;SEQ ID NOs:132 and 23, respectively; SEQ ID NOs:133 and 23, respectively; SEQ ID NOs:134 and 23, respectively; SEQ ID NOs:135 and 23, respectively; SEQ ID NOs:136 and 23, respectively; SEQ ID NOs:137 and 23, respectively; SEQ ID NOs:22 and 138, respectively; SEQ ID NOs:22 and 139, respectively; SEQ ID NOs:22 and 140, respectively; SEQ ID NOs:22 and 141, respectively; SEQ ID NOs:22 and 142, respectively; SEQ ID NOs:22 and 143, respectively; SEQ ID NOs:22 and 144, respectively; SEQ ID NOs:22 and 145, respectively; SEQ ID NOs:22 and 146, respectively; SEQ ID NOs:22 and 147, respectively; SEQ ID NOs:22 and 148, respectively; SEQ ID NOs:22 and 149, respectively; SEQ ID NOs:22 and 150, respectively; SEQ ID NOs:22 and 151, respectively; SEQ ID NOs:22 and 152, respectively; SEQ ID NOs:22 and 153, respectively; SEQ ID NOs:154 and 6, respectively; or SEQ ID NOs:154 and 153, respectively.

[0010] In some aspects, provided herein is an antigen-binding domain that specifically binds to human CD98hc, wherein the antigen-binding domain comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 5, 22, 24, 26 or 28.

[0011] In some aspects, provided herein is an antigen-binding domain that specifically binds to human CD98hc, wherein the antigen-binding domain comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 114, 115, 116, 117, 118, 119, 120, 22, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, or 154.

[0012] In some aspects, provided herein is an antigen-binding domain that specifically binds to human CD98hc, wherein the antigen-binding domain comprises a VH and a VL, wherein the VL comprises the amino acid sequence of SEQ ID NO: 6 or 23.

[0013] In some aspects, provided herein is an antigen-binding domain that specifically binds to human CD98hc, wherein the antigen-binding domain comprises a VH and a VL, wherein the VL comprises the amino acid sequence of SEQ ID NO: 121, 122, 123, 124, 125, 126, 127, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, or 153.

[0014] In some aspects, the antigen-binding domain comprises a VH and VL comprising the amino acid sequences of: SEQ ID NOs: 5 and 6, respectively; SEQ ID NOs: 22 and 23, respectively; SEQ ID NOs: 24 and 23, respectively; SEQ ID NOs: 26 and 23, respectively; or SEQ ID NOs: 28 and 23, respectively.

[0015] In some aspects, the antigen-binding domain comprises a VH and VL comprising the amino acid sequences of: SEQ ID NOs: 114 and 23, respectively; SEQ ID NOs: 115 and 23, respectively; SEQ ID NOs: 116 and 23, respectively; SEQ ID NOs: 117 and 23, respectively; SEQ ID NOs: 118 and 23, respectively; SEQ ID NOs:119 and 23, respectively; SEQ ID NOs:120 and 23, respectively; SEQ ID NOs:22 and 121, respectively; SEQ ID NOs:22 and 122, respectively; SEQ ID NOs:22 and 123, respectively; SEQ ID NOs:22 and 124, respectively; SEQ ID NOs:22 and 125, respectively; SEQ ID NOs:22 and 126, respectively; SEQ ID NOs:22 and 127, respectively; SEQ ID NOs:116 and 123, respectively; SEQ ID NOs:128 and 23, respectively; SEQ ID NOs:129 and 23, respectively;SEQ ID NOs:130 and 23, respectively; SEQ ID NOs:131 and 23, respectively; SEQ ID NOs:132 and 23, respectively; SEQ ID NOs:133 and 23, respectively; SEQ ID NOs:134 and 23, respectively; SEQ ID NOs:135 and 23, respectively; SEQ ID NOs:136 and 23, respectively; SEQ ID NOs:137 and 23, respectively; SEQ ID NOs:22 and 138, respectively; SEQ ID NOs:22 and 139, respectively; SEQ ID NOs:22 and 140, respectively; SEQ ID NOs:22 and 141, respectively; SEQ ID NOs:22 and 142, respectively; SEQ ID NOs:22 and 143, respectively; SEQ ID NOs:22 and 144, respectively; SEQ ID NOs:22 and 145, respectively; SEQ ID NOs:22 and 146, respectively; SEQ ID NOs:22 and 147, respectively; SEQ ID NOs:22 and 148, respectively; SEQ ID NOs:22 and 149, respectively; SEQ ID NOs:22 and 150, respectively; SEQ ID NOs:22 and 151, respectively; SEQ ID NOs:22 and 152, respectively; SEQ ID NOs:22 and 153, respectively; SEQ ID NOs:154 and 6, respectively; or SEQ ID NOs:154 and 153, respectively.

[0016] In some aspects, the antigen-binding domain is capable of crossing the blood brain barrier (BBB).

[0017] In some aspects, the antigen-binding domain binds human CD98hc with an affinity of 10 nM to 100 nM.

[0018] In some aspects, inding domain binds human CD98hc with an affinity of 10 nM to 500 nM.

[0019] In some aspects, inding domain binds human CD98hc with an affinity of 10 nM to 1000 nM.

[0020] In some aspects, the antigen-binding domain binds to cynomolgus monkey CD98hc.

[0021] In some aspects, the antigen-binding domain binds cynomolgus monkey CD98hc with an affinity of 10 nM to 1000 nM. In some aspects, the affinity is measured by high throughput surface plasmon resonance (SPR) detection.

[0022] In some aspects, the antigen-binding domain binds cynomolgus monkey CD98hc with an affinity of 10 nM to 500 nM. In some aspects, the affinity is measured by high throughput surface plasmon resonance (SPR) detection.

[0023] In some aspects, the antigen-binding domain binds cynomolgus monkey CD98hc with an affinity of 10 nM to 100 nM. In some aspects, the affinity is measured by high throughput surface plasmon resonance (SPR) detection.

[0024] 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.

[0025] In some aspects, the antigen-binding domain is internalized in blood-brain barrier epithelial cells. In some aspects, the blood-brain barrier epithelial cells are HCMEC / D3 cells.

[0026] In some aspects, the antigen-binding domain does not reduce cell-surface expression of CD98hc on HCMEC / D3 cells by more than 20% relative to cell-surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control.

[0027] In some aspects, the antigen-binding domain does not increase cell-surface expression of CD98hc on HCMEC / D3 cells by more than 50% relative to cell-surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control.

[0028] In some aspects, the antigen-binding domain accumulates at least 3-fold more than an isotype control in vessel-depleted mouse brain.

[0029] In some aspects, the antigen-binding domain accumulates at least 5-fold more than an isotype control in vessel-depleted mouse brain.

[0030] In some aspects, the antigen-binding domain accumulates at least 10-fold or at least 20- fold more than an isotype control in vessel-depleted mouse brain.

[0031] In some aspects, the antigen-binding domain has at least a 5-fold increase in brain concentration ratio over an isotype control 24 hours after administration to a mouse.

[0032] In some aspects, the antigen-binding domain comprises a VH and a VL on a single polypeptide chain.

[0033] In some aspects, the antigen-binding domain comprises a single-chain fragment variable (scFv).

[0034] In some aspects, the scFv is in the orientation VH-linker-VL.

[0035] In some aspects, the scFv is in the orientation VL-linker-VH.

[0036] In some aspects, the linker is about 5 to about 25 amino acids.

[0037] In some aspects, the linker is about 10 to about 25 amino acids.

[0038] In some aspects, the linker is about 15 to about 25 amino acids.

[0039] In some aspects, the linker comprises the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 48) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 49).

[0040] In some aspects, the scFv comprises the amino acid sequence of any one of SEQ ID NOs: 21, 39, 40, 41 or 42.

[0041] In some aspects, the antigen-binding domain comprises a VH on a first polypeptide and a VL on a second polypeptide.

[0042] In some aspects, the antigen-binding domain is a murine, chimeric, humanized, or human antigen-binding domain. In some aspects, the antigen-binding domain is a humanized antigen-binding domain.

[0043] 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) the VH CDR1, VH CDR2, and VH CDR3 of any of the antigen-binding domains disclosed herein or (ii) the VH of any of the antigen-binding domains disclosed herein. In some aspects, the VHH is capable of crossing the blood brain barrier (BBB).

[0044] In some aspects, provided herein is a complex or fusion protein comprising any of the antigen-binding domains disclosed herein and a heterologous protein or peptide.

[0045] In some aspects, the heterologous protein or peptide comprises the amino acid sequence of beta-secretase 1 (BACE1), amyloid beta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), 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 expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4- Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), or Transmembrane Protein 106B (TMEM106b), clusterin (APOJ), Reelin, ubiquitin protein ligase E3A (UBE3A), Tripeptidyl Peptidase 1 (CLN2 / TPP1), Alpha-L-Iduronidase (IDUA), Iduronate 2-Sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N- acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), N- sulfoglucosamine sulfohydrolase (SGSH), or a portion thereof.

[0046] In some aspects, the heterologous protein or peptide comprises the amino acid sequence of beta-secretase 1 (BACE1), amyloid beta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), 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 expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4- Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), or Transmembrane Protein 106B (TMEM106b), clusterin (APOJ), Reelin, very low density lipoprotein receptor (VLDLR), apolipoprotein E receptor 2 (APOER2; also known as low- density lipoprotein receptor-related protein 8 (LRP8)), ubiquitin protein ligase E3A (UBE3A), Tripeptidyl Peptidase 1 (CLN2 / TPP1), Alpha-L-Iduronidase (IDUA), Iduronate 2-Sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N- acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), N- sulfoglucosamine sulfohydrolase (SGSH), or a portion thereof.

[0047] In some aspects, the heterologous protein or peptide comprises the amino acid sequence of β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), clusterin (APOJ), Reelin, Tripeptidyl Peptidase 1 (CLN2 / TPP1), or Alpha-L-Iduronidase (IDUA), Iduronate 2-Sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha- glucosaminide N-acetyltransferase (HGSNAT), N-acetyl-alpha-glucosaminidase (NAGLU), N- sulfoglucosamine sulfohydrolase (SGSH), or ubiquitin protein ligase E3A (UBE3A) or a variantor portion thereof or (b) specifically binds to beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, 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 expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), or Transmembrane Protein 106B (TMEM106b).

[0048] In some aspects, provided herein is an antibody comprising any of the antigen-binding domains disclosed herein.

[0049] In some aspects, provided herein is an antibody or antigen-binding fragment thereof that binds to the same human CD98hc epitope as any of the antigen-binding domains disclosed herein.

[0050] In some aspects, provided herein is an antibody or antigen-binding fragment thereof that competitively inhibits binding of any of the antigen-binding domains disclosed herein to human CD98hc.

[0051] In some aspects, provided herein is multi-specific protein comprising a first antigen- binding domain that is any of the antigen-binding domains disclosed herein to a second antigen- binding domain. In some aspects, the second antigen-binding domain specifically binds to a CNS antigen.

[0052] In some aspects, provided herein is a multi-specific protein comprising a first antigen- binding domain that is disclosed herein linked to an antibody or antigen-binding domain that binds to a cancer antigen.

[0053] In some aspects, the antibody or antigen-binding domain that binds to a cancer antigen is selected from the group consisting of rituximab, cetuximab, trastuzumab, pertuzumab, bevacizumab, nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab.

[0054] In some aspects, the antibody or antigen-binding domain that binds to a cancer antigen is selected from the group consisting of a version of rituximab comprising reduced effector function, a version of cetuximab comprising reduced effector function, a version of trastuzumab comprising reduced effector function, a version of pertuzumab comprising reduced effectorfunction, a version of bevacizumab comprising reduced effector function, a version of nivolumab comprising reduced effector function, a version of pembrolizumab comprising reduced effector function, a verison of atezolizumab comprising reduced effector function, a verison of avelumab comprising reduced effector function, and a verison of durvalumab comprising reduced effector function.

[0055] In some aspects, the reduced effector function comprises a mutation of LALA-P331S and / or LALA-P329S.

[0056] In some aspects, the antibody or antigen-binding domain that binds to a cancer antigen is a verison of rituximab comprising reduced effector function.

[0057] In some aspects, the antibody or antigen-binding domain that binds to a cancer antigen is rituximab.

[0058] In some aspects, the multi-specific protein comprises the amino acid sequences of SEQ ID NOs:161-163.

[0059] In some aspects, provided herein is a multi-specific protein comprising any of the antigen-binding domains disclosed herein linked to an antibody or antigen-binding fragment thereof. In some aspects, the antibody or antigen-binding fragment thereof specifically binds to a CNS antigen.

[0060] In some aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region.

[0061] In some aspects, any of the antigen-binding domains disclosed herein is linked, optionally via an amino acid linker, to the C-terminus of the heavy chain constant region.

[0062] In some aspects, the multi-specific protein is bispecific.

[0063] In some aspects, the multi-specific protein is bivalent, trivalent, or tetravalent.

[0064] In some aspects, the multi-specific protein is bivalent.

[0065] In some aspects, the multi-specific protein is trivalent. In somem aspects, the trivalent protein comprises one of the antigen-binding domain that binds to human CD98hc and two antigen-binding domains that bind to a CNS antigen.

[0066] In some aspects, the multi-specific protein is tetravalent. In some aspects, 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.

[0067] In some aspects, provided herein is a multi-specific protein that is trivalent and bi- specific and comprises any of the antigen-binding domains disclosed herein linked to an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two lightchains, and wherein the antigen-binding domain is an scFv linked, optionally via an amino acid linker, to the C-terminus of one of the two antibody heavy chains.

[0068] In some aspects, provided herein is a multi-specific protein that is tetravalent and bi- specific and comprises two of any of the antigen-binding domains disclosed herein, and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, wherein each of the two antigen-binding domains is an scFv, Fab, or VHH, wherein one of the two antigen-binding domains is linked, optionally via an amino acid linker, to the C- terminus of one of the antibody heavy chains, and wherein the other antigen-binding domain is linked, optionally via an amino acid linker, to the C-terminus of the other antibody heavy chain.

[0069] In some aspects, the antibody or antigen-binding fragment thereof comprises a constant region comprising a knob mutation and a constant region comprising a hole mutation.

[0070] In some aspects, the antigen-binding domain is linked, optionally via an amino acid linker, to the constant region comprising a hole mutation.

[0071] In some aspects, the antigen-binding domain is linked, optionally via an amino acid linker, to the constant region comprising a knob mutation.

[0072] In some aspects, the amino acid linker is a glycine-serine linker. In some aspects, the glycine-serine linker comprises the amino acid sequence (GGGGS)x3 (SEQ ID NO: 50).

[0073] In some aspects, the amino acid linker is a glycine-serine linker. In some aspets, the glycine-serine linker comprises the amino acid sequence (GGSGG)x3 (SEQ ID NO: 51).

[0074] In some aspects, the CNS antigen is a brain antigen.

[0075] In some aspects, the CNS antigen is not CD98hc.

[0076] In some aspects, the antibody or antigen-binding fragment thereof comprises a mutation that reduces effector function. In some aspects, 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.

[0077] In some aspects, the antibody or antigen-binding fragment thereof comprises a constant region comprising a knob mutation and a mutation that reduces effector function. In some aspects, 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.

[0078] In some aspects, the antibody or antigen-binding fragment thereof comprises a constant region comprising a hole mutation and a mutation that reduces effector function. In some aspects, 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.

[0079] In some aspects, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

[0080] 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.

[0081] In some aspects, the multi-specific 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 KDof about 3 nM to about 225 nM.

[0082] In some aspects, the multi-specific protein binds cynomolgus monkey CD98hc with a KDof about 27 nM to about 1430 nM. In some aspects, the multi-specific protein binds cynomolgus monkey CD98hc with a KDof about 28 nM to about 651 nM. In some aspects, the multi-specific protein binds cynomolgus monkey CD98hc with a KD of about 53 nM to about 859 nM.

[0083] In some aspects, the multi-specific protein is internalized in blood-brain barrier epithelial cells greater than 10-fold as compared to internalization by an isotype control. In some aspects, the blood-brain barrier epithelial cells are HCMEC / D3 cells.

[0084] In some aspects, the multi-specific protein does not reduce cell-surface expression of CD98hc on HCMEC / D3 cells by more than 20% relative to cell-surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control.

[0085] In some aspects, the multi-specific protein does not increase cell-surface expression of CD98hc on HCMEC / D3 cells by more than 50% relative to cell-surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control.

[0086] In some aspects, the multi-specific protein accumulates at least 3-fold, at least 5-fold, at least 10-fold, or at least 20-fold more than an isotype control in vessel-depleted mouse brain.

[0087] In some aspects, the multi-specific protein has at least a 5-fold increase in brain concentration ratio over an isotype control 24 hours after administration to a mouse.

[0088] In some aspects, the CNS antigen is beta-secretase 1 (BACE1), amyloid beta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophinreceptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), sialic acid binding Ig-like lectin 11 (Siglec11), glycoprotein nonmetastatic melanoma protein B (GPNMB), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains 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.

[0089] In some aspects, the CNS antigen is beta-secretase 1 (BACE1), amyloid beta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), sialic acid binding Ig-like lectin 11 (Siglec11), glycoprotein nonmetastatic melanoma protein B (GPNMB), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains 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, reelin, very low density lipoprotein receptor (VLDLR), apolipoprotein E receptor 2 (APOER2; also known as low-density lipoprotein receptor-related protein 8 (LRP8)), or ADAM1.

[0090] In some aspects, the CNS antigen is beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid bindingIg-like lectin 9 (Siglec9), sialic acid binding Ig-like lectin 11 (Siglec11), glycoprotein nonmetastatic melanoma protein B (GPNMB), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), MS4A4E, Transmembrane Protein 106B (TMEM106b), CR1, ABCA1, ABCA7, HLA-DR1, HLA-DR5, IL1RAP, TREML2, IL-34, SORL1, or ADAM1In some aspects, any of the complexes or fusion proteins, antibodies or antigen-binding fragments thereof, or the multi- specific proteins disclosed herein is capable of crossing the BBB.

[0091] In some aspects, the complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein is linked to an imaging agent.

[0092] 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 any of the multi-specific proteins disclosed herein, 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.

[0093] 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 any of the multi-specific proteins disclosed 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.

[0094] In some aspects, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation.

[0095] In some aspects, the ratio of the first, second, and third polynucleotides is about 1:3:6.

[0096] In some aspects, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0097] In some aspects, provided herein is a composition comprising a first polynucleotide and a second polynucleotide, wherein the first and second polynucleotides encode any of the multi- specific proteins disclosed herein, wherein the first polynucleotide encodes a heavy chain and the antigen-binding domain that bind to human CD98hc, and wherein the second polynucleotide encodes a light chain.

[0098] In some aspects, provided herein is a host cell comprising any of the compositions disclosed herein.

[0099] In some aspects, provided herein is an isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain of any of the antigen-binding domains disclosed herein.

[0100] In some aspects, provided herein is an isolated polynucleotide comprising a nucleic acid molecule encoding the light chain variable region of any of the antigen-binding domains disclosed herein.

[0101] In some aspects, provided herein is an isolated vector comprising any of the polynucleotides disclosed herein.

[0102] In some aspects, provided herein is an isolated vector comprising a nucleic acid molecule encoding the heavy chain variable region of any of the antigen-binding domains disclosed herein and a nucleic acid molecule encoding the light chain variable region of the antigen-binding domain.

[0103] In some aspects, provided herein is a host cell comprising any of the polynucleotides disclosed herein or any of the vectors disclosed herein.

[0104] In some aspects, the host cell is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, BMT10 cell, plant cell, insect cell, and human cell in tissue culture.

[0105] In some aspects, provided herein is a method of producing an antigen-binding domain or multi-specific protein comprising culturing any of the host cells disclosed herein so that the antigen-binding domain or multi-specific protein is produced. In some aspects, the method further comprises isolating the antigen-binding domain or multi-specific protein from the culture.

[0106] In some aspects, provided herein is an isolated antigen-binding domain or multi-specific protein thereof produced by any of the methods disclosed herein.

[0107] In some aspects, provided herein is an antigen-binding domain, an antibody comprising the antigen-binding domain, a complex or fusion protein, or a multi-specific protein further comprising a cytotoxic drug.

[0108] In some aspects, the cytotoxic drug is a microtubule disrupting agent, optionally wherein the microtubule disrupting agent is monomethyl auristatin E (MMAE).

[0109] In some aspects, provided herein is an antibody drug conjugate (ADC) comprising a drug and (i) the antigen-binding domain, (ii) the antibody comprising the antigen-binding domain, (iii) the complex or fusion protein, or (iv) the multi-specific protein.

[0110] In some aspects, the ADC binds to tissue factor (TF), human epidermal growth factor receptor (HER2), B7-H4, or Nectin-4.

[0111] In some aspects, the ADC comprises a microtubule disrupting agent, optionally wherein the microtubule disrupting agent is monomethyl auristatin E (MMAE).

[0112] In some aspects, the ADC comprises a version of tisotumab vedotin, disitamab vedotin, felmetatug vedotin, enfortumab vedotin, or trastuzumab deruxtecan with reduced effector function.

[0113] In some aspects, provided herein is a pharmaceutical composition comprising (i) any of the antigen-binding domains, complexes, fusion proteins, antibodies or antigen-binding fragments thereof, or multi-specific proteins disclosed herein and (ii) a pharmaceutically acceptable carrier.

[0114] In some aspects, the concentration of the complex, fusion protein, antibody or an antigen-binding fragment thereof, or multi-specific protein is increased in the brain following administration to a subject as compared to an isotype control.

[0115] In some aspects, administration increases delivery of the complex, fusion protein, antibody or antigen-binding fragment thereof, multi-specific protein, or pharmaceutical composition into the brain by at least 50%, at least 100%, at least 200%, at least 500% or at least 1000% as compared to an isotype control.

[0116] In some aspects, provided herein is a method of treating a neurological disease or disorder in a subject comprising administering any of the complexes, fusion proteins, antibodies or antigen-binding fragments thereof, multi-specific proteins, or pharmaceutical compositions disclosed herein to the subject.

[0117] In some aspects, administration increases delivery of the complex, fusion protein, antibody or antigen-binding fragment thereof, multi-specific protein, or pharmaceutical composition into the brain by at least 50%, at least 100%, at least 200%, at least 500% or at least 1000% as compared to an isotype control.

[0118] In some aspects, administration increases delivery of the complex, fusion protein, antibody or antigen-binding fragment thereof, multi-specific protein, or pharmaceutical composition into the frontal cortex, the entorhinal cortex and / or the hippocampus.

[0119] In some aspects, the neurological disease or disorder is selected from a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation.

[0120] 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).

[0121] In some aspects, the dementia is frontotemporal dementia (FTD).

[0122] In some aspects, the neurological disease or disorder is Alzheimer’s disease.

[0123] In some aspects, the Alzheimer’s disease is early onset Alzheimer’s disease, prodromal Alzheimer’s disease, mild Alzheimer’s disease, or late onset Alzheimer’s disease.

[0124] In some aspects, the neurological disease or disorder is Parkinson’s disease.

[0125] In some aspects, the neurological disease or disorder is frontal temporal epilepsy.

[0126] In some aspects, the neurological disease or disorder is autism.

[0127] In some aspects, the neurological disease or disorder is lissencephaly.

[0128] In some aspects, provided herein is a method of treating a lysosomal storage disease in a subject, comprising administering any of the complexes or fusion proteins disclosed herein to the subject.

[0129] 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.

[0130] In some aspects, provided herein is a method of transporting a complex, fusion protein, antibody or an antigen-binding fragment thereof, or multi-specific protein across the BBB of a subject, comprising administering to the subject any of the complexes or fusion proteins, the antibodies or antigen-binding fragments thereof, the multi-specific proteins, or the pharmaceutical compositions disclosed herein to the subject.

[0131] In some aspects, the concentration of the complex, fusion protein, antibody or an antigen-binding fragment thereof, or multi-specific protein is increased in the brain following administration as compared to an isotype control.

[0132] In some aspects, the concentration of the complex, 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% as compared to an isotype control.

[0133] In some aspects, administration of the complex, fusion protein, antibody or an antigen- binding fragment thereof, or multi-specific protein does not result in reticulocyte count reduced in the subject by more than 10%, as compared to administration of an isotype control.

[0134] In some aspects, administration of the complex, fusion protein, antibody or an antigen- binding fragment thereof, or multi-specific protein does not result in reticulocyte count reduction in the subject, as compared to an isotype control.

[0135] In some aspects, provided herein is a method of increasing the concentration of a CNS binding antigen in the CSF of a subject, comprising administering any of the multi-specific proteins disclosed herein to the subject, wherein the concentration of the CNS binding antigen is increased as compared to administering the CNS binding antigen alone to the subject.

[0136] In some aspects, provided herein is a method of imaging a CNS antigen within a subject, comprising administering to the subject any of the complexes, fusion proteins, antibodies or antigen-binding fragments thereof, or multi-specific proteins disclosed herein and locating the imaging agent within the subject.

[0137] In some aspects, provided herein is s method of detecting a CNS antigen in vitro, comprising contacting an in vitro sample with any of the complexes, fusion proteins, antibodies or antigen-binding fragments thereof, or multi-specific proteins disclosed herein and locating the imaging agent within the sample.

[0138] In some aspects, provided herein is a use of any of the complexes or fusion proteins, the antibodies or antigen-binding fragments thereof, the multi-specific proteins, or the pharmaceutical compositions disclosed herein in any of the methods disclosed herein.

[0139] In some aspects, any of the complexes or fusion proteins, the antibodies or antigen- binding fragments thereof, the multi-specific proteins, or the pharmaceutical compositions disclosed herein are for use in any of the methods disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0140] FIG.1A shows exemplary formats for complexes disclosed herein comprising an anti- CD98hc antigen-binding domain and a heterologous polypeptide, such as an enzyme. FIG.1A includes examples of 1+1 formats ((i)-(iv)), 2+1 formats ((v)-(vi)), and 2+2 formats ((vii)-(viii)). The 1+1 format in FIG.1A (i) shows an enzyme–Fc domain – anti-CD98hc scFv fusion protein. The 1+1 format in FIG.1A (ii) shows an anti-CD98hc antigen-binding domain (Fv) – Fc domain – enzyme complex. The 1+1 format in FIG.1A (iii) shows an anti-CD98hc antigen-bindingdomain (Fv) – Fc region (i.e. a Fc domain in a hole format and a second Fc domain in a knob format) – enzyme complex. The 1+1 format in FIG.1A (iv) shows a complex comprising an enzyme linked to the N-terminal of a Fc domain in a knob format, and an anti-CD98hc antigen- binding domain (Fv) linked to the N-terminal of a Fc domain in a hole format. The 2+1 format in FIG.1A (v) shows a complex comprising an anti-CD98hc antigen-binding domain (Fv) linked to the N-terminal of a Fc domain in a knob format, an enzyme linked to the C-terminal of the Fc domain in the knob format, and an enzyme linked to the C-terminal of a Fc domain in a hole format. The 2+1 format in FIG.1A (vi) shows a complex comprising an enzyme linked to the N- terminal of a Fc domain in a knob format, an enzyme linked to the N-terminal of a Fc domain in a hole format, and an anti-CD98hc scFv linked to the C-terminal of the Fc domain in the knob format. The 2+2 format in FIG.1A (vii) shows a complex comprising an anti-CD98hc antibody and an enzyme linked to the C-terminal of both Fc domains of the anti-CD98hc antibody. The 2+2 format in FIG.1A (viii) shows a complex comprising an enzyme linked to the N-terminal of a Fc domain in a knob format, an anti-CD98hc scFv linked to the C-terminal of the Fc domain in the knob format, an enzyme linked to the N-terminal of a Fc domain in a hole format, and an anti-CD98hc scFv linked to the C-terminal of the Fc domain in the hole format.

[0141] FIG.1B shows exemplary formats for bispecific complexes disclosed herein. FIG.1B (i) shows an exemplary 2+1 bispecific format; FIG.1B (ii) shows an exemplary 1+1 format; and FIG.1B (iii) shows another exemplary 1+1 format with two different VHH domains.

[0142] FIG.2 shows a mvFc-scFv schematic for a bivalent and bispecific antibody. This mvFc-scFv antibody example contains one scFv that targets CD98hc and one heavy chain and light chain pair that bind to a different target. See Example 9.

[0143] FIG.3A shows antibody levels in vessel-depleted brain fraction at 24 hours post 20 mg / kg i.v. injection of CD98hc.04.048 and CD98hc.04.064.1e antibodies. See Example 12.

[0144] FIG.3B shows serum antibody levels in vessel-depleted brain fraction at 24 hours post 20 mg / kg i.v. injection of CD98hc.04.048 and CD98hc.04.064.1e antibodies. See Example 12.

[0145] FIGs.4A and 4B show cellular uptake of humanized and engineered CD98hc.04.048 antibody variants. Anti-CD98hc mvFc-scFv antibodies with an isotype control Fab were incubated for 2 hours and cell uptake / binding was detected by immunofluorescent staining with anti-human IgG labeled with Alexafluor-488. Nuclei were stained with DAPI. Images were taken at 40x by confocal, and 2 z-planes 1 μm apart were merged. Two to four fields were imaged per well and representative fields are shown in FIG.4B and quantified in FIG 4A. See Example 15.

[0146] FIGs.5A and 5B show the brain uptake of mvFc-scFv antibodies as measured by antibody levels in vessel-depleted brain fraction at T=1 day, T=2 days, and T=4 days post 20 mg / kg iv injection of mvFc-scFv antibodies. FIG.5A shows antibody levels measured as ng antibody per mg of total protein (as determined by BCA analysis) in the vessel-depleted brain lysates FIG.5B shows fold change of antibody level of anti-CD98hc mvFc-scFv antibodies as compared to the format matched control (iso.iso.mvFc). See Example 16.

[0147] FIG.6 shows antibody level in serum of hCD98hc+ / + mice at T=1 hour, 24 hour, 48 hour, and 96 hour timepoints after 20 mg / kg i.v. injection, determined by Gyros assay. See Example 16.

[0148] FIGs.7A and 7B show increased brain uptake of CD98hc.04.048.WH1 in mice (FIG. 7A) and fold-change compared to isotype control antibody (FIG.7B). See Example 21.

[0149] FIG.8 shows immunohistochemistry analysis of brain tissues of mice administered CD98hc.04.048.WH1 compared to isotype control antibody. See Example 22.

[0150] FIG.9 shows no significant changes in red blood cell count in mice following administration of CD98hc.04.048.WH1 compared to isotype control antibody. See Example 23.

[0151] FIG.10A shows increased brain uptake in mice of CD98hc.04.048.WH1 antibody variants of the present disclosure. See Example 26.

[0152] FIG.10B shows increased brain uptake in mice of CD98hc.04.048.WH1 antibody variants of the present disclosure, shown as fold-change compared to isotype control antibody. See Example 26.

[0153] FIG.11 shows no decrease in amino acid uptake in cells treated with CD98hc.04.048.WH1 antibody variants of the present disclosure. See Example 27.

[0154] FIGs.12A and 12B show an increase in CD98hc.04.048.WH1 antibody (Figure 12A) and anti-GPNMB / CD98hc.04.048.WH1 multi-specific antibody (Figure 12B) levels in mouse brain compared to that observed with the corresponding isotype control antibody. See Example 28.

[0155] FIG.13 shows exemplary formats of multi-specific proteins comprising (i) an antigen- binding domain that specifically binds to human CD98hc and (ii) rituximab with LALA-P331S.

[0156] FIGs.14A and 14B show levels of brain uptake of various anti-CD98hc.04.048.WH1 monovalent Fab variants of the present disclosure presented as ng Ab / mg total protein and fold- change over control, respectively. See Example 30.

[0157] FIG.15A shows serum clearance of antibodies containing an anti- CD98hc.04.048.WH1 binding domain following 3 weekly doses in mice. See Example 32.

[0158] FIG.15B shows increased levels of antibodies containing an anti-CD98hc.04.048.WH1 binding domain in vessel-depleted mouse brain fractions compared to that observed with an isotype control antibody. See Example 32.

[0159] FIG.16A shows serum pharmacokinetic (PK) measurements of various anti- CD98hc.04.048.WH1 antibody variants in mice dosed with 20 mg / kg. See Example 34.

[0160] FIG.16B shows serum pharmacokinetic (PK) measurements of various anti- CD98hc.04.048.WH1 antibody variants in mice dosed with 3 mg / kg. See Example 34.

[0161] FIG.16C shows antibody concentrations in vessel-depleted brain samples from mice following administration of various anti-CD98hc.04.048.WH1 antibody variants at 20 mg / kg. See Example 34.

[0162] FIG.16D shows antibody concentrations in vessel-depleted brain samples from mice following administration of various anti-CD98hc.04.048.WH1 antibody variants at 3 mg / kg. See Example 34. DETAILED DESCRIPTION OF THE PRESENT DISCLOSURE

[0163] The present disclosure relates to antigen-binding domains that specifically bind to human CD98 heavy chain (CD98hc), and complexes, fusion proteins, antibodies, and antigen- binding fragments thereof comprising such antigen-binding domains, methods of making and using such antigen-binding domains, complexes, fusion proteins, antibodies, and antigen-binding fragments thereof; pharmaceutical compositions comprising such antigen-binding domains, complexes, fusion proteins, antibodies, and antigen-binding fragments thereof; nucleic acids encoding such antigen-binding domains, complexes, fusion proteins, antibodies, and antigen- binding fragments thereof; and host cells comprising nucleic acids encoding such antigen- binding domains, complexes, fusion proteins antibodies, and antigen-binding fragments thereof.

[0164] Exemplary anti-CD98hc antigen-binding domains and fusion proteins, antibodies, and antigen-binding fragments thereof comprising such antigen-binding domains, methods of making the same, methods of measuring the affinity and activity of the same, and methods of using the same are provided in International Application No. PCT / US2023 / 071238, filed July 28, 2023, which is herein incorporated by reference.

[0165] In some aspects, a multi-specific binding protein of the present disclosure with a “1+1 multi-specific binding protein format” comprises a CD98hc antigen-binding domain in a bivalent, bi-specific format comprising (i) one antigen-binding domain that binds to humanCD98hc and (ii) one antigen binding domain that binds to a CNS antigen that is not CD98hc. Such a “1+1” format can also comprise an Fc domain or Fc region.

[0166] In some aspects, a complex of the present disclosure has a “1+1 format” comprises (i) an antigen-binding domain that binds to human CD98hc; and (ii) a protein or polypeptide useful for protein replacement therapy or an enzyme or fragment thereof useful for enzyme replacement therapy. Such a “1+1” format can also comprise an Fc domain or Fc region.

[0167] In some aspects, a multi-specific binding protein of the present disclosure with a “2+1 multi-specific binding protein format” comprises a trivalent, bi-specific format comprising (i) an antigen-binding domain that binds to human CD98hc, and (ii) an antibody comprising two antigen-binding domains that bind to a CNS antigen that is not CD98hc, wherein the antibody comprises two heavy chains and two light chains; wherein the antigen-binding domain that binds to human CD98hc is linked to the C-terminus of one of the two antibody heavy chains.

[0168] In some aspects, a multi-specific binding protein of the present disclosure with a “2+1 multi-specific binding protein format” comprises a trivalent, bi-specific format comprising (i) an antigen-binding domain that bind to a CNS antigen that is not CD98hc, and (ii) an antibody comprising two antigen-binding domains that binds to CD98hc, wherein the antibody comprises two heavy chains and two light chains; wherein the antigen-binding domain that binds to the CNS antigen that is not human CD98hc is linked to the C-terminus of one of the two antibody heavy chains.

[0169] In some aspects, a complex of the present disclosure has a “2+1 format” comprising (i) an antigen-binding domain that binds to human CD98hc, (ii) two copies of a protein or polypeptide useful for protein replacement therapy or an enzyme or fragment thereof useful for enzyme replacement therapy. Such a “2+1” format can also comprise an Fc domain or Fc region.

[0170] In some aspects, a multi-specific binding protein of the present disclosure with a “2+2 multi-specific binding protein format” comprises a tetravalent, bi-specific format comprising (i) two antigen-binding domains that bind to human CD98hc and (ii) an antibody comprising two antigen-binding domains that bind to a CNS antigen that is not CD98hc, 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.

[0171] In some aspects, a multi-specific binding protein of the present disclosure with a “2+2 multi-specific binding protein format” comprises a tetravalent, bi-specific format comprising (i)two antigen-binding domains that bind to a CNS antigen that is not human CD98hc and (ii) an antibody comprising two antigen-binding domains that bind to human CD98hc, wherein the antibody comprises two heavy chains and two light chains; wherein one antigen-binding domain that binds to a CNS antigen that is not 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 a CNS antigen that is not human CD98hc is linked to the C-terminus of the other of the two antibody heavy chains.

[0172] In some aspects, a complex of the present disclosure has a “2+2” format comprising (i) two antigen-binding domains that bind to human CD98hc and (ii) two copies of a protein useful for protein replacement therapy or an enzyme or fragment thereof useful for enzyme replacement therapy. Such a “2+2” format can also comprise an Fc domain or Fc region.

[0173] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000). Definitions

[0174] The terms “central nervous system” and “CNS” refer to the complex of nerve tissues that control bodily function and includes the brain and spinal cord.

[0175] The terms “blood brain barrier” and “BBB” refer to a network of brain capillary endothelial cells that are closely sealed by tight junctions and characterized by low levels of non- specific paracellular and transcellular transport.

[0176] The terms “BBB target”, “BBB protein”, “BBB receptor”, and “BBB antigen” refer to a target / protein / receptor / antigen expressed on blood brain barrier cells (e.g., TfR or CD98hc). In some aspects, an antigen-binding domain (e.g., in an antibody, scFv, or Fab) that binds to the BBB target / protein / receptor / antigen allows transportation of a molecule or compound associated with said antigen-binding domain across the BBB.

[0177] A “central nervous system antigen” or “CNS antigen” is an antigen expressed in the CNS, including the brain, which can be targeted with an antibody or small molecule. Examples of such antigens include, without limitation: beta-secretase 1 (BACE1), amyloid beta (Abeta),epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interleukin 6 receptor (IL6R), TNF receptor 1 (TNFR1), interleukin 1 beta (IL1β)), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), glycoprotein nonmetastatic melanoma protein B (GPNMB), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), ubiquitin protein ligase E3A (UBE3A), or Transmembrane Protein 106B (TMEM106b).

[0178] A “brain antigen” is a CNS antigen expressed in the brain.

[0179] The terms “CD98hc,” “CD98hc polypeptide,” and “CD98hc protein” are used interchangeably herein to refer to any native CD98hc from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (cynos)) and rodents (e.g., mice and rats), unless otherwise indicated. CD98hc is also referred to as 4F2 cell-surface antigen heavy chain, 4F2hc, 4F2 heavy chain antigen, lymphocyte activation antigen 4F2 large subunit, solute carrier family 3 member 2, and CD98. 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 both wild-type sequences and naturally occurring variant sequences, e.g., splice variants or allelic variants. In some aspects, the term encompasses “full-length,” unprocessed CD98hc, as well as any form of CD98hc that results from processing in the cell. In some aspects, the CD98hc is human CD98hc. As used herein, the term “human CD98hc” refers to a polypeptide with the amino acid sequence of SEQ ID NO: 43. MELQPPEASIAVVSIPRQLPGSHSEAGVQGLSAGDDSELGSHCVAQTGLELLASGDPLPS ASQNAEMIETGSDCVTQAGLQLLASSDPPALASKNAEVTGTMSQDTEVDMKEVELNEL EPEKQPMNAASGAAMSLAGAEKNGLVKIKVAEDEAEAAAAAKFTGLSKEELLKVAGSP GWVRTRWALLLLFWLGWLGMLAGAVVIIVRAPRCRELPAQKWWHTGALYRIGDLQAF QGHGAGNLAGLKGRLDYLSSLKVKGLVLGPIHKNQKDDVAQTDLLQIDPNFGSKEDFD SLLQSAKKKSIRVILDLTPNYRGENSWFSTQVDTVATKVKDALEFWLQAGVDGFQVRDI ENLKDASSFLAEWQNITKGFSEDRLLIAGTNSSDLQQILSLLESNKDLLLTSSYLSDSGSTGEHTKSLVTQYLNATGNRWCSWSLSQARLLTSFLPAQLLRLYQLMLFTLPGTPVFSYGD EIGLDAAALPGQPMEAPVMLWDESSFPDIPGAVSANMTVKGQSEDPGSLLSLFRRLSDQ RSKERSLLHGDFHAFSAGPGLFSYIRHWDQNERFLVVLNFGDVGLSAGLQASDLPASAS LPAKADLLLSTQPGREEGSPLELERLKLEPHEGLLLRFPYAA (SEQ ID NO: 43)

[0180] As used herein, the terms “antibody” and “immunoglobulin” are used interchangeably and refer to an antibody molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing (e.g., a glycoprotein), through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term “antibody” encompasses monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multi-specific (e.g., bi- specific) antibodies, and any other immunoglobulin molecule so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy-chain constant regions referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of antibodies have different and well-known subunit structures and three-dimensional configurations. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0181] The terms “anti-CD98hc antibody,” “antibody that binds to CD98hc,” and “antibody that specifically binds CD98hc” refer to an antibody that is capable of binding CD98hc with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in 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 as measured, e.g., by a radioimmunoassay (RIA). In certain aspects, an antibody that binds to CD98hc has a dissociation constant (KD) of < 10 μΜ, < 1 μΜ, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10-8M or less, e.g., from 10-8M to 10-13M, e.g., from 10-9M to 10-13M). In certain aspects, an anti-CD98hc antibody binds to an epitope of CD98hc that is conserved among CD98hc from different species.

[0182] The term “antibody fragment” refers to a portion of an antibody. An “antigen-binding fragment” of an antibody refers to a portion of an antibody that binds to an antigen. An antigen- binding fragment of an antibody can comprise the antigenic determining regions of an antibody (e.g., the complementarity determining regions (CDRs)). Examples of antigen-binding fragmentsof antibodies 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 can be monovalent or multi-valent (e.g., bi-valent). An antigen-binding fragment of an antibody can be monospecific or multi-specific (e.g., bi-specific.) An antigen-binding fragment of an antibody can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans or can be artificially produced.

[0183] 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” can comprise the antigenic determining regions of an antibody (e.g., the complementarity determining regions (CDRs)). An antibody or antigen-binding fragment thereof (including mono-specific and multi- specific (e.g., bi-specific) antibodies or antigen-binding fragments thereof can comprise an antigen-binding domain.

[0184] 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 useful as a diagnostic agent, a therapeutic agent, or for transporting a molecule or compound across the BBB. In one aspect, the extent of binding of an anti-CD98hc antigen- binding domain to an unrelated, non-CD98hc polypeptide is less than about 10% of the binding of the antigen-binding domain to CD98hc as measured, e.g., by a radioimmunoassay (RIA). In certain aspects, an antibody that binds to CD98hc has a dissociation constant (KD) of < 0.1 μΜ, < 1 μΜ, <10 μM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10-8M or less, e.g., from 10-8M to 10-13M, e.g., from 10-9M to 10-13M). In certain aspects, an anti- CD98hc antigen-binding domain binds to an epitope of CD98hc that is conserved among CD98hc from different species.

[0185] The terms “full-length antibody,” “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant regions can be native sequence constant regions (e.g., human native sequence constant regions) or amino acid sequence variants thereof. In some cases, the intact antibody can have one or more effector functions. The C-terminal lysine (residue 447 according to the EU numbering system) of an intact antibody can be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acidencoding a heavy chain of the antibody. Accordingly, a composition of “full-length antibodies,” “intact antibodies,” or “whole antibodies” can comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

[0186] “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 intra-chain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) 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. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

[0187] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire light chain along with the variable region domain of the heavy chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab’)2fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab’ fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab’)2 antibody fragments originally were produced as pairs of Fab’ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0188] The Fc fragment comprises the carboxy-terminal portions of both heavy chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.

[0189] “Fv” is the minimum antibody fragment which comprises a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanates six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0190] “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some aspects, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding.

[0191] The term “diabodies” refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10) residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the variable domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.

[0192] As used herein, the terms “variable region” or “variable domain” are used interchangeably and are common in the art. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in a sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In some aspects, the variable region is a human variable region. In some aspects, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In some aspects, the variable region is a primate (e.g., non- human primate) variable region. In some aspects, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs). The term “Kabat numbering” and like terms are recognized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody or an antigen-bindingfragment thereof. In certain aspects, CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Kabat numbering scheme as 35A and 35B) (CDRH1), amino acid positions 50 to 65 (CDRH2), and amino acid positions 95 to 102 (CDRH3). Using the Kabat numbering system, 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 refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol.196:901-917 (1987)). The end of the Chothia CDRH1 loop, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, 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 Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software. In some aspects, the CDRs can be “contact” CDRs. The “contact” CDRs are based on an analysis of the available complex crystal structures. The residues from each of these CDRs are noted below. Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

[0193] CDRs can comprise “extended CDRs” as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH. The variable-domain residues are numbered according to Kabat et al., supra, for each of these extended-CDR definitions.

[0194] The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody.

[0195] As used herein, the term “heavy chain” when used in reference to an antibody can refer to any distinct type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (µ), based on the amino acid sequence of the constant region, which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., 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.

[0196] The terms “VL” and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody.

[0197] As used herein, the term “light chain” when used in reference to an antibody can refer to any distinct type, e.g., kappa (κ) or lambda (λ) based on the amino acid sequence of the constant regions. Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.

[0198] As used herein, the term “constant region” is a region of an antibody that is not the variable region of the antibody, e.g., a carboxyl terminal portion of a light and / or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain. In certain aspects, an antibody or antigen-binding fragment comprises a constant region or portion thereof that is sufficient for antibody-dependent cell- mediated cytotoxicity (ADCC).

[0199] A “constant domain” means a domain within a constant region that is capable of forming an immunoglobulin fold. Constant domains include the CH1, CH2, CH3, and CL domains.

[0200] The term “monoclonal” when referring to an antibody or antigen-binding fragment thereof refers to a homogeneous antibody or antigen-binding fragment population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal” antibody or antigen-binding fragment thereof encompasses both intact and full-length monoclonal antibodies as well as antibodyfragments (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv) mutants, fusion proteins or complexes comprising an antibody or antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, a “monoclonal” antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0201] The term “chimeric” antibodies or antigen-binding fragments thereof refers to antibodies or antigen-binding fragments thereof wherein the amino acid sequence is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies or antigen-binding fragments thereof derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies or antigen-binding fragments thereof derived from another (usually human) to avoid eliciting an immune response in that species.

[0202] The term “humanized” antibody or antigen-binding fragment thereof refers to forms of non-human (e.g., murine) antibodies or antigen-binding fragments that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies or antigen-binding fragments thereof are human immunoglobulins in which residues from the complementarity determining regions (CDRs) are replaced by residues from the CDRs of molecule originating from a non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability (“CDR grafted”) (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). The humanized antibody or antigen-binding fragment thereof can be further modified by the substitution of additional residues either in the Fv framework region and / or within the replaced non-human residues to refine and optimize the specificity, affinity, and / or capability of the antibody or antigen-binding fragment thereof. In general, the humanized antibody or antigen- binding fragment thereof will comprise VH and VL that comprise substantially all of at least one, and typically two or three, of the CDR regions that correspond to the non-human immunoglobulin, whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody or antigen-binding fragment thereof can also comprise at least a portion of an immunoglobulin constant region or Fc region, typically that of a human immunoglobulin. Examples of methods used to generate humanizedantibodies are described in U.S. Pat.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.

[0203] 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 gene locus, where such antibody or antigen-binding fragment is made using any technique known in the art. This definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0204] “Framework” or “FR” residues are those variable-domain residues other than the CDR residues as herein defined.

[0205] An “acceptor human framework” as used herein is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence thereof, or it can comprise pre-existing amino acid sequence changes. In some aspects, the number of pre-existing amino acid changes are 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. Where pre-existing amino acid changes are present in a VH, in some aspects those changes occur at only three, two, or one of positions 71H, 73H and 78H; for instance, the amino acid residues at those positions can by 71A, 73T and / or 78A. In some aspects, the VL acceptor human framework is identical in sequence to the VLhuman immunoglobulin framework sequence or human consensus framework sequence.

[0206] A “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VLor VHframework sequences. Generally, the selection of human immunoglobulin VLor VHsequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include for the VL, the subgroup can be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al., supra. Additionally, for the VH, the subgroup can be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra.

[0207] An “amino-acid modification” at a specified position, e.g., of an antibody of the present disclosure, refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent the specified residue. Insertion “adjacent” to a specified residuemeans insertion within one to two residues thereof. The insertion can be N-terminal or C- terminal to the specified residue. In some aspects, an amino acid modification is a substitution.

[0208] 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.

[0209] As used herein, a “Fc region” or “fragment crystallizable region” is composed of two or more polypeptides, each being an antibody heavy chain fragment and each containing at least one (e.g., two or three) heavy chain constant domains. In some aspects, an Fc region is composed of two heavy chain fragments, each containing a CH2 domain and a CH3 domain. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a Fc region may not contain any K447 residues, may contain at least one polypeptide containing a K447 residue, or may only contain polypeptides that include a K447 residue. Suitable native-sequence Fc regions for use in the present disclosure include human IgG1, IgG2, IgG3 and IgG4. In a native antibody, an Fc region refers to the region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. However, as used herein, an Fc region can be modified to increase, decrease, or eliminate interaction with Fc receptors and / or proteins of the compliment system. In native IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody’s two heavy chains. However, as used herein, the two or more polypeptides in an “Fc region” do not need to have identical sequences. In some aspects, an “Fc region” comprises a first polypeptide comprising an Fc domain (e.g., IgG1 Fc domain) with a knob mutation and a second polypeptide comprising an Fc domain (e.g., IgG1 Fc domain) with a hole mutation. In native IgM and IgE antibody isotypes, the Fc region contains three heavy chain constant domains (CH domains 2–4) in each polypeptide chain.

[0210] The term “Fc domain” refers to one or more constant region domains within an Fc region, such as a CH2 or CH3 domain, in a single polypeptide. In some aspects, the Fc domain includes at least one amino acid deletion, addition, or substitution as compared to the amino acid sequence of a native Fc domain, such as by including a set of “knob-into-hole” deletions,additions, or substitutions or including amino acid deletions, additions, or substitutions to effect electrostatic steering of the Fc domain to favor attractive interactions among different polypeptide chains. In some aspects, the Fc domain is in a “knob” format. In some aspects, the Fc domain is in a “hole” format.

[0211] 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 a native sequence human IgG1 Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well.

[0212] A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, in some aspects two or more amino acid substitution(s). In some aspects, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and in some aspects from about one to about five amino acid substitutions compared to a native sequence Fc region or in the Fc region of the parent polypeptide. In some aspects, the variant Fc region possesses at least 80% homology with a native sequence Fc region and / or with an Fc region of a parent polypeptide, at least 90% homology therewith, or at least 95% homology therewith.

[0213] “Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. In some aspects, an FcR is a native sequence human FcR. In some aspects, a FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (“ITAM”) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (“ITIM”) in its cytoplasmic domain. Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. FcRs can also increase the serum half-life of antibodies.

[0214] As used herein, a “complex” refers to one or more proteins comprising connected parts. The parts can be connected e.g., via a peptide bond (e.g., in a fusion protein), a linker (e.g., a peptide linker), or via noncovalent protein-protein interactions such as disulfide bonds (e.g., in anantibody). Exemplary parts that can be included in a complex include a protein useful for protein replacement therapy or an enzyme useful for enzyme replacement therapy, an antigen-binding domain, antibody or antigen-binding fragment thereof that binds to a CNS antigen, an antigen- binding domain than specifically binds to CD98hc, an Fc region, and / or an Fc domain. Accordingly, non-limiting examples of a “complex” comprising an antigen-binding domain and a protein or enzyme useful for replacement therapy include (a) a fusion protein comprising the antigen-binding domain and the protein or enzyme useful for replacement therapy in a single polypeptide chain (e.g., as shown in Figure 1A(i)), and (b) three proteins connected via noncovalent protein-protein interactions, wherein the first protein contains the protein or enzyme useful for replacement therapy and an Fc domain, the second protein contains a VH of the antigen-binding domain, and the third protein contains a VL of the antigen-binding domain (e.g., as shown in Figure 1A(iv)). Formats of other exemplary complexes are provided in Figures 1A, 1B, and 2.

[0215] “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody or antigen-binding fragment thereof and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is calculated from the quotient of koff / kon, whereas KAis calculated from the quotient of kon / koff. konrefers to the association rate constant of, e.g., an antibody or antigen-binding fragment thereof to an antigen, and koffrefers to the dissociation rate constant of, e.g., an antibody or antigen-binding fragment thereof from an antigen. The kon and koff can be determined by techniques known to one of ordinary skill in the art, such as BIAcore®or KinExA. Dissociation constants may also be determined through any analytical technique, including any biochemical or biophysical technique such as ELISA, surface plasmon resonance (SPR), bio-layer interferometry (see, e.g., Octet System by ForteBio), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped- flow analysis, and colorimetric or fluorescent protein melting analyses. (See, e.g., Estep et al, (2013) MAbs 5(2):270-8.)

[0216] With regard to the binding of an antibody to a target molecule, the term “specific binding” or “specifically binds” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a KD for the target of about any of 10-4M or lower, 10-5M or lower, 10-6M or lower, 10-7M or lower, 10-8M or lower, 10-9M or lower, 10-10M or lower, 10-11M or lower, 10-12M or lower or a KD in the range of 10-4M to 10-6M or 10-6M to 10-10M or 10-7M to 10-9M. As will be appreciated by the skilled artisan, affinity and KD values are inversely related. A high affinity for an antigen is measured by a low KD value. In some aspects, the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0217] The term “linker” or “linked” refers to the covalent linkage between two polypeptides or two heterologous molecules. In some aspects, a linker is a chemical linker. In some aspects, the linker comprises a peptide bond, and the two polypeptides or two heterologous molecules are linked to each other either directly to or via one or more additional amino acids. A glycine linker is one that comprises one or more glycines but no other amino acids, e.g., GGGG (SEQ ID NO: 45). A glycine-rich linker is one that comprises one or more glycines and can contain other amino acids as long as glycine is the predominant species in the linker e.g., GGGNGG, wherein N is any amino acid (SEQ ID NO: 46). A glycine-serine linker is one which contains both glycine and serine in any proportion, e.g., GGGS (SEQ ID NO: 47). Similarly, a proline linker is one that comprises one or more prolines but no other amino acids. A proline-rich linker is one that comprises one or more prolines and can contain other amino acids so long as proline is the predominant species in the linker.

[0218] 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 with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary,to achieve the maximum percent sequence identity, and not considering any conservative substitutions as identical matches. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM(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.

[0219] The term “epitope” includes any determinant capable of being bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets that antigen, and when the antigen is a polypeptide, includes specific amino acids that directly contact the antibody. Most often, epitopes reside on polypeptides, but in some instances, can reside on other kinds of molecules, such as nucleic acids. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of polypeptides and / or macromolecules.

[0220] An antibody that “binds to the same epitope” as a reference antibody refers to an antibody that contacts the same amino acid residues on the antigen as the reference antibody. The ability of an antibody to bind to the same epitope as a reference antibody can be determined using peptide scanning mutagenesis or high throughput alanine scanning mutagenesis. In the latter methodology, a comprehensive mutation library of antigen, or a portion thereof (e.g., the extracellular domain), can be generated by mutating each individual amino acid residue to alanine (or if the amino acid residue is alanine, then to another residue such as serine) and testing each mutant for binding to a target antibody or antigen-binding fragment thereof.

[0221] An antibody is said to “competitively inhibit” binding of a reference antibody to a given epitope if it preferentially binds to that epitope or an overlapping epitope such that it blocks, to some degree, binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays. 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%.

[0222] A polypeptide, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in aform not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some aspects, an antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

[0223] As used herein, “substantially pure” refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0224] The term “expression system” refers to one or more nucleic acid molecules comprising coding sequence and control sequence(s) in operable linkage, along with a host cell and / or other in vitro transcription and translation machinery, such that one or more proteins encoded by the nucleic acid molecule(s) are capable of being produced.

[0225] The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors,” or simply, “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.

[0226] “Polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.

[0227] A “host cell” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology orin genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of this invention. In some aspects, the host cell is an isolated host cell.

[0228] “Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.

[0229] As used herein, the term “treatment” refers to clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of progression, ameliorating or palliating the pathological state, and remission or improved prognosis of a particular disease, disorder, or condition. An individual is successfully “treated”, for example, if one or more symptoms associated with a particular disease, disorder, or condition are mitigated or eliminated.

[0230] The terms “administer,” “administering,” “administration,” and the like, as used herein, refer to methods that can be used to deliver a drug, e.g., an anti-human antibody or antigen- binding fragment thereof, to the desired site of biological action.

[0231] An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. An effective amount can be provided in one or more administrations. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and / or prolonging survival. An effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” can be considered in the context of administering one or more therapeutic agents, and a single agent can be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result can be or is achieved.

[0232] 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., 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.

[0233] As used herein, administration “in conjunction” or “in combination” with another compound or composition includes simultaneous administration and / or administration at different times. Administration in conjunction also encompasses administration as a co- formulation or administration as separate compositions, 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 a part of the same treatment regimen.

[0234] A “neurological disorder” as used herein refers to a disease or disorder which affects the CNS and / or which has an etiology in the CNS. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, an ocular disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, seizure, behavioral disorders, and a lysosomal storage disease.

[0235] A “Lysosomal storage disorder” or (LSD) as used herein refers to an inherited metabolic disease characterized by the accumulation of substrates, such as undigested or partially digested macromolecules, in excess in various cells of organs, which ultimately results in cellular dysfunction and clinical abnormalities. LSDs have been defined as deficiencies in lysosomal function generally classified by the accumulated substrate and include sphingolipidoses, oligosaccharidoses, mucolipidoses, mucopolysaccharidoses, lipoprotein storage disorders, neuronal ceroid lipofuscinoses, and others. LSDs may also include other deficiencies or defects in proteins that result in accumulation of macromolecules, such as proteins necessary for normal post-translational modification of lysosomal enzymes, or proteins important for proper lysosomal trafficking. LSDs are diseases caused by defects in single genes. Enzyme defects cause nearly seventy percent of the LSDs, and the rest are defects in enzyme activator or associated proteins.

[0236] “Protein replacement therapy” or “PRT” refers to a medical treatment that supplements or replaces a protein in a patient in whom that particular protein is deficient or absent.

[0237] An “enzyme replacement therapy enzyme” or “ERT enzyme” refers to an enzyme that is deficient in a lysosomal storage disorder. An “ERT enzyme variant” refers to a functional variant, including allelic and splice variants, of a wild-type ERT enzyme or a fragment thereof, where 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 thecorresponding wild-type ERT enzyme or fragment thereof, e.g., when assayed under identical conditions. A “catalytically active fragment” of an ERT enzyme refers to a portion of a full- length ERT enzyme or a variant thereof, where 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 variant thereof, e.g., when assayed under identical conditions.

[0238] As used herein, the terms “about” and “approximately,” when used to modify a numeric value or numeric range, indicate that deviations of up to 10% above and down to 10% below the value or range remain within the intended meaning of the recited value or range. It is understood that wherever aspects are described herein with the language “about” or “approximately” a numeric value or range, otherwise analogous aspects referring to the specific numeric value or range are also provided.

[0239] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly indicates otherwise. For example, reference to an “antibody” is a reference to from one to many antibodies, such as molar amounts, and includes equivalents thereof known to those skilled in the art, and so forth.

[0240] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and / or “consisting essentially of” are also provided. In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially of” are open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art aspects. Anti-CD98hc Antigen-Binding Domains

[0241] Provided herein are antigen-binding domains that specifically bind to human CD98hc.

[0242] Such antigen-binding domains can be capable of crossing the blood brain barrier (BBB) and capable of transporting other agents (e.g., therapeutically active agents) associated with the antigen-binding domain across the BBB. Accordingly, in some aspects, provided herein are antigen-binding domains that specifically bind to human CD98hc that are capable of being internalized in BBB epithelial cells, such as HCMEC / D3 cells.

[0243] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the six CDRs of an antibody listed in Tables 9 and 10 (i.e., the three VH CDRs of theantibody listed in Table 9 and the three VL CDRs of the same antibody listed in Table 10) or an antibody listed in Tables 15 and 16 (i.e., the three VH CDRs of the antibody listed in Table 15 and the three VL CDRs of the same antibody listed in Table 16), or the six CDRs of an antibody listed in Table 8 or 14.

[0244] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the six CDRs of an antibody listed in Table 8 or 14. In some aspects, the CDRs of such an antigen-binding domain can be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No.7,709,226). Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34, the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97. The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, 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).

[0245] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the six Chothia CDRs of an antibody listed in Table 8 or 14. In some aspects, such as an antigen-binding domain that specifically binds to human CD98hc comprises one or more CDRs, in which the Chothia and Kabat CDRs have the same amino acid sequence. In some aspects, provided herein are antigen-binding domains that specifically binds to human CD98hc and comprise combinations of Kabat CDRs and Chothia CDRs.

[0246] In some aspects, the CDRs of an antigen-binding domain that specifically binds to human CD98hc can be determined according to MacCallum RM et al., (1996) J Mol Biol 262: 732-745. See also, e.g., 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 are antigen-binding domains that specifically bind to human CD98hc and comprise VH and VL CDRs of an antibody listed in Table 8 or 14 as determined by the method in MacCallum RM et al.

[0247] In some aspects, the CDRs of an antigen-binding domain that specifically binds to human CD98hc can be determined according to the AbM numbering scheme, which refers to AbM hypervariable regions, which represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (Oxford Molecular Group, Inc.). In some aspects, provided herein are antigen-binding domains that specifically bind to human CD98hc and comprise VH and VL CDRs of an antibody listed in Table 8, 13, 22 or 25 as determined by the AbM numbering scheme.

[0248] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the six IMGT CDRs of an antibody listed in Table 8 or 14 according to the IMGT numbering system as described in Lefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999) Nucleic Acids Res 27: 209-212. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97.

[0249] In some aspects, an antigen-binding domain that specifically binds to human CD98hc provided herein is described by its VL domain alone, or its VH domain alone, or by its 3 VL CDRs alone, or its 3 VH CDRs alone. See, for example, Rader C et al., (1998) PNAS 95: 8910- 8915, which is incorporated herein by reference in its entirety, describing the humanization of the mouse anti-αvβ3 antibody by identifying a complementing light chain or heavy chain, respectively, from a human light chain or heavy chain library, resulting in humanized antibody variants having affinities as high or higher than the affinity of the original antibody. See also Clackson T et al., (1991) Nature 352: 624-628, which is incorporated herein by reference in its entirety, describing methods of producing antibodies that bind a specific antigen by using a specific VL domain (or VH domain) and screening a library for the complementary variable domains. The screen produced 14 new partners for a specific VH domain and 13 new partners for a specific VL domain, which were strong binders, as determined by ELISA. See also Kim SJ & Hong HJ, (2007) J Microbiol 45: 572-577, which is incorporated herein by reference in its entirety, describing methods of producing antibodies that bind a specific antigen by using a specific VH domain and screening a library (e.g., human VL library) for complementary VL domains; the selected VL domains in turn could be used to guide selection of additional complementary (e.g., human) VH domains.

[0250] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the VH of an antibody listed in Table 8 or 14.

[0251] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the VL of antibody listed in Table 8 or 14.

[0252] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the VH and the VL of an antibody listed in Table 8 (i.e., the VH of the antibody listed in Table 8 and the VL of the same antibody listed in the Table 8) or Table 14 (i.e., the VH of the antibody listed in Table 14 and the VL of the same antibody listed in the Table 14).

[0253] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 80% identical to a VH amino acid sequence of an antibody in Table 8 or 14 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 or 14. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Table 8 or 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0254] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 85% identical to a VH amino acid sequence of an antibody in Table 8 or 14 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 or 14. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Table 8 or 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0255] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 90% identical to a VH amino acid sequence of an antibody in Table 8 or 14 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 or 14. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Table 8 or 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0256] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 95% identical to a VH amino acid sequence of an antibody in Table 8 or 14 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 or 14. In some aspects, the antigen-binding domain that specifically binds to humanCD98hc also comprises the CDRs of the antibody in Table 8 or 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0257] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 96% identical to a VH amino acid sequence of an antibody in Table 8 or 14 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 or 14. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Table 8 or 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0258] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 97% identical to a VH amino acid sequence of an antibody in Table 8 or 14 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 or 14. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Table 8 or 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0259] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 98% identical to a VH amino acid sequence of an antibody in Table 8 or 14 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 or 14. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Table 8 or 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0260] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 99% identical to a VH amino acid sequence of an antibody in Table 8 or 14 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 or 14. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Table 8 or 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0261] 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 or 14 and a VL amino acid sequence of the same antibody in Table 8 or 14.

[0262] In some aspects, provided herein is an antigen-binding domain that competitively inhibits binding to CD98hc of as an antibody comprising a VH amino acid sequence of an antibody in Table 8 or 14 and a VL amino acid sequence of the same antibody in Table 8 or 14.

[0263] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises a VH and a VL on a single polypeptide chain (e.g., a VH and VL in Table 8 or 14). In some aspects, the antigen-binding domain comprises an scFv. The scFv can comprise a VH that is N-terminal to a VL or a VL that is N-terminal to a VH. The scFv can comprise a linker, e.g., between a VH and a VL. Accordingly, the scFv can be in the orientation VH-linker-VL or VL- linker-VH. Such a linker can be about 5 to about 25 amino acids in length. Such a linker can be about 5 to about 20 amino acids in length. Such a linker can be about 10 to about 25 amino acids in length. Such a linker can be about 10 to about 20 amino acids in length. Such a linker can be, e.g., a glycine linker, a glycine-rich linker, or a glycine-serine linker. Such a linker can comprise the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 48). Such a linker can comprise the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 49).

[0264] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises a VH on a first polypeptide and a VL on a second polypeptide (e.g., a Fab).

[0265] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the antigen-binding fragment of a heavy chain only antibody (e.g., a VHH or nanobody).

[0266] In some aspects, an antigen-binding domain that specifically binds to human CD98hc is a murine antigen-binding domain. In some aspects, an antigen-binding domain that specifically binds to human CD98hc is a chimeric antigen-binding domain. In some aspects, an antigen- binding domain that specifically binds to human CD98hc is a humanized antigen-binding domain. In some aspects, an antigen-binding domain that specifically binds to human CD98hc is a human antigen-binding domain

[0267] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc also binds to cynomolgus monkey CD98hc.

[0268] In certain aspects, an antibody that binds to CD98hc has a dissociation constant (KD) of about 10 nM to about 1500 nM, about 100 nM to about 500 nM, about 500 nM to about 10 μM, or less than about 20 µM, less than about 15 µM, less than about 12 µM, less than about 10 µM, less than about 7.5 µM, less than about 5 µM, less than about 2.5 µM, less than about 1 μΜ, less than about 10 μM, less than about 100 nM, less than about 10 nM, less than about 1 nM, less thanabout 0.1 nM, less than about 0.01 nM, or less than about 0.001 nM (e.g., 10-8M or less, e.g., from 10-8M to 10-13M, e.g., from 10-9M to 10-13M).

[0269] In some aspects, the anti-CD98hc 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 sequences comprising the amino acid sequences of SEQ ID NOs: 70, 31, 12, 16, 17, and 18, respectively.

[0270] In some aspects, the anti-CD98hc antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 22 and 23, respectively.

[0271] In some aspects, the anti-CD98hc 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 sequences comprising the amino acid sequences of SEQ ID NOs: 70, 31, 12, 93, 17, and 18, respectively.

[0272] In some aspects, the anti-CD98hc antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 22 and 123, respectively.

[0273] In some aspects, the anti-CD98hc 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 sequences comprising the amino acid sequences of SEQ ID NOs: 70, 75, 12, 16, 17, and 18, respectively.

[0274] In some aspects, the anti-CD98hc antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 115 and 23, respectively.

[0275] In some aspects, the anti-CD98hc 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 sequences comprising the amino acid sequences of SEQ ID NOs: 70, 80, 12, 16, 17, and 18, respectively.

[0276] In some aspects, the anti-CD98hc antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 120 and 23, respectively.

[0277] In some aspects, an antigen-binding domain provided herein specifically binds 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, e.g., using the CarterraLSA platform. In some aspects, an antigen-binding domain provided herein that specifically binds 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 1nM, 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.

[0278] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of less than about 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 less than about 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 less than about 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.

[0279] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 18 nM to about 87 nm.

[0280] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of less than about 100 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 100 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 200 nM to about 1500 nM. In some aspects, an antigen- binding domain provided herein specifically binds to human CD98hc with an affinity of about 300 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 400 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 500 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 600 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 700 nM to about 1500 nM. In some aspects, an antigen- binding domain provided herein specifically binds to human CD98hc with an affinity of about 800 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 900 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1000 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1100 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds tohuman CD98hc with an affinity of about 1200 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1300 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1400 nM to about 1500 nM.

[0281] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 100 nM to about 200 nM. In some aspects, an antigen- binding domain provided herein specifically binds to human CD98hc with an affinity of about 200 nM to about 300 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 300 nM to about 400 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 400 nM to about 500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 500 nM to about 600 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 600 nM to about 700 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 700 nM to about 800 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 800 nM to about 900 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 900 nM to about 1000 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1000 nM to about 1100 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1100 nM to about 1200 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1200 nM to about 1300 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1300 nM to about 1400 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1400 nM to about 1500 nM.

[0282] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 24 nM to about 1330 nM. In some aspects, an antigen- binding domain provided herein specifically binds to human CD98hc with an affinity of about 21 nM to about 826 nM.

[0283] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 58 nM to about 447 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 51 nM to about 709 nM. In some aspects, the affinity is measured using surface plasmon resonance (BIACORE®). See Example 25.

[0284] In some aspects, an antigen-binding domain provided herein with a lower affinity (e.g., an affinity of up to 1 µM) can have improved transport across the blood brain barrier and into the brain parenchyma, e.g., as a result of the fact that they would not remain associated with blood vessels for as long a time (e.g., as compared to an antigen-binding domain with a higher affinity such as the parental WH1). In some aspects, an antigen-binding domain provided herein with a lower affinity (e.g., an affinity of up to 1 µM) can have an improved safety profile (e.g., as compared to an antigen-binding domain with a higher affinity such as the parental WH1).

[0285] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to human CD98hc with an ELISA OD450 of at least 0.45. In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to cynomolgus CD98hc with an ELISA OD450of at least 0.45. In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to human CD98hc with an ELISA OD450 of at least 0.45 and binds to cynomolgus CD98hc with an ELISA OD450 of at least 0.45.

[0286] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to cynomolgus CD98hc with an affinity of less than about 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 less than about 200 nM (e.g., 10 µM to 200 nM, 5 µM to 200 nM, 1 µM to 200 nM, 10 µM to 250 nM, 1 nM to 200 nM or 3 nM to 200 nM), or an affinity of less than about 150 nM (e.g., 10 µM to 150 nM, 5 µM to 150 nM, 1 µM to 150 nM, 10 µM to 250 nM, 1 nM to 150 nM or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance.

[0287] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to cynomolgus 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, 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 200 nM, 10 µM to 250 nM, 1 nM to 200 nM or 3 nM to 200 nM), or an affinity of no more than 150 nM (e.g., 10 µM to 150 nM, 5 µM to 150 nM, 1 µM to 150 nM, 10 µM to 250 nM, 1 nM to 150 nM or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, e.g., using the Carterra LSAplatform. In some aspects, an antigen-binding domain provided herein that specifically binds to cyno CD98hc binds to cyno 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.

[0288] In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 46 nM to about 539 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 100 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 200 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 300 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 400 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 500 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 600 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 700 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 800 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 900 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1000 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1100 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1200 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1300 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1400 nM to about 1500 nM.

[0289] In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 100 nM to about 200 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 200 nM to about 300 nM. In some aspects, an antigen-binding domain providedherein specifically binds to cynomolgus CD98hc with an affinity of about 300 nM to about 400 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 400 nM to about 500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 500 nM to about 600 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 600 nM to about 700 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 700 nM to about 800 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 800 nM to about 900 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 900 nM to about 1000 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1000 nM to about 1100 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1100 nM to about 1200 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1200 nM to about 1300 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1300 nM to about 1400 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1400 nM to about 1500 nM.

[0290] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to each of human CD98hc and cynomolgus CD98hc with an affinity of less than about 250 nM (e.g., 1 µM to 250 nM, 1 nM to 250 nM or 3 nM to 250 nM), an affinity of less than about 200 nM (e.g., 1 µM to 200 nM, 1 nM to 200 nM or 3 nM to 200 nM), or an affinity of less than about 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.

[0291] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to each of human CD98hc and cynomolgus 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, e.g., using the Carterra LSA platform. In some aspects, an antigen-bindingdomain provided herein that specifically binds to human CD98hc binds to each of human CD98hc and cynomolgus 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.

[0292] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 18 nM to about 87 nM and binds to cynomolgus CD98hc with an affinity of about 46 nM to about 539 nM.

[0293] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 24 nM to about 1330 nM and binds to cynomolgus CD98hc with an affinity of about 27 nM to about 1430 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 21 nM to about 826 nM and binds to cynomolgus CD98hc with an affinity of about 28 nM to about 651 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 57 nM to about 533 nM and binds to cynomolgus CD98hc with an affinity of about 52 nM to about 859 nM.

[0294] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc does not reduce cell surface expression of CD98hc on HCMED / D3 cells by more than 20% relative to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western blot or FACS.

[0295] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc does not increase cell surface expression of CD98hc on HCMED / D3 cells by more than 50% relative to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western blot or FACS.

[0296] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc does not reduce cell surface expression of CD98hc on HCMED / D3 cells by more than 20% relative to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control and does not increase cell surface expression of CD98hc on HCMED / D3 cells by more than 50% relative to cell surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western Blot or FACS.

[0297] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc accumulates at least 1-fold more than an isotype control in vessel-depleted mouse brain. In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc accumulates at least 1.5-fold more than an isotype control in vessel-depleted human CD98hc knock-in mouse brain after peripheral injection. In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc accumulates at least 3- fold more than an isotype control in vessel-depleted mouse brain. In some aspects, an antigen- binding domain provided herein that specifically binds to human CD98hc accumulates at least 5- fold more than an isotype control in vessel-depleted mouse brain. In some aspects, an antigen- binding domain provided herein that specifically binds to human CD98hc accumulates at least 10-fold more than an isotype control in vessel-depleted mouse brain. In some aspects, an antigen- binding domain provided herein that specifically binds to human CD98hc accumulates at least 20-fold more than an isotype control in vessel-depleted mouse brain.

[0298] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc has at least a 5-fold increase in brain concentration ratio over an isotype control 24 hours after administration to a mouse.

[0299] Also provided herein are antigen-binding domains that bind to the same epitope of CD98hc as a CD98hc antigen-binding domain provided herein. Also provided herein are antigen- binding domains that competitively inhibit binding to CD98hc of CD98hc antigen-binding domain provided herein. Agents Comprising Anti-CD98hc Antigen-Binding Domains

[0300] Provided herein are agents (e.g., fusion proteins, complexes, multi-specific (e.g., bispecific) proteins, antibodies, antigen-binding fragments thereof, etc.) comprising an antigen- binding domain that specifically binds to human CD98hc. Complexes and Fusion Proteins

[0301] In some aspects, a complex or fusion protein provided herein comprises an antigen- binding domain that specifically binds to human CD98hc and a heterologous protein or polypeptide. In some aspects, the heterologous protein is a protein or polypeptide or fragment thereof useful in protein replacement therapy (PRT). In some aspects, the heterologous polypeptide is an enzyme (e.g., an enzyme for use in enzyme replacement therapy (ERT)) or a catalytically active fragment thereof. In some 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 a complex or fusion protein provided herein is a growth factor. In some aspects, the heterologous polypeptide in a complex fusion protein provided herein is a decoy receptor. In some aspects, the heterologous polypeptide in a complex or fusion protein provided herein is progranulin (PGRN), prosaposin (PSAP), or survival motor neuron protein(SMN). In some aspects, the heterologous protein is an enzyme selected from ubiquitin protein ligase E3A (UBE3A), α-L Iduronidase (IDUA), Iduronate-2-sulphatase (IDS), N- acetylgalactoslamine-6-sulphatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N- acetylgalactosamine-4-sulphatase (arylsulphatase B; ARSB), acid sphingomyelinase (ASM), β- glucocerebrosidase (GCase or GBA), galactosylceramide beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta-hexosaminidase B, arylsulphatase A, beta- galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme replacement thereof, such as larodinase, idursulphase, elosulphase alpha or galsuphase, or a variant thereof, or a catalytically active fragment thereof. In some aspects, the heterologous protein is a protein or an enzyme selected from clusterin (APOJ), Reelin, Tripeptidyl Peptidase 1 (CLN2 / TPP1), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan- alpha-glucosaminide N-acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), α-L Iduronidase (IDUA), Iduronate-2-sulphatase (IDS), N-acetylgalactoslamine-6- sulphatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4- sulphatase (arylsulphatase B; ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramide beta-galactosidase, glucosylceramidase, beta- hexosaminidase A, beta-hexosaminidase B, arylsulphatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme replacement thereof, such as larodinase, idursulphase, elosulphase alpha or galsuphase, or a variant thereof, or a catalytically active fragment thereof.

[0302] In some aspects, the heterologous protein is from ubiquitin protein ligase E3A (UBE3A), α-L Iduronidase (IDUA), Iduronate-2-sulphatase (IDS), N-acetylgalactoslamine-6- sulphatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4- sulphatase (arylsulphatase B; ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramide beta-galactosidase, glucosylceramidase, beta- hexosaminidase A, beta-hexosaminidase B, arylsulphatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme replacement thereof, such as larodinase, idursulphase, elosulphase alpha or galsuphase, or a variant thereof, or a catalytically active fragment thereof. In some aspects, the heterologous protein is a protein or an enzyme selected from progranulin (PGRN), prosaposin (PSAP), clusterin (APOJ), Reelin, Tripeptidyl Peptidase 1 (CLN2 / TPP1), Alpha-L-Iduronidase (IDUA), Iduronate 2-Sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha- glucosaminide N-acetyltransferase (HGSNAT), N-acetyl-alpha-glucosaminidase (NAGLU), α-LIduronidase (IDUA), Iduronate-2-sulphatase (IDS), N-acetylgalactoslamine-6-sulphatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulphatase (arylsulphatase B; ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramide beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta- hexosaminidase B, arylsulphatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme replacement thereof, such as larodinase, idursulphase, elosulphase alpha or galsuphase, or a variant thereof, or a catalytically active fragment thereof N-sulfoglucosamine sulfohydrolase (SGSH), ubiquitin protein ligase E3A (UBE3A), or a variant or portion thereof.

[0303] In some aspects, the heterologous protein in the fusion protein is N-terminal to the antigen-binding domain that specifically binds to human CD98hc. In some aspects, the heterologous protein or polypeptide in the fusion protein is C-terminal to 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 connected via a peptide bond. In some aspects, the heterologous fusion protein and the antigen- binding domain that specifically binds to human CD98hc are connected via a linker, e.g., a peptide linker. In some aspects, the fusion protein comprises an antigen-binding domain and a heterologous protein or polypeptide and an Fc portion. In some aspects, the antigen-binding domain and the heterologous protein or polypeptide are linked to the N-terminus of the Fc portion of the fusion protein. In other aspects, the antigen-binding domain is linked to the N- terminus of the Fc portion and the heterologous protein or polypeptide is linked to the C-terminus of the Fc portion of the fusion protein. In other aspects, the antigen-binding domain is linked to the C-terminus of the Fc portion and the heterologous protein or polypeptide is linked to the N- terminus of the Fc portion of the fusion protein.

[0304] In some aspects, the heterologous polypeptide in a complex or fusion protein provided herein is β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), clusterin (APOJ), Reelin, Tripeptidyl Peptidase 1 (CLN2 / TPP1), Alpha-L-Iduronidase (IDUA), Iduronate 2-Sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha- glucosaminide N-acetyltransferase (HGSNAT), N-acetyl-alpha-glucosaminidase (NAGLU), or N-sulfoglucosamine sulfohydrolase (SGSH), or ubiquitin protein ligase E3A (UBE3A), or a variant or portion thereof.

[0305] In some aspects, a complex provided herein comprises an antigen-binding domain that specifically binds to human CD98hc and a polynucleic acid. In some aspects, a complexprovided herein comprises an antigen-binding domain that specifically binds to human CD98hc and a small interfering RNA (siRNA). In some embodiments, a complex provided herein comprises an antigen-binding domain that specifically binds to human CD98hc and an siRNA that targets tau (such as an siRNA that targets tau mRNA). Bispecific and Multi-specific Proteins

[0306] In some aspects, an antibody or antigen-binding fragment thereof provided herein comprises an antigen-binding domain that specifically binds to human CD98hc. In some aspects, an antibody or antigen-binding fragment thereof comprises 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 thereof that bind to the same epitope of CD98hc as a CD98hc antigen-binding domain provided herein. Also provided herein are antibodies or antigen-binding fragments thereof that competitively inhibit binding to CD98hc of a CD98hc antigen-binding domain provided herein.

[0307] In some aspects, a multi-specific protein provided herein comprises a first antigen- binding domain that binds to human CD98hc and a second antigen-binding domain. The first antigen-binding domain that binds to human CD98hc can be any antigen-binding domain that binds to human CD98hc provided herein. The second antigen-binding domain can be an antigen- binding domain that specifically binds to a CNS antigen or a brain antigen. In some aspects, the CNS antigen or brain antigen is not CD98hc.

[0308] In some aspects, a multi-specific protein provided herein comprises an antigen-binding domain that binds to human CD98hc linked to an antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof can bind a CNS antigen or brain antigen. In some aspects, the CNS antigen or brain antigen is not CD98hc. In some aspects, such a multi- specific protein can be in a 2+1 multi-specific binding format or a 2+2 multi-specific binding format.

[0309] In some aspects, a multi-specific protein provided herein comprises a CD98hc antigen- binding domain that is an scFv linked to an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains. In some aspects, the scFv is linked to the C-terminus of one of the two antibody heavy chains, e.g., via a protein linker.

[0310] In some aspects, the multi-specific protein comprises 1) an antigen-binding domain that binds CD98hc, 2) a second antigen-binding domain that binds a different CNS or brain antigen,and 3) an Fc region, wherein the CD98hc antigen-binding domain and the second antigen- binding domain are connected or linked to an Fc region of the multi-specific protein. In other aspects, the multi-specific protein comprises 1) an antigen-binding domain that comprises a heavy chain variable region and binds CD98hc, 2) a second antigen binding domain that comprises a heavy-chain variable region and binds a different CNS or brain antigen, and 3) an Fc region, wherein the CD98hc antigen-binding domain and the second antigen-binding domain are connected or linked to the Fc region of the multi-specific protein. In some aspects, the multi- specific protein comprises an antigen-binding domain that binds CD98hc, a second antigen- binding domain that binds 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 connected or linked to the N-terminus of the Fc portion of the multi-specific protein. In other aspects, the CD98hc antigen-binding domain is connected or linked to the N-terminus of an Fc portion of the multi-specific protein and the second antigen-binding domain is linked to the C-terminus of the Fc portion of the multi-specific protein. In other aspects, the CD98hc antigen-binding domain is connected or linked to the C-terminus of an Fc portion of the multi-specific protein and the second antigen-binding domain is linked to the N-terminus of the Fc portion of the multi-specific protein.

[0311] In some aspects, a multi-specific protein provided herein comprises two copies of a CD98hc antigen-binding domain that is an scFv and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, wherein one of the two copies of the antigen-binding domain is linked to the C-terminus of one of the antibody heavy chains, and wherein the other copy of the antigen-binding domain is linked to the C-terminus of the other antibody heavy chain. In some aspects, the scFvs are linked to heavy chains via a protein linker.

[0312] As provided herein, a complex, fusion protein, antibody, or antigen-binding fragment thereof, or multi-specific protein provided herein can be multi-specific, e.g., bi-specific. Many different formats and uses of bi-specific binding molecules are known in the art (reviewed in, e.g., Kontermann; Drug Discov Today, 2015 July; 20(7):838-47; MAbs, 2012 March-April; 4(2):182-97). A bispecific protein according to the present invention is not limited to any particular bispecific format or method of producing it. Accordingly, bispecific proteins of the present disclosure can include various configurations having a first antigen-binding domain that binds to human CD98hc and a second antigen-binding domain, e.g., that binds to a CNS antigen or a brain antigen.

[0313] Examples of bispecific molecules that can be used in the present disclosure include, e.g., (i) a single antibody that has two arms comprising different antigen-binding domains; (ii) a single chain antibody that has specificity to two different epitopes, e.g., via two scFvs linked in tandem by an extra peptide linker; (iii) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (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 resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vi) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (vii) a so-called “dock and lock” molecule, based on the “dimerization and docking domain” in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (viii) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fab-arm; and (ix) a diabody. Other examples of antibody structures are described in WO2019 / 246288, which is incorporated by reference.

[0314] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein is multivalent (e.g., bivalent). In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein is trivalent (e.g., in the 2+1 format or 2+1 multi-specific binding format). In some aspects, a trivalent format comprises a single CD98hc antigen-binding domain provided herein and two antigen-binding domains that bind to a CNS antigen or a brain antigen. The two antigen-binding domains that bind to a CNS antigen or a brain antigen can comprise the same amino acid sequence or can comprise different amino acid sequences. In some aspects, the CD98hc antigen-binding domain is an scFv. In some aspects, the CD98hc antigen-binding domain is a VHH.

[0315] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein is tetravalent (e.g., in the 2+2 format or 2+2 multi-specific binding format). In some aspects, a tetravalent format comprises two CD98hc antigen-binding domains provided herein and two antigen-binding domains that bind to a CNS antigen or a brain antigen. The two CD98hc antigen-binding domains can comprise the same amino acid sequence or can comprise different amino acid sequences. In some aspects, the two CD98hc antigen-binding domains comprise the same amino acid sequence. In some aspects, oneor both of the CD98hc antigen-binding domains is an scFv. The two antigen-binding domains that bind to a CNS antigen or a brain antigen can comprise the same amino acid sequence or can comprise different amino acid sequences.

[0316] A complex, fusion protein, antibody or antigen-binding fragment thereof, or multi- specific protein provided herein can comprise a linker, e.g., linking a CD98hc antigen-binding domain to a heterologous protein, antibody or antigen-binding fragment thereof, or other antigen- binding domain. The linker can be e.g., a glycine linker, a glycine-rich linker, or a glycine-serine linker. The linker can comprise the amino acid sequence (GGGGS)x3 (SEQ ID NO: 50). The linker can comprise the amino acid sequence (GGSGG)x3 (SEQ ID NO: 51). The linker can comprise the amino acid sequence GGSGG (no repeats) (SEQ ID NO: 52). The linker can be 1 to 20 amino acids in length.

[0317] A complex, fusion protein, antibody or antigen-binding fragment thereof, or multi- specific protein provided herein can comprise a constant region. In some aspects, a CD98hc antigen-binding domain provided herein is linked to the constant region, e.g., the C-terminus of the constant region. In some aspects, a constant domain is a human constant domain. In some aspects, a constant domain is a murine, rat, rabbit, or monkey (e.g., cynomolgus) constant domain. The constant region can be a heavy chain constant region. The constant region can be a human constant region. The constant region can be a human heavy chain constant region. The constant region can be an IgG constant region. The constant region can be an IgG1 constant region. The constant region can be an IgG2 constant region. The constant region can be an IgG4 constant region. The constant region can be a human IgG constant region. The constant region can be a human IgG1 constant region. The constant region can be a human IgG2 constant region. The constant region can be a human IgG4 constant region.

[0318] In some aspects a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein comprises a heavy chain and a light chain. With respect to the heavy chain, in some aspects, the heavy chain of an 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 can comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (µ) heavy chain. In some aspects, the heavy chain comprises a human gamma (γ) heavy chain constant region. In some aspects, the heavy chain of comprises the amino acid sequence of an IgG1 heavy chain constant region. In some aspects, the heavy chain comprises the amino acid sequence of an IgG2 (e.g., IgG2a or IgG2b) heavy chain constant region. In some aspects, the heavy chain comprises the amino acid sequence of an IgG4 heavy chain constant region. Withrespect to 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.

[0319] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein comprises constant regions comprising the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein comprises constant regions comprising the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule. In some aspects, the constant regions comprise the amino acid sequences of the constant regions of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule.

[0320] Non-limiting examples of human constant region sequences have been described in e.g., U.S. Patent No.5,693,780 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91- 3242).

[0321] In some aspects, a constant region provided herein comprises a knob mutation. In some aspects, a constant region provided herein comprises a hole mutation. Accordingly, in some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi- specific protein provided herein can comprise a constant region comprising a knob mutation and a constant region comprising a hole mutation. Bispecific or Multi-specific Proteins that Bind to CD98hc and to Cancer Antigens

[0322] In some aspects, a bispecific or multispecific protein provided herein comprises an antigen-binding domain that specifically binds to human CD98hc and an antigen-binding domain that specifically binds to a cancer antigen. Such bispecific or multi-specific proteins can comprise, for example, an antibody or antigen-binding fragment thereof that binds to a target expressed on the surface of a cell (e.g., a tumor cell) in the CNS and an anti-CD98hc antigen- binding domain. The anti-CD98hc antigen-binding domain can facilitate crossing the BBB and the antibody or antigen-binding fragment thereof can target and induce cell death of the tumorcells. The bispecific or multi-specific protein can be a bispecific or multi-specific protein with reduced effector function. Exemplary formats of a multi-specific protein comprising an antigen- binding domain that specifically binds to human CD98hc and to a cancer antigen is shown in Figure 13.

[0323] In some aspects, the bispecific or multi-specific proteins provided herein comprises an antigen-binding domain that binds to CD98hc and an antibody or antigen-binding fragment thereof that binds to CD20. In some aspects, the antibody or antigen-binding fragment thereof that binds to CD20 is rituximab. In some aspects, the antibody or antigen-binding fragment thereof that binds to CD20 is a version of rituximab with reduced effector function. In some aspects, the antibody or antigen-binding fragment thereof that binds to CD20 (e.g., rituximab) contains a LALA-P331S and / or LALA-P329S mutation and has reduced effector function. Such bispecific and multi-specific proteins can be used for treating chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma, which has manifested in the CNS and / or brain.

[0324] In some aspects, the bispecific or multi-specific proteins provided herein comprises an antigen-binding domain that binds to CD98hc and an antibody or antigen-binding fragment thereof that binds to epidermal growth factor receptor (EGFR). In some aspects, the antibody or antigen-binding fragment thereof that binds to EGFR is cetuximab. In some aspects, the antibody or antigen-binding fragment thereof that binds to EGFR is a version of cetuximab with reduced effector function. In some aspects, the antibody or antigen-binding fragment thereof that binds to EGFR (e.g., cetuximab) contains a LALA-P331S and / or LALA-P329S mutation and has reduced effector function. Such bispecific and multi-specific proteins can be used for treating head and neck cancer and colorectal cancer, which has metastasized to the CNS and / or brain.

[0325] In some aspects, the bispecific or multi-specific proteins provided herein comprises an antigen-binding domain that binds to CD98hc and an antibody or antigen-binding fragment thereof that binds to HER2. In some aspects, the antibody or antigen-binding fragment thereof that binds to HER2 is trastuzumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to HER2 is a version of trastuzumab with reduced effector function. In some aspects, the antibody or antigen-binding fragment thereof that binds to HER2 (e.g., trastuzumab) contains a LALA-P331S and / or LALA-P329S mutation and has reduced effector function. Such bispecific and multi-specific proteins can be used for treating breast cancer and gastric cancer, which has metastasized to the CNS and / or brain. In some aspects, the antibody or antigen- binding fragment thereof that binds to HER2 is pertuzumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to HER2 is a version of pertuzumab with reducedeffector function. In some aspects, the antibody or antigen-binding fragment thereof that binds to HER2 (e.g., pertuzumab) contains a LALA-P331S and / or LALA-P329S mutation and has reduced effector function. Such bispecific and multi-specific proteins can be used for treating breast cancer, which has metastasized to the CNS and / or brain.

[0326] In some aspects, the bispecific or multi-specific proteins provided herein comprises an antigen-binding domain that binds to CD98hc and an antibody or antigen-binding fragment thereof that binds to vascular endothelial growth factor (VEGF). In some aspects, the antibody or antigen-binding fragment thereof that binds to VEGF is bevacizumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to VEGF is a version of bevacizumab with reduced effector function. In some aspects, the antibody or antigen-binding fragment thereof that binds to VEGF (e.g., bevacizumab) contains a LALA-P331S and / or LALA-P329S mutation and has reduced effector function. Such bispecific and multi-specific proteins can be used for treating colorectal cancer, lung cancer, breast cancer, kidney cancer, ovarian cancer, cervical cancer, and brain cancer, which has manifested or metastasized to the CNS and / or brain.

[0327] In some aspects, the bispecific or multi-specific proteins provided herein comprises an antigen-binding domain that binds to CD98hc and an antibody or antigen-binding fragment thereof that binds to programmed cell death protein 1 (PD-1). In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-1 is nivolumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-1 is a version of nivolumab with reduced effector function. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-1 (e.g., nivolumab) contains a LALA-P331S and / or LALA-P329S mutation and has reduced effector function. Such bispecific and multi-specific proteins can be used for treating non-small cell lung cancer (NSCLC), renal cell carcinoma, gastric cancer, gastroesophageal junction cancer, esophageal cancer, melanoma, urothelial carcinoma, pleural mesothelioma, head and neck cancer, hepatocellular carcinoma, colorectal cancer, and Hodgkin lymphoma, which has manifested or metastasized to the CNS and / or brain. In some aspects, the antibody or antigen- binding fragment thereof that binds to PD-1 is pembrolizumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-1 is a version of pembrolizumab with reduced effector function. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-1 (e.g., pembrolizumab) contains a LALA-P331S and / or LALA-P329S mutation and has reduced effector function. Such bispecific and multi-specific proteins can be used for treating melanoma, NSCLC, head and neck cancer, Hodgkin lymphoma, B-cell lymphoma, urothelial cancer, bladder cancer, colorectal cancer, gastric cancer, gastroesophageal junction cancer,cervical cancer, biliary tract cancer, Merkel cell carcinoma, renal cell carcinoma, endometrial carcinoma, squamous cell carcinoma, and breast cancer, which has manifested or metastasized to the CNS and / or brain.

[0328] In some aspects, the bispecific or multi-specific proteins provided herein comprises an antigen-binding domain that binds to CD98hc and an antibody or antigen-binding fragment thereof that binds to programmed death-ligand 1 (PD-L1). In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-L1 is atezolizumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-L1 is a version of atezolizumab with reduced effector function. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-L1 (e.g., atezolizumab) contains a LALA-P331S and / or LALA-P329S mutation and has reduced effector function. Such bispecific and multi-specific proteins can be used for treating non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), hepatocellular carcinoma, melanoma, and alveolar soft part sarcoma (ALPS), which has metastasized to the CNS and / or brain. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-L1 is avelumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-L1 is a version of avelumab with reduced effector function. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-L1 (e.g., avelumab) contains a LALA-P331S and / or LALA-P329S mutation and has reduced effector function. Such bispecific and multi-specific proteins can be used for treating urothelial cancer, Merkel cell carcinoma, and renal cell carcinoma, which has metastasized to the CNS and / or brain. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-L1 is durvalumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-L1 is a version of durvalumab with reduced effector function. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-L1 (e.g., durvalumab) contains a LALA-P331S and / or LALA- P329S mutation and has reduced effector function. Such bispecific and multi-specific proteins can be used for treating non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), biliary tract cancer, and hepatocellular carcinoma, which has metastasized to the CNS and / or brain. Fc Domains and Regions

[0329] A complex, fusion protein, antibody or antigen-binding fragment thereof, or multi- specific protein provided herein can comprise an Fc domain or region or fragment thereof. In some aspects, an Fc domain or region is of IgG class, the IgM class, or the IgA class. In someaspects, an Fc domain or region or fragment thereof is an IgG Fc domain or region or fragment thereof. In some aspects, an Fc domain or region or fragment thereof is a human IgG Fc domain or region or fragment thereof. In some aspects, an Fc domain or region or fragment thereof is a human IgG1 Fc domain or region or fragment thereof. In some aspects, an Fc domain or region or fragment thereof is a human IgG2 Fc domain or region or fragment thereof. In some aspects, an Fc domain or region or fragment thereof is a human IgG4 Fc domain or fragment thereof.

[0330] In some aspects provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided comprises a modified Fc domain or region 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) R. J. 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), C226S, C229S, E233P, L234V, L234F, L235E (McEarchern et al., (2007) Blood, 109:1185-1192), P331S (Sazinsky et al., (2008) Proc Natl Acad Sci USA 2008, 105:20167-20172), S267E, L328F, A330L, M252Y, S254T, E430G, and / or T256E, where the amino acid position is 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.

[0331] In some aspects, the Fc domain or fragment thereof is a wildtype IgG2. In some aspects, the modified Fc domain or fragment thereof is a modified IgG2 Fc comprising one or more modifications. For example, in some aspects, the modified IgG2 Fc comprises one or more amino acid substitutions (e.g., relative to a wild-type IgG2 Fc).

[0332] In some aspects, the Fc domain or fragment thereof is a wildtype IgG4. In some aspects, the modified Fc domain or fragment thereof is a modified IgG4 Fc comprising one or more modifications. For example, in some aspects, the modified IgG4 Fc comprises one or more amino acid substitutions (e.g., relative to a wild-type IgG4 Fc). In some aspects, the one or more aminoacid substitutions in the modified IgG4 Fc are IgG4-S228P or IgG4-S228P / L235E, where the amino acid position is according to the EU numbering convention.

[0333] In some aspects provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein is a bi-specific complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein. Bi-specific molecules include, e.g., a kappa-lambda body, a dual-affinity re-targeting molecule (DART), a knob-in-hole antibody, a strand-exchange engineered domain body (SEEDbody), and a DuoBody. In some aspects, a bispecific complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein comprises a knob mutation and a hole mutation. In some aspects, the knob mutation comprises the amino acid substitution T366W according to EU numbering. In some aspects, the hole mutation comprises the amino acids substitutions T366S, L368A, and Y407V according to EU numbering.

[0334] In some aspects provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein comprises a mutation to promote heterodimerization of Fc regions. In some aspects, a dimerized Fc region of a bispecific provided herein is formed by Fc regions that contain amino acid mutations, substitutions, additions, or deletions to promote heterodimerization in which different polypeptides comprising different Fc domains can dimerize to yield a heterodimeric Fc region configuration. In some aspects, a bispecific of the present disclosure comprises a first Fc sequence comprising a first CH3 region, and a second Fc sequence comprising a second CH3 region, wherein the sequences of the first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions

[0335] Methods to promote heterodimerization of Fc domains include amino acid deletions, additions, or substitutions of the amino acid sequence of the Fc domain, such as by including a set of “knob-into-hole” deletions, additions, or substitutions or including amino acid deletions, additions, or substitutions to effect electrostatic steering of the Fc to favor attractive interactions among different polypeptide chains. Methods for promoting heterodimerization of complementary Fc polypeptides have been previously described in, for example, Ridgway et al, 1996, Protein Eng, 9:617-621; Merchant et al, 1998, Nature Biotechnol, 16:677-681; Moore et al, 2011, MAbs, 3:546-557; Von Kreudenstein et al, 2013, 5:646-654; Gunasekaran et al, 2010, J Biol Chem, 285:19637-19464; Leaver-Fay et al, 2016, Structure, 24:641-651; Ha et al, 2016, Frontiers in Immunology, 7:1; Davis et al, 2010, Protein Eng Des Sel, 23:195-202;WO1996 / 027011; WO1998 / 050431; WO2006 / 028936; WO2009 / 089004; 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.

[0336] In some aspects, complementary Fc polypeptides of an Fc heterodimer include a mutation to alter charge polarity across the Fc dimer interface such that co-expression of electrostatically matched Fc domains support favorable attractive interactions, thereby promoting desired Fc heterodimer formation; whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation (Guneskaran et al, 2010, J Biol Chem, 285:19637-19646). When co-expressed in a cell, association between the polypeptide chains is possible but the chains do not substantially self-associate due to charge repulsion.

[0337] Additionally, complementary Fc polypeptides of an Fc heterodimer include “knob-into- hole” configurations to promote heterodimerization of two Fc polypeptides. “Knob-into-hole” technology is described in e.g., U.S. Pat. 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 involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis. In some aspects, a knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two subunits of the Fc domain. In some aspects, the subunit of the Fc domain comprising the knob modification additionally comprises the amino acid substitution S354C, and the subunit of the Fc domain comprising the hole modification additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, thus further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).Thus, in such configurations, a first Fc polypeptide comprises amino acid modifications to form the “knob” and a second Fc polypeptide comprises amino acid modifications to form the “hole” thus forming an Fc heterodimer comprising complementary Fc polypeptides.

[0338] Exemplary paired amino acid modifications of complementary Fc polypeptides of an Fc heterodimeric configuration are set forth below in the table below (EU numbering). Table 1: Exemplary paired Fc modifications for heterodimeric Fc domains

[0339] Some agents provided herein comprise 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, e.g., WO 93 / 16185; and U.S. Patent Nos.5571894 and 5587458. For discussion of Fab and F(ab’)2fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No.5869046.

[0340] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent and / or bispecific. See, for example, EP404097; WO 1993 / 01161; Hudson et al. Nat. Med.9:129- 134 (2003). Triabodies and tetrabodies are also described in Hudson et al. Nat. Med.9:129-134 (2003). Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. Some aspects, a single-domain antibody is a human single-domain antibody (see, e.g., U.S. Patent No. 6248516).

[0341] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0342] As provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein can be chimeric. Certain chimeric antibodies are described, e.g., 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 a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody.

[0343] As provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein can be humanized. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some aspects, a humanized antibody is substantially non-immunogenic in humans. In some aspects, a humanized antibody has substantially the same affinity for a target as an antibody from another species from which the humanized antibody is derived. See, e.g., U.S. Pat. No.5530101, 5693761; 5693762; and 5585089. In some aspects, amino acids of an antibody variable domain that can be modified without diminishing the native affinity of the antigen-binding domain while reducing its immunogenicity are identified. See, e.g., U.S. Pat. Nos.5766886 and 5869619. Generally, a humanized antibody comprises one or more variable domains in which CDRs (or portions thereof) are derived from a non-human antibody, and framework regions (FRs) (or portions thereof) are derived from human antibody sequences. A humanized antibody can 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 (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0344] Humanized antibodies and methods of making them are reviewed, for example, in Almagro et al. Front. Biosci.13:1619-1633 (2008), and are further described, e.g., in US Patent Nos.5821337, 7527791, 6982321, and 7087409. Human framework regions that can be used for humanization include but are not limited to: framework regions selected using the “best- fit” method (see, e.g., Sims et al. J. Immunol.151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variableregions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al., J. Immunol.151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson Front. Biosci.13:1619-1633 (2008)); and framework regions 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)).

[0345] As provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein can be human. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk et al. Curr. Opin. Pharmacol.5:368-74 (2001) and Lonberg Curr. Opin. Immunol. 20:450-459 (2008).

[0346] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. One can engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci in anticipation that such mice would produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can preserve the large variable gene diversity as well as the proper regulation of antibody production and expression. By exploiting the mouse machinery for antibody diversification and selection and the lack of immunological tolerance to human proteins, the reproduced human antibody repertoire in these mouse strains can yield high affinity fully human antibodies against any antigen of interest, including human antigens. Using the hybridoma technology, antigen-specific human MAbs with the desired specificity can be produced and selected. Certain exemplary methods are described in U.S. Pat. No.5545807, EP 546073, and EP 546073. See also, for example, U.S. Patent Nos.6075181 and 6150584 describing XENOMOUSE™ technology; U.S. Patent No.5770429 describing HUMAB® technology; U.S. Patent No.7041870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007 / 0061900, describing VELOCIMOUSE® technology. Human variable regions from intact antibodies generated by such animals can be further modified, e.g., by combining with a different human constant region.

[0347] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol.133:3001 (1984) and Boerner et al. J. Immunol.147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al. Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additionalmethods include those described, for example, in U.S. Patent No.7189826 (describing 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 generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0348] In some aspects provided herein, an antibody is a human antibody isolated by in vitro methods and / or screening combinatorial libraries for antibodies with the desired activity or 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), and the like. In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. Ann. Rev. Immunol.12: 433-455 (1994). For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. See also Sidhu et al. J. Mol. Biol. 338(2): 299-310, 2004; Lee et al. J. Mol. Biol.340(5): 1073-1093, 2004; Fellouse Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al. J. Immunol. Methods 284( -2):119- 132 (2004). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al. EMBO J.12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers comprising random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom et al. J. Mol. Biol., 227: 381-388, 1992. Patent publications describing human antibody phage libraries include, for example: US Patent No. 5750373, and US Patent Publication Nos.2007 / 0292936 and 2009 / 0002360. Antibodies isolatedfrom human antibody libraries are considered human antibodies or human antibody fragments herein.

[0349] As provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein can comprise an antigen-binding domain that binds to a CNS antigen or a brain antigen. In some aspects, the CNS antigen or brain antigen can be beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), or Transmembrane Protein 106B (TMEM106b). In some aspects, the CNS antigen or brain antigen can be beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), ubiquitin protein ligase E3A (UBE3A), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33, sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), sialic acid binding Ig-like lectin 11 (Siglec11), glycoprotein nonmetastatic melanoma protein B (GPNMB), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), MSA4A4E, Transmembrane Protein 106B (TMEM106b), CR1, ABCA1, ABCA7, HLA-DR1, HLA-DR5, IL1RAP, TREML2, IL-34, SORL1, and ADAM1.

[0350] In some aspects, the CNS antigen or brain antigen can be beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha- synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebrosidase (GCase or GBA), progranulin (PGRN),Prosaposin (PSAP), ubiquitin protein ligase E3A (UBE3A), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33, sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), sialic acid binding Ig-like lectin 11 (Siglec11), glycoprotein nonmetastatic melanoma protein B (GPNMB), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), MSA4A4E, Transmembrane Protein 106B (TMEM106b), CR1, ABCA1, ABCA7, HLA-DR1, HLA-DR5, IL1RAP, TREML2, IL-34, SORL1, Reelin, very low density lipoprotein receptor (VLDLR), apolipoprotein E receptor 2 (APOER2; also known as low-density lipoprotein receptor-related protein 8 (LRP8)), and ADAM1.

[0351] In some aspects, the CNS antigen or brain antigen can be beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha- synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, 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 expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), or Transmembrane Protein 106B (TMEM106b).

[0352] In some aspects, the CNS or brain antigen is on a cancer cell within the central nervous system. In some aspects, the CNS or brain antigen is a cell surface target on a hematological 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 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), GD2, GD3, GloboH (globohexasylceramide), gp100, GPNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B,PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, ROR1, TF (tissue factor), and TROP2.

[0353] As provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein can comprise an antigen-binding domain that binds to a CNS antigen or a brain antigen. As provided herein, a multi-specific protein provided herein can comprise an antigen-binding domain that binds to a CNS antigen or a brain antigen. The antigen-binding domain that binds to a CNS antigen or a brain antigen can comprise a VH and a VL. Exemplary CNS antigen-binding VH and VL sequences are provided below. Additional VH and VL and antigen-binding domain sequences are found in US2017 / 0224702, US 2018 / 0002433, US 2021 / 0236634, and US 2021 / 0238265, each of which is herein incorporated by reference in its entirety. Table 2: Exemplary CNS Antigens

[0354] As provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein can be capable of crossing the CD98hc as a result of the fact that the anti-CD98 antigen-binding domain in the complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein is capable of crossing the CD98hc.

[0355] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein is internalized in blood-brain barrier epithelial cells greater than 10-fold as compared to internalization by an isotype control. The blood-brain barrier endothelial cells can be, e.g., HCMEC / D3 cells.

[0356] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein does not reduce cell-surface expression of CD98hc on HCMEC / D3 cells by more than 20% relative to cell-surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western blot or FACS.

[0357] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein does not increase cell-surface expression of CD98hc on HCMEC / D3 cells by more than 50% relative to cell-surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western blot or FACS.

[0358] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein does not reduce cell-surface expression ofCD98hc on HCMEC / D3 cells by more than 20% relative to cell-surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control and does not increase cell-surface expression of CD98hc on HCMEC / D3 cells by more than 50% relative to cell-surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western blot or FACS.

[0359] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein accumulates at least 1.5-fold more than an isotype control in vessel-depleted mouse brain. In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein accumulates at least 2-fold more than an isotype control in vessel-depleted mouse brain. In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi- specific protein provided herein accumulates at least 3-fold more than an isotype control in vessel-depleted mouse brain. In some aspects, a complex, fusion protein, antibody or antigen- binding fragment thereof, or multi-specific protein provided herein accumulates at least 5-fold more than an isotype control in vessel-depleted mouse brain. In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein accumulates at least 10-fold more than an isotype control in vessel-depleted mouse brain. In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi- specific protein provided herein accumulates at least 20-fold more than an isotype control in vessel-depleted mouse brain.

[0360] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein has at least a 5-fold increase in brain concentration ratio over an isotype control 24 hours after administration to a mouse.

[0361] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein binds human CD98hc with an equilibrium dissociation constant (KD) of about 18 nM to about 87 nM. In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein binds cynomolgus monkey CD98hc with a KD of about 46 nM to about 531 nM. In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein binds human CD98hc with an equilibrium dissociation constant (KD) of about 18 nM to about 87 nM and binds cynomolgus monkey CD98hc with a KDof about 46 nM to about 531 nM.Antibody Drug Conjugates Comprising Anti-CD98hc Antigen-Binding Domains

[0362] In some aspects, provided herein are antibody drug conjugates (ADCs) comprising an anti-CD98hc antigen-binding domain. Such ADCs can comprise, for example, an antibody or antigen-binding fragment thereof that binds to a target expressed on the surface of a cell (e.g., a tumor cell) in the CNS, a cytotoxic drug, and an anti-CD98hc antigen-binding domain. In some aspects, such an ADC further comprises a linker. For example, the linker can conjugate the cytotoxic drug to the antibody or antigen-binding fragment thereof that binds to a target expressed on the surface of a cell (e.g., a tumor cell) in the CNS. The anti-CD98hc antigen- binding domain can facilitate crossing the BBB; the antibody or antigen-binding fragment thereof can target the cytotoxic drug to cells that express the target (e.g., tumor cells); and the cytotoxic drug can kill the tumor cells. In some aspects, such ADCs have effector function. In some aspects, such ADCs have reduced effector function. In some aspects, the ADCs comprise an antibody or antigen-binding fragment with LALA-P331S and / or LALA-P329S. Such ADCs can be useful, e.g., for the treatment of CNS cancers, glioblastoma, and brain metastatis of a solid cancer.

[0363] In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, an antibody or antigen-binding fragment thereof that binds to tissue factor (TF), and a cytotoxic drug. In some aspects, an ADC provided herein comprises an anti- CD98hc antigen-binding domain provided herein, tisotumab or antigen-binding fragment thereof, and a cytotoxic drug, e.g., monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, tisotumab or antigen- binding fragment thereof with reduced effector function, and a cytotoxic drug, e.g., monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen- binding domain provided herein, an antibody or antigen-binding fragment thereof that binds to to a target expressed on the surface of a cell (e.g., a tumor cell) in the CNS, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen- binding domain provided herein, tisotumab or antigen-binding fragment thereof, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain and tisotumab vedotin. Such ADCs can be useful, e.g., for the treatment of glioblastoma.

[0364] In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, an antibody or antigen-binding fragment thereof that binds to humanepidermal growth factor receptor (HER2), and a cytotoxic drug, e.g., monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, trastuzumab or antigen-binding fragment thereof, and a cytotoxic drug. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, trastuzumab or antigen-binding fragment thereof, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, trastuzumab or antigen-binding fragment thereof with reduced effector function, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain and disitamab vedotin. Such ADCs can be useful, e.g., for the treatment of brain metastasis of breast cancer or other solid tumors.

[0365] In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, an antibody or antigen-binding fragment thereof that binds to human B7-H4, and a cytotoxic drug e.g., monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, brentuximab or antigen-binding fragment thereof, and a cytotoxic drug. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, brentuximab or antigen-binding fragment thereof, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, brentuximab or antigen-binding fragment thereof with reduced effector function, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain and felmetatug vedotin. Such ADCs can be useful, e.g., for the treatment of brain metastasis. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, an antibody or antigen-binding fragment thereof that binds to human nectin-4, and a cytotoxic drug e.g., monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, enfortumab or antigen- binding fragment thereof, and a cytotoxic drug. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, enfortumab or antigen- binding fragment thereof, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, enfortumab or antigen-binding fragment thereof with reduced effector function, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain and enfortumab vedotin. Such ADCs can be useful, e.g., for the treatment of brain metastasis.

[0366] In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, an antibody or antigen-binding fragment thereof that binds to human HER2, and a cytotoxic drug e.g., deruxtecan. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, trastuzumab or antigen- binding fragment thereof, and a cytotoxic drug. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, trastuzumab or antigen- binding fragment thereof, and deruxtecan. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain provided herein, trastuzumab or antigen-binding fragment thereof with reduced effector function, and deruxtecan. In some aspects, an ADC provided herein comprises an anti-CD98hc antigen-binding domain and trastuzumab deruxtecan. Such ADCs can be useful, e.g., for the treatment of brain metastasis of breast cancer or other solid tumors. Polynucleotides and Methods of Making Anti-CD98hc Antigen-Binding Domains and Agents Comprising the Same

[0367] In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein or a domain thereof described herein, and vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells).

[0368] In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically bind to human CD98hc provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the light chain of an antigen-binding domain that specifically bind to human CD98hc provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically bind to human CD98hc provided herein and a nucleic acid molecule encoding the light chain of an antigen-binding domain that specifically bind to human CD98hc provided herein.

[0369] In some aspects, combinations or compositions of polynucleotides are provided herein. In some aspects, a combination or composition comprises a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode a multi-specific protein provided herein, e.g., 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 provided herein, and the third polynucleotide encodes a light chain. In some aspects, the antigen-binding domains that bind to human CD98hc is an scFv. In some aspects, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation. In some aspects, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0370] In some aspects, a combination or composition comprises a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode a multi-specific 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 scFvs. 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.

[0371] In some aspects, a combination or composition comprises a first polynucleotide and a second polynucleotide, wherein the first and second polynucleotides encode a multi-specific protein provided herein, wherein the first polynucleotide encodes a heavy chain and an antigen- binding domain that bind to human CD98hc provided herein, and wherein the second polynucleotide encodes a light chain.

[0372] Also provided herein are polynucleotides comprising a nucleotide sequence encoding an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein, that are optimized, e.g., by codon / RNA optimization, replacement with heterologous signal sequences, and / or elimination of mRNA instability elements. Methods to generate optimized nucleic acids for recombinant expression by introducing codon changes (e.g., a codon change that encodes the same amino acid due to the degeneracy of the genetic code) and / or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Patent Nos.5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly.

[0373] A polynucleotide comprising a nucleotide sequence encoding an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen- binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein, can be generated from 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 hybridizable to the 3’ and 5’ ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising, e.g., the sequence encoding the light chain and / or heavy chain of an antigen-binding domain, antibody, or antigen-binding fragment thereof. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to generate an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein.

[0374] Polynucleotides provided herein can be, e.g., in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and DNA can be double- stranded or single-stranded. If single stranded, DNA can be the coding strand or non-coding (anti-sense) strand. In some aspects, the polynucleotide is a cDNA or a DNA lacking one more endogenous introns. In some aspects, a polynucleotide is a non-naturally occurring polynucleotide. In some aspects, a polynucleotide is recombinantly produced. In some aspects, the polynucleotides are isolated. In some aspects, the polynucleotides are substantially pure.

[0375] In some aspects, polynucleotides provided herein are in the form of RNA. In some aspects, polynucleotides provided herein are in the form of RNA encoding a complex or fusion protein provided herein. In some aspects, a polynucleotide provided herein is a synthetic messenger RNA (mRNA). In some aspects, the synthetic mRNA has at least one nucleoside modification. In some aspects, the at least one nucleoside modification is selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4- thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl- uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl- pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1- methyl-1-deaza-pseudouridine, dihydrouridine, 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-pseudoisocytidine, 2-thio-cytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl- 1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5- methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2- aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2- aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, 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.

[0376] In some aspects, provided herein are polynucleotides encoding a complex or fusion protein comprising an antigen-binding protein provided herein and a heterologous polypeptide. In some aspects, the heterologous polypeptide comprises an antigen binding domain that binds to beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), glycoprotein nonmetastatic melanoma protein B (GPNMB), Paired immunoglobin like type 2 receptor alpha (PILRA), MembraneSpanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains 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 a complex or fusion protein comprising an antigen-binding domain provided herein and a heterologous polypeptide. In some aspects, the heterologous polypeptide comprises the amino acid sequence of beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β-glucocerebrosidase (GBA), progranulin (PGRN), Prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), glycoprotein nonmetastatic melanoma protein B (GPNMB), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), or Transmembrane Protein 106B (TMEM106b), or a portion thereof. In some aspects, the multi-specific protein comprises an antigen binding domain that binds to ubiquitin protein ligase E3A (UBE3A). In some aspects, the polynucleotide is mRNA (e.g., synthetic mRNA).

[0377] In some aspects, the heterologous polypeptide comprises an antigen binding domain that binds to beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, 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 expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), or Transmembrane Protein 106B (TMEM106b). In some aspects, the polynucleotide is mRNA (e.g., synthetic mRNA).

[0378] In some aspects, disclosed herein are polynucleotides encoding a complex or fusion protein disclosed herein comprising a heterologous polypeptide. In some aspects, the heterologous polypeptide is an ERT enzyme or an ERT enzyme variant, or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide comprises β-glucocerebrosidase (GBA), progranulin (PGRN), Prosaposin (PSAP), or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide in a complex or fusion protein provided herein is a growth factor. In some aspects, the heterologous polypeptide in a complex or fusion protein provided herein is a decoy receptor.In some aspects, the heterologous polypeptide in a complex or fusion protein provided herein is progranulin (PGRN), prosaposin (PSAP), or survival motor neuron protein (SMN). In some aspects, the heterologous protein is an enzyme selected from α-L Iduronidase (IDUA), Iduronate-2-sulphatase (IDS), N-acetylgalactoslamine-6-sulphatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulphatase (arylsulphatase B; ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramide beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta- hexosaminidase B, arylsulphatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme replacement thereof, such as larodinase, idursulphase, elosulphase alpha or galsuphase, or a variant thereof, or a catalytically active fragment thereof. In some aspects, the heterologous protein is an enzyme selected from clusterin (APOJ), Reelin, ubiquitin protein ligase E3A (UBE3A), Tripeptidyl Peptidase 1 (CLN2 / TPP1), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N- acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), α-L Iduronidase (IDUA), Iduronate-2-sulphatase (IDS), N-acetylgalactoslamine-6-sulphatase (GALNS), N- sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulphatase (arylsulphatase B; ARSB), acid sphingomyelinase (ASM), β-glucocerebrosidase (GCase or GBA), galactosylceramide beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta- hexosaminidase B, arylsulphatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme replacement thereof, such as larodinase, idursulphase, elosulphase alpha or galsuphase, or a variant thereof, or a catalytically active fragment thereofα-L Iduronidase (IDUA), Iduronate-2-sulphatase (IDS), N- acetylgalactoslamine-6-sulphatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N- acetylgalactosamine-4-sulphatase (arylsulphatase B; ARSB), acid sphingomyelinase (ASM), β- glucocerebrosidase (GCase or GBA), galactosylceramide beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta-hexosaminidase B, arylsulphatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme replacement thereof, such as larodinase, idursulphase, elosulphase alpha or galsuphase, or a variant thereof, or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide in a complex or fusion protein provided herein is β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), clusterin (APOJ), Reelin, Tripeptidyl Peptidase 1 (CLN2 / TPP1), or Alpha-L-Iduronidase (IDUA), Iduronate 2-Sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), N-sulfoglucosamine sulfohydrolase (SGSH), ubiquitin protein ligase E3A (UBE3A), or a variant or portion thereof. In some aspects, the polynucleotide is mRNA (e.g., synthetic mRNA).

[0379] In some aspects, the heterologous polypeptide in a complex or fusion protein provided herein is β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), clusterin (APOJ), Reelin, very low density lipoprotein receptor (VLDLR), apolipoprotein E receptor 2 (APOER2; also known as low-density lipoprotein receptor-related protein 8 (LRP8)), Tripeptidyl Peptidase 1 (CLN2 / TPP1), or Alpha-L-Iduronidase (IDUA), Iduronate 2-Sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N- acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), N- sulfoglucosamine sulfohydrolase (SGSH), ubiquitin protein ligase E3A (UBE3A), or a variant or portion thereof. In some aspects, the polynucleotide is mRNA (e.g., synthetic mRNA).

[0380] In some aspects, disclosed herein are polynucleotides encoding a complex or fusion protein disclosed herein comprising a heterologous polypeptide. In some aspects, the heterologous polypeptide is an ERT enzyme or an ERT enzyme variant, or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide comprises β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), clusterin (APOJ), Reelin, Tripeptidyl Peptidase 1 (CLN2 / TPP1), or Alpha-L-Iduronidase (IDUA), Iduronate 2-Sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), N-sulfoglucosamine sulfohydrolase (SGSH), or ubiquitin protein ligase E3A (UBE3A), or a variant or portion thereof.

[0381] In certain aspects, provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein, for recombinant expression in a host cell, e.g., in a mammalian host cell. A vector for the productionof the antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein, can be produced, e.g., by recombinant DNA technology using techniques well known in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein, operably linked to a promoter. Such vectors can, for example, include the nucleotide sequence encoding the constant region of an antigen-binding domain, antibody or antigen-binding fragment thereof (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464), and variable domains of the antigen-binding domain, antibody or antigen-binding fragment thereof can be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains. In some aspects, the vector is gene therapy vector (e.g., an AAV or lentiviral vector).

[0382] In certain aspects, provided herein are expression systems comprising polynucleotides comprising nucleotide sequences encoding an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi- specific protein described herein, or a domain thereof described herein. An expression system can be included on a vector. An expression system can also be integrated into a host cell chromosome. In some aspects, an expression system is a cell free expression system. In some aspects, an expressions system comprises a host cell comprising a polynucleotide and / or vector provided herein.

[0383] Accordingly, also provided herein are cells, e.g., host cells, comprising polynucleotides and / or vectors for recombinantly expressing an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi- specific protein described herein, or a domain thereof described herein. In some aspects, for the expression of double-chained antigen-binding proteins, vectors encoding both the heavy and light chains, individually, can be co-expressed in the host cell for expression of the entire immunoglobulin. In some aspects, a host cell contains two different vectors, a first vector comprising a polynucleotide encoding a heavy chain of an antigen-binding protein described herein, and a second vector comprising a polynucleotide encoding a light chain of an antigen- binding protein. In some aspects, a first host cell comprises a first vector comprising apolynucleotide encoding a heavy chain, and a second host cell comprises a second vector comprising a polynucleotide encoding a light chain. In some aspects, provided herein is a population of host cells comprising such first host cell and such second host cell.

[0384] In some aspects, provided herein are methods for producing an antigen-binding domain that specifically binds to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein in a host cell. In some aspects, provided herein are methods for producing a single chain antigen- binding domain that specifically binds to human CD98hc, an Fc domain, and a heterologous protein or polypeptide, as described herein in a host cell. In some aspects, provided herein are methods for producing a single chain antigen-binding domain that specifically binds to human CD98hc, an Fc domain, and a second antigen-binding domain, as described herein in a host cell. An expression vector can be transferred to a cell (e.g., host cell) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce an antigen-binding domain that specifically binds to human CD98hc, complex, fusion protein, antibody, antigen- binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein.

[0385] A variety of host-expression vector systems can be utilized to express an antigen- binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein (see, e.g., U.S. Patent No.5,807,715). Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid)containing coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH3T3, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB / 20 and BMT10 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In some aspects, cells for expressing an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein are CHO cells, for example CHO cells from the CHO GS System™ (Lonza). In some aspects, cells for expressing an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein as described herein are human cells, e.g., human cell lines. In some aspects, a mammalian expression vector is pOptiVEC™ or pcDNA3.3. In some aspects, bacterial cells, such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells) are used for the expression of an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein. For example, mammalian cells such as Chinese hamster ovary (CHO) cells in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-105; and Cockett MI et al., (1990) Biotechnology 8: 662-667). In some aspects, an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein is produced by CHO cells or NS0 cells.

[0386] In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can contribute to the function of the protein. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO,HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells.

[0387] Once an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein has been produced by recombinant expression, it can be purified by any method known in the art for purification, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein can be fused to heterologous polypeptide sequences to facilitate purification.

[0388] In some aspects, once an antigen-binding domain that specifically binds to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein is isolated or purified. Generally, an isolated antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein is one that is substantially free of other proteins. For example, in some aspects, a preparation of Once an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein is substantially free of cellular material and / or chemical precursors. Pharmaceutical Compositions

[0389] Provided herein are compositions comprising an antigen-binding domain that specifically binds to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein. In some aspects, the antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen- binding fragment thereof, or multi-specific protein having the desired degree of purity is present in a formulation comprising, e.g., 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 recipients at the dosages and concentrations employed. Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can comprise antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0390] In some aspects, a pharmaceutical composition comprises an antigen-binding domain that specifically bind to human CD98hc, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, and a pharmaceutically acceptable carrier (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). Pharmaceutical compositions described herein are, in some aspects, for use as a medicament. The compositions to be used for in vivo administration can be sterile. This is readily accomplished by filtration through, e.g., sterile filtration membranes.

[0391] Also provided herein are pharmaceutical compositions comprising a polynucleotide encoding an antigen-binding domain that specifically binds to human CD98hc, a complex, a fusion protein, an antibody or antigen-binding fragment thereof, or a multi-specific protein described herein. In some aspects, the polynucleotide is RNA. In some aspects, the polynucleotide is synthetic mRNA. In some aspects, the pharmaceutical composition comprising a polynucleotide further comprises a lipid-based transfection reagent.

[0392] A pharmaceutical composition described herein can be used to exert a biological effect(s) in vivo or in vitro. For example, a pharmaceutical composition described herein can be used to cross a blood brain barrier, e.g., in a subject.

[0393] In some aspects, a pharmaceutical composition provided herein is used to treat diseases or conditions such as a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation. In some aspects, a pharmaceutical composition provided herein is used to treat diseases or conditions such as Alzheimer’s disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman’s syndrome, Liddle syndrome, Parkinson’s disease, Pick’s disease, Paget’s disease, cancer, and traumatic brain injury. In some aspects, a pharmaceutical composition provided herein is used to treat frontotemporal dementia.

[0394] In some aspects, a pharmaceutical composition provided herein is formulated for intravenous administration. In some aspects, a pharmaceutical composition provided herein is formulated for subcutaneous administration. Methods of Using Anti-CD98hc Antigen-Binding Domains and Agents Comprising the Same

[0395] Antigen-binding domains that specifically bind to human CD98hc, complexes, fusion proteins, antibodies, antigen-binding fragments thereof, and multi-specific proteins comprising such antigen-binding domains as provided herein can advantageously be transported across a blood brain barrier. Accordingly, provided herein are methods of administering or transporting an antigen-binding protein that specifically binds to human CD98hc or a complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein comprising an antigen- binding protein that specifically binds to human CD98hc 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 complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein comprising an antigen-binding protein that specifically binds to human CD98hc.

[0396] In view of the ability of antigen-binding domains that specifically bind to human CD98hc, complexes, fusion proteins, antibodies, antigen-binding fragments thereof, and multi- specific proteins comprising such antigen-binding domains as provided herein to be transported across a blood brain barrier, they can be used to treat a neurological disease or disorder. In some aspects, a method of treating a neurological disease or disorder in a subject comprises administering to the subject an antigen-binding protein that specifically binds to human CD98hc or a complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein comprising an antigen-binding protein that specifically binds to human CD98hc. The neurological disease or disorder can be, for example, a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, or CNS inflammation. The neurological 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 disease and supranuclear palsy), a prion disease (such as bovine spongiform encephalopathy,scrapie, Creutz-feldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, a nervous system heterodegenerative disorders (such as Canavan disease, Huntington’s disease, neuronal ceroid-lipofuscinosis, Alexander’s disease, Tourette’s syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (such as Pick’s disease, and spinocerebellar ataxia), cancer of the CNS and / or brain (such as glioblastoma or brain metastases resulting from cancer elsewhere in the body), Alzheimer’s disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), limbic-predominant age-related TDP-43 encephalopathy (LATE), cystic fibrosis, Angelman’s syndrome, Liddle syndrome, Parkinson’s disease, Pick’s disease, Paget’s disease, cancer, or traumatic brain injury. In some aspects, the neurological disease or disorder is dementia. In some aspects, the neurological disease or disorder is frontotemporal dementia. In some aspects, the neurological disease or disorder is Alzheimer’s disease. In some aspects, the neurological disease or disorder is Parkinson’s disease. In some aspects, the neurological disease or disorder is frontal temporal epilepsy. In some aspects, the neurological disease or disorder is autism. In some aspects, the neurological disease or disorder is lissencephaly.

[0397] In some aspects, provided herein is a method of treating a lysosomal storage disease with a complex or fusion protein disclosed 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.

[0398] Antigen-binding domains that specifically bind to human CD98hc, complexes, fusion proteins, antibodies, antigen-binding fragments thereof, and multi-specific proteins comprising such antigen-binding domains as provided herein can be used to detect an antigen (e.g., a CNS antigen or a brain antigen). Antigen-binding domains that specifically bind to human CD98hc and complexes, fusion proteins, antibodies, antigen-binding fragments thereof, and multi-specific proteins comprising such antigen-binding domains for such purposes can be labeled. Exemplary labels include, for example, radioisotopes (e.g.,64CU) and fluorescent labels. Accordingly, methods of detecting an antigen using an antigen-binding protein that specifically binds to human CD98hc or a complex, fusion protein, antibody, antigen-binding fragment thereof, or multi- specific protein comprising an antigen-binding protein that specifically binds to human CD98hc described herein are provided. In some aspects, a method of detecting an antigen in the CNS(e.g., brain) of a subject comprises administering an antigen-binding protein that specifically binds to human CD98hc or a complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein comprising an antigen-binding protein that specifically binds to human CD98hc to the antigen in the CNS (e.g., brain). Such methods can further comprise, e.g., performing Positron emission tomography (PET) imaging on the subject. In some aspects, disclosed herein is a method of detecting a CNS antigen in vitro, comprising contacting an in vitro sample with a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein disclosed herein and locating the imaging agent within the sample.

[0399] Antigen-binding domains that specifically bind to human CD98hc and complexes, fusion proteins, antibodies, antigen-binding fragments thereof, and multi-specific proteins comprising such antigen-binding domains as provided herein can be used for prognostic, diagnostic, monitoring, and / or screening applications, including in vivo applications well known and standard to the skilled artisan and based on the present description. In some aspects, provided herein is an antigen-binding protein that specifically binds to human CD98hc or a complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein comprising an antigen-binding protein that specifically binds to human CD98hc for use as a diagnostic. In some aspects, the antigen-binding protein that specifically binds to human CD98hc or a complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein comprising an antigen-binding protein that specifically binds to human CD98hc comprises a detectable label.

[0400] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0401] The present disclosure will be more fully understood by reference to the following Examples. They should not, however, be construed as limiting the scope of the present disclosure. All citations throughout the disclosure are hereby expressly incorporated by reference. EXAMPLES Example 1: Production of Avi-His tagged variants of CD98 Heavy Chain (CD98hc)

[0402] To generate Avi-His tagged variants of the extra-cellular domain (ECD) of human, cynomolgus macaque (cyno), and mouse CD98 heavy chain (CD98hc), the following methods were used. Mammalian expression of human, cyno and mouse variants of BBB receptor antigens (SEQ ID NOs: 65-67) was performed by cloning synthetic genes based on cDNA intomammalian expression vectors, followed by transient transfection and expression in Expi293 cells. Constructs included a heterologous signal peptide and C-terminal Avi-His tag to allow for purification and biotinylation. Briefly, plasmids encoding the antigens were transfected using the Expifectamine 293 Transfection kit (ThermoFisher A14524) according to the manufacturer’s specifications. Five days after transfection, the culture supernatants were harvested, clarified by centrifugation, and purified using HisPur Ni-NTA resin (Thermo Scientific 88223) in a drip- column format.200mL of culture supernatant was filtered using 0.2 µm filtration unit and 3mL of resin slurry in PBS was added to the filtered supernatant. The sample was incubated overnight with shaking at 4ºC, and on the following day, the beads were loaded onto 20 mL drip columns, washed with 10 mL of His-Select wash buffer (Millipore Sigma H5288) and eluted with 5mL of His-Pur elution buffer (Millipore Sigma H5413). Eluate was buffer exchanged into PBS using Amicon Ultra-15 centrifugal filter units (Millipore UFC9010). Quantification of the antibody concentration was determined by measuring the absorbance at 280 nm using the Nanodrop 8000 (ThermoFisher). Purity of the antigens was determined by SDS-PAGE. The antigens were analyzed with size exclusion chromatography (SEC) for aggregation. Some antigens were biotinylated using a BirA biotin-protein ligase kit (AVIDITY), according to the manufacturer’s instructions. Table 3. Avi-His tagged variants of CD98hc ECDExample 2: Production of CHO cell lines over-expressing CD98hc

[0403] Several cell lines were generated for screening the binding of anti-CD98hc antibodies, as described herein. Briefly, pLenti-EF1a constructs each expressing full length human CD98hc or mouse CD98hc (SEQ ID NOs: 68-69, respectively) were used to generate CHO cells stably expressing human CD98hc or mouse CD98hc. Lentiviral constructs (Genecopoeia) were used to express human CD98hc with puromycin selection and mouse CD98hc with GFP as a reporter. Lentivirus was generated from transfection of 293T cells using the ViraSafe™ Lentiviral Packaging System (CellBiolabs VPK-206). Subsequently, the supernatant containing lentivirus was used to transduce Chinese Hamster Ovary (CHO) cells. At 2 days post transduction, puromycin was added to the medium as a selection pressure for the cells expressing human CD98hc. CHO cells expressing mouse CD98hc were FACS sorted for positive GFP signal on the FACS Aria system (BD Biosciences). The resulting CHO cells stably expressing human CD98hc and mouse CD98hc were analyzed for cell surface expression by flow cytometry.Table 4. Full Length CD98hc SequencesExample 3: Generation of CD98hc humanized mice

[0404] A humanized mouse line expressing the human extracellular domain of CD98hc was generated (Taconic Biosciences GmbH (Germany)). CRISPR was used to replace the mouse ECD with the human version, while retaining the mouse intracellular and transmembrane portions under the control of the mouse promoter. Brain slices from these mice were evaluated byIHC and / or Western Blot to confirm expression and localization of the human ECD in vivo (data not shown). Example 4: Generation of anti-CD98hc hybridoma antibodies

[0405] In order to obtain antibodies against CD98hc, the following procedures 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 injections of purified extracellular domain polypeptides of human, cyno, and / or mouse CD98hc (obtained as described above in Example 1) with or without adjuvant. After a total of 6-8 injections and three days following the final boost, the lymph nodes were harvested from the mice for hybridoma cell line generation.

[0406] Sera from the animals were analyzed for reactivity to CD98hc by FACS on CHO cells overexpressing human or mouse CD98hc (as obtained in Example 2) and by ELISA against human, cyno and mouse CD98hc Avi-His polypeptides (as described below). Lymphocytes from animals whose sera demonstrated strong binding to CHO cells overexpressing human or mouse CD98hc were isolated and fused with myeloma fusion partners to produce mouse hybridoma cells via electrofusion (Hybrimune, BTX, Holliston, MA) and incubated at 37°C, 5% CO2, overnight in Clonacell-HY Medium C (Stemcell Technologies, Vancouver, BC, Canada, Cat# 03803).

[0407] The fused hybridomas were recovered overnight, resuspended in ClonaCell-HY Medium C with anti-mouse IgG Fc-FITC and mixed into Clonacell-HY Medium D. The cells were plated into Nunc OmniTrays (Thermo Fisher Scientific, Rochester, NY) and allowed to grow at 37°C, 5% CO2 for 8 days. The Clonepix 2 (Molecular Devices, Sunnyvale, CA) system was used to select and transfer IgG positive colonies into 96-well plates with high glucose DMEM culture media containing 10% Fetal Clone 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, Cat# 03805).

[0408] In total, 531 IgG positive hybridoma clones were isolated. After six days of culture, tissue culture supernatants from the hybridomas were screened by FACS on CHO cells overexpressing human or mouse CD98hc (as described below).Example 5: Screening of anti-CD98hc antibody hybridoma supernatants by FACS

[0409] The IgG positive hybridoma supernatants were initially screened for their ability to differentially bind CHO cells overexpressing human or mouse CD98hc cells compared to CHO parental cells by FACS. Overexpressing cells were harvested, washed, and labeled with various concentrations and combinations of CellTrace cell proliferation dyes CFSE and Violet (ThermoFisher, Cat#C34554 and Cat#34557, respectively) to create uniquely barcoded cell populations. Barcoded cells were aliquoted into 96-well U-bottom plates and incubated with 50μl of hybridoma cell culture supernatant or 1μg / ml of commercially available purified mouse anti-human CD98hc monoclonal antibody (Sigma Aldrich, Cat# SAB 4700503) and rat anti- mouse CD98hc antibody (Bio-Rad Laboratories, Hercules, CA, Cat# MCA2684) on ice for 30 minutes. After this primary incubation, the supernatants were removed via centrifugation, the cells were washed twice with 175μl of ice-cold FACS buffer (PBS + 1% FBS + 2mM EDTA), and the cells were then further incubated on ice for 20 minutes with anti-mouse IgG Fc- allophycocyanin (APC) or anti-rat IgG Fc-APC (Jackson Labs, Cat# 115-136-071 and Cat # 112- 136-071, respectively), diluted 1:1000 in FACS buffer. Following this secondary antibody incubation, the cells were again washed twice with ice-cold FACS buffer and resuspended in a final volume of 50μl of FACS buffer containing 0.25μl / well propidium iodide (BD Biosciences, Cat#556463). Binding intensity on cells was analyzed using the FACS Canto system (BD Biosciences), with sorting gates drawn to exclude dead (i.e., propidium iodide-positive) cells. For each barcoded cell population, the ratio of APC Mean Fluorescence Intensity (MFI) divided by secondary antibody MFI was calculated for each anti-CD98hc hybridoma supernatant tested.

[0410] A total of 78 hybridoma clones displayed an MFI ratio greater than 2-fold for binding to CHO cells stably overexpressing human CD98hc.77 of these clones also bound with an MFI ratio greater than 5-fold for binding to HCMEC / D3 endothelial cells endogenously expressing human CD98hc. None of the hybridoma clones showed binding to CHO cells stably overexpressing mouse CD98hc. Data are shown in Table 5 for a subset of the FACS positive clones.Table 5. FACS MFI values of unique anti-CD98hc antibodies identified from hybridoma campaignsExample 6: Screening of anti-CD98hc antibody hybridoma supernatants by recombinant CD98hc protein binding assay

[0411] Hybridoma culture supernatants from 78 hybridomas (obtained from Example 5) were screened for their ability to bind Avi-His-tagged human, cyno, and mouse CD98hc (prepared as described in Example 1) and compared to binding to Avi-His-tagged human transferrin receptor (TfR), a control protein. Briefly, 96-well polystyrene plates were coated with 5µg / ml of streptavidin (Thermo Fisher, Cat# PI21125) in coating buffer (0.05M carbonate buffer, pH 9.6, Sigma, Cat# C3041) overnight at 4oC. Coated plates were then blocked with ELISA diluent (PBS + 0.5% BSA + 0.05% Tween20) for one hour. Blocking buffer was removed and human, cyno, or mouse Avi-His-tagged CD98hc and Avi-His-tagged human TfR polypeptides were added at 1µg / ml in ELISA diluent and captured for 1 hour at room temperature. After washing three times with 300μl of PBST (PBS + 0.05% Tween20, Thermo 28352), the hybridoma cell culture supernatants or 1μg / ml of commercially available purified mouse anti-human CD98hc monoclonal antibody (Sigma Aldrich, Cat# SAB 4700503) and rat anti-mouse CD98hc antibody (Bio-Rad Laboratories, Hercules, CA, Cat# MCA2684) were added (50µl / well) to each well. After 30 minutes of incubation at room temperature, the plates were washed three times with 300μl of PBST. Anti-mouse IgG Fc-HRP or anti-rat IgG Fc-HRP (Jackson Immunoresearch, Cat#115-035-071 and 112-036-071, respectively) secondary antibodies were diluted 1:5000 in ELISA diluent, added to each well at 50μl / well, and incubated for 30 minutes at roomtemperature with shaking. After a final set of washes (3x300μl in PBST), 50μl / well of BioFx TMB substrate (Surmodics, Eden Praire, MN, Cat#TMBW-1000-01) was added to the wells. The reaction was then quenched after 5-10 mins with 50μl / well of 2N sulfuric acid. The plates were read for absorbance at 450nm on a SpectraMax M5 (Molecular Devices, Sunnyvale, CA) using SoftMax Pro software. ELISA data for selected antibodies is shown in Table 4 below.

[0412] From this hybridoma supernatant screen, all 78 of the anti-CD98hc hybridoma clones had OD450values greater than 0.45 to human and cyno CD98hc. None of the clones bound to mouse CD98hc. Table 6. ELISA OD450values of unique anti-CD98hc antibodies identified from hybridoma campaigns 3 and 4.Example 7: In Vitro Internalization of Anti-CD98hc Antibodies into a Blood-Brain Barrier Endothelial Cell Line

[0413] Supernatants from the 78 anti-CD98hc hybridoma clones were purified using ProPlus Phytip columns (Biotage, Uppsala, Sweden, Cat# PTH 91-20-07) on a Hamilton STAR platform (Hamilton Company, Reno, NV). Briefly, antibodies from the supernatants were captured by protein A coupled on resin-packed tips, washed twice with PBS, eluted with Pierce IgG elution buffer (ThermoFisher, Cat# 21004), and neutralized with 1M Tris-HCl pH8 to a final pH of 6.0. The concentrations of the purified antibodies were determined by measuring the absorbance at 280 nm using the Nanodrop 8000 (ThermoFisher). Hybridoma purified antibodies were then tested for their ability to internalize into endothelial cells.

[0414] Internalization into endothelial cells at the blood-brain barrier is the first stage of transcytosis across the BBB and into the brain. To identify anti-CD98hc antibodies with ability tointernalize, HCMEC / D3 cells (a cell line derived from human brain endothelial cells; Weksler B. et al.) were seeded at 2.5*10^4 cells / well in a black wall clear bottom 96-well plate (#3904, Corning). The next day, cells were treated with 6 μg / ml of anti-CD98hc antibodies pre- conjugated with an equal concentration of pHrodo-Red labeling reagent (Z25612, Invitrogen) in 100 μl of culture medium (EBM2, Lonza). A human IgG isotype and an anti-CD98hc reference antibody with known internalization ability were included in the assay as negative and positive controls, respectively.

[0415] Plates were then placed into an IncuCyte machine (live-cell analysis system), and images were captured every 2 hours over a 24 hour time course. Images were then processed and analyzed using IncuCyte software. Internalization data at the 24 hour timepoint (pHrodo Red positive area µm2 / image) are shown in Table 5 as relative fold-change to isotype hIgG1. Table 7. Internalization fold-change of purified unique anti-CD98hc antibodies identified from hybridoma campaigns relative to isotype hIgG1Example 8: Molecular Cloning of Anti-CD98hc Antibodies

[0416] Anti-CD98hc antibodies from the 78 hybridomas described above were cloned as follows for recombinant expression. Anti-CD98hc antibodies were selected based on FACS positive data (described above in Example 5).1-2x105hybridoma cells were harvested, washed with PBS, and resuspended in 200μl of RNAlater (Invitrogen, Cat#. AM7021). Samples were stored at -80°C and sent to Abterra Biosciences (San Diego, CA) for RNA extraction, cDNA synthesis and sequencing. Briefly, the variable regions of IgG / IgM, IgK, and IgL were amplified using proprietary primers in a 5’ RACE strategy. Hybridoma variable region amplicons were sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA). Reads from the hybridomas were processed through Abterra’s Reptor analysis pipeline. Out of the 78 hybridoma clones, 63 had unique sequences.

[0417] Exemplary unique amino acid sequences of the variable heavy chains and variable light chains of anti-CD98hc antibodies are provided below in Table 8. Tables 9 and 10 provide theCDR sequences (Kabat) for each of the unique VH and VL sequences in Table 8. The antibody CD98hc.04.064.1e is a humanized version of CD98hc04.064, that was humanized in the manner described in Example 13. Table 8. Unique VH / VL sequences derived from hybridoma campaignsTable 9. CDR Heavy Chain sequences derived from hybridoma campaignsTable 10. CDR Light Chain sequences derived from hybridoma campaignsExample 9: Generation of Antibodies in mvFc-scFv Format

[0418] From the panel of 63 unique anti-CD98hc hybridoma clones, a subset of antibodies were selected for further reformatting based on various criteria: 1) the antibodies covered a broad range of affinities based on ELISA and FACS binding assays, 2) the antibodies covered a broad range of internalization and cell uptake activity, 3) the antibodies were phylogenetically diverse from each other within the hybridoma sequences obtained, and 4) the antibodies had similar binding kinetics to human and cyno CD98hc.

[0419] The variable regions of the selected clones were cloned into a monovalent Fc-scFv format (mvFc-scFv) as shown in Figure 2. The anti-CD98hc scFv VH and VL domains are connected by a 20 amino acid linker, GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 48) (Bird et al, 1988), while the scFv is connected to the C-terminus of the mvFc by a 5 amino acid linker, GGSGG (SEQ ID NO: 52). Exemplary anti-CD98hc scFv amino acid sequences in the orientation VH-linker-VL are provided below in Table 11.

[0420] The cloning of the mvFc-scFv is described as follows. DNA sequences encoding the mvFc-scFv antibody were prepared by either expression construct synthesis (Twist Biosciences) or by gene synthesis and cloned into the expression vector pcDNA3.4 (ThermoFisher). The corresponding light chain expression constructs were also generated. Table 11. scFv Sequences of Humanized and Engineered antibodies

[0421] Recombinant antibodies were generated via transient transfection with Expi293 cells (ThermoFisher Scientific, #A14635) at 2x106cells / mL density with Expifectamine 293 / plasmid DNA complex according to manufacturer’s protocol (ThermoFisher Scientific). Clarified supernatants were harvested and purified using protein A affinity chromatography, and if necessary, further purified by cation exchange chromatography on an AKTA Pure (Cytiva) to remove product-related impurities. The purified antibodies were dialyzed into PBS, pH7.4. Analytical characterization was performed by absorbance at 280nm, CE-SDS, size-exclusion chromatography, and endotoxin measurement. Example 10: Affinity Characterization of mvFc-scFv Antibodies

[0422] Binding kinetics of anti-CD98hc mvFc-scFv antibodies to human and cyno CD98hc were evaluated using a Carterra LSA instrument (Carterra, Salt Lake City, UT). Briefly, a HC200M sensor chip was loaded with goat anti-human Fc antibody (Jackson ImmunoResearch, Cat 109-005-098) via sulfo-NHS (Thermo Scientific 24510) / EDC (Thermo Scientific PG82079) coupling and remaining capture surfaces blocked with 1M Ethanolamine (Carterra, 3628). Anti- CD98hc mvFc-scFvs were captured onto individual spots on the sensor chip (n=4 spots per antibody). Eight serially diluted concentrations of human and cyno CD98hc (0.2 – 5000 nM) were injected over all spots for five minutes of association, followed by 10 minutes of dissociation with regeneration performed using 10mM glycine-HCl pH 1.7. Data was processed and analyzed using NextGenKIT high throughput kinetics analysis software (Carterra). The equilibrium dissociation constants (KD) were calculated from the fitted association and dissociation rate constants. The values were combined, means and standard deviation calculated, and graphs prepared using GraphPad Prism. The KD values are summarized in Table 12 below.Table 12. Binding Affinities of Selected mvFc-scFv Variants to Human and Cyno CD98hcExample 11: Cell uptake of mvFc-scFv antibodies

[0423] To determine relevant cellular uptake and localization of anti-CD98hc antibodies, wild- type HCMEC / D3 cells were used in an acute 2-hour incubation study. Cells were seeded onto black 96-well plates (Revvity) and allowed to adhere for an additional 24-48 hours. Anti-CD98hc antibodies were then incubated at 200 nM or 40 nM at 37°C in HCMEC / D3 growth media.

[0424] After 2 hours, cells were washed with room temperature PBS and fixed with PFA. Cells were then washed with PBS followed by simultaneous permeabilization and block for 1 hour at RT. Secondary detection antibody (anti-human IgG AlexaFluor-488 or 647, Jackson ImmunoResearch) was incubated overnight at 4°C along with DAPI. The next day cells were washed 2x in PBS and imaged using the Operetta High Content Imaging system (Revvity). Mean fluorescent intensities (MFI) per cell or total area of signal per cell, per field, per well were quantified and recorded for relative comparisons between antibody variants. The MFI of each sample was normalized by subtracting the MFI of the isotype control.

[0425] The cell uptake data is summarized in Table 13. All of the anti-CD98hc antibodies tested had a concentration dependent cellular uptake in the hCMEC / D3 cells, with CD98hc.04.048 having the highest uptake at both concentrations tested. The isotype control did not show any staining. Antibodies with in vitro cell uptake were screened in vivo for brain uptake. Table 13. Normalized Mean Fluorescent Intensities (MFI) of anti-CD98hc mvFc-scFv AntibodiesExample 12: In Vivo Brain Uptake of anti-CD98hc Antibodies

[0426] To establish in vivo proof of concept for brain penetration of anti-CD98hc antibodies, huCD98hc ECD+ / +knock-in homozygous mice in the C57Bl / 6 background at 7-9 weeks of agewere dosed with antibodies at 20 mg / kg via intravenous injection of the tail vein. Blood samples were collected at 1 hour, 4 hours and 24 hours post-dose and processed to serum to determine peripheral clearance of the injected antibodies over time. Animals were sacrificed 24 hours post- dose, terminally bled, perfused and whole brains were harvested. Subsequently, homogenized brain tissue samples were centrifuged to separate the vessel portion from the parenchyma portion and were frozen on dry ice and stored at −80 °C until analysis. Total protein concentration in the lysates was determined by BCA Assay (Pierce #23225). The validity of this method has been confirmed using western blot analysis, in which markers of brain endothelial cells were absent in the parenchymal portions.

[0427] Antibody concentrations in the brain and serum samples were measured using an MSD (Meso Scale Discovery) assay. Antibody concentrations were normalized to the total protein concentration in the lysate and then graphed as fold-change over the control antibody.

[0428] Brain uptake data for CD98hc.04.048 is summarized in Figure 3A. In this experiment, all anti-CD98hc antibodies tested showed increased brain uptake at the 24 hr timepoint, with CD98hc.04.064.1e showing ~3.5X enrichment and CD98hc.04.048 showing a 5.5X-fold enrichment compared to a format matched isotype control. Serum PK data for this experiment is shown in Figure 3B. An increase in serum clearance was seen for both antibodies containing anti- CD98hc binding domains at the 24 hr timepoint. This enhancement in serum clearance was modest and not substantially different between CD98hc.04.064.1e and CD98hc.04.048 antibodies. Example 13: Humanization and Engineering of CD98hc.04.048 Antibodies

[0429] The CD98hc.04.048 antibody was chosen for humanization based on its function in cell uptake assay, kinetic profile, antibody expression profile and in vivo brain uptake.

[0430] One of the most common methods of humanizing a non-human antibody is to transplant the CDRs from a non-human antibody onto a human antibody acceptor framework. Frequently, such CDR transplantation results in attenuation or complete loss of affinity of the humanized antibody due to perturbation in its framework. As a result, certain residues from the mouse framework may need to be retained to replace the human residues at the corresponding positions (back mutations) in order to restore attenuated or lost affinity. Therefore, it is crucial to precisely predict the residues to be retained in the context of the selected human antibody germline acceptor framework that can retain functions and paratopes of the humanized antibody. Inaddition, retained or improved thermal stability and solubility are desired for good manufacturability and downstream development.

[0431] Structure-based antibody modeling was applied in the process of humanizing anti- CD98hc mouse monoclonal antibodies (mAbs) utilizing the BioMOE module of MOE (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada). Briefly, VH and VL sequences of the mouse mAb sequence to be humanized were compared to human VL, VH, LJ, HJ functional germline sequences taken from IMGT (http: / / www.imgt.org / ). Pseudo-genes and ORFs were excluded. Per one mouse mAb (query), five most similar VL and five most similar VH germline sequences were selected and combined with the most similar VJ and HJ genes, producing 25 humanized sequences. The CDRs to be transplanted onto the human framework were defined according to the AbM definition (http: / / www.bioinf.org.uk / abs / #cdrdef).

[0432] Two humanized sequences were selected based on the frequency of their VH and VL frameworks in human repertoire. The query and the humanized sequences were used to create Fv homology models. The BioMOE module or the Antibody Modeler module of MOE (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada) was utilized to create Fv homology models. AMBER10:EHT force field was used for energy minimization through the entire antibody homology modeling process. Based on the Fv homology models, molecular descriptors such as interaction energy between VL and VH, coordinate-based isoelectric point (3D pI), hydrophobic patch, and charged surface area were calculated, analyzed, and sorted by scoring metrics provided by MOE. These molecular descriptors were utilized to prioritize the humanized mAbs for downstream experimental procedures, including protein expression, purification, and binding affinity test, and functional assays.

[0433] The BioMOE module of MOE provides a tool, Mutation Site Properties, to visualize and classify potential residues for back-mutation. Back-mutation is defined as amino acid substitution which is reverted to the original query sequence replacing the humanized sequence. Using this tool, the original query (reference) was compared individually to the selected humanized variants for both the primary amino acid sequence and the 3D structure of the 3D Fv homology model.

[0434] The changes between the reference and the humanized variant were classified based on amino acid type difference, interaction potential with CDR residues, impact potential for VL / VH pairing, and potential change in hydrophobic and charged surface area in and near the CDRs.

[0435] Mutations that are near the CDRs or the VL / VH interface, have a significant charge difference or contain strong H-bond interactions were individually evaluated and the significantly disrupting mutations were reverted back to the original query residues. As a result, humanized sequences may contain up to five back mutations.

[0436] Using this method, CD98hc.04.048 was humanized to generate CD98hc.04.048.WH1, the sequence of which is listed in Tables 14-16 below. Additional engineering was carried out to tune the affinity and remove potential liability sites. In one approach, a potential Asp isomerization site at position 69, was removed by replacing Asp 69 and Ser 70 separately with different amino acid residues. By using overlap extension polymerase chain reactions (Higuchi, et al), 23 engineered variants were generated as described above. The purified antibodies were characterized in vitro for affinity and cellular uptake. Selected sequences of the engineered antibodies with favorable properties are listed in Tables 14-16 below. Table 14. VH and VL Sequences of Humanized and Engineered antibodiesTable 15. CDR Heavy Chain Sequences of Humanized and Engineered VariantsTable 16. CDR Light Chain Sequences of Humanized and Engineered VariantsTable 17. VH and VL Sequences of Humanized and Engineered VariantsExample 14: Binding Kinetics of Humanized and Engineered Variants

[0437] Binding kinetics of the CD98hc.04.048 humanized and engineered antibodies to human and cyno CD98hc described above were evaluated using the Biacore T200 (Cytiva). Briefly, antibodies were diluted to 10 μg / mL and captured using a Cytiva Anti-Fab (Cytiva, # 28958325) surface on a CM4 chip that was prepared by amine coupling according to the instrument manufacturer’s recommendations. The captured antibodies were tested for their ability to bind human and cyno CD98hc recombinant proteins as follows.

[0438] The human and cyno CD98hc analytes were diluted in running buffer (HBS-EP+, Teknova, #8022, with 0.5 mg / mL BSA, MP Biomedicals LLC, #820451) to a concentration of 333 nM, and then diluted 3-fold serially to 111, 37, and 12.3 nM. Each sample was injected for 90 seconds to allow association, followed by dissociation in buffer alone for 5 minutes. Each sample injection was followed by three 30-second injections of 10 mM glycine pH 2.1 to regenerate the chip. Fresh antibody was captured at the beginning of each cycle.

[0439] Data were analyzed using Biacore evaluation software to generate kinetic constants. The equilibrium dissociation constants (KD) were calculated from the fitted association and dissociation rate constants (k-on and k-off) for each of the anti-CD98hc antibodies. The KD values are summarized in Table 18 below.Table 18. Kinetic Rate Constants for Humanized and Engineered Variants to Human and Cyno CD98hcExample 15: In Vitro Cell Uptake of Humanized and Engineered Variants

[0440] The humanized and engineered variants were screened for cellular uptake and localization on wild-type HCMEC / D3 cells as described in the example above. Antibodies were tested in a titration series starting at 1000 nM and serially diluted 4-fold down to 0.06 nM. The cell uptake data is summarized in Figure 4A and 4B. All of the anti-CD98 antibodies tested displayed concentration dependent cellular uptake in the hCMEC / D3 cells, and the isotype control did not show any staining. Example 16: In Vivo Brain Uptake of Humanized anti-CD98hc Antibodies

[0441] Brain uptake of the humanized anti-CD98hc antibodies CD98hc.04.064.1e and CD98hc.04.048.WH1 was assayed in a time course study in huCD98hc ECD+ / + knock-in homozygous mice. The animal study was conducted as described in Example 12, where the antibodies were dosed at 20 mg / kg via intravenous injection of the tail vein. The animal takedowns were conducted at 24, 48 and 96 hours post IV injection.

[0442] Antibody concentrations in brain samples were measured in duplicates using the MSD method described above. Test article levels in hCD98hc KI + / + mice serum were measured using a custom Gyrolab assay based on a stepwise sandwich format with a biotin labeled Goat anti- human IgG (Catalog No.2049-08; Southern Biotech) as the capturing reagent and an Alexafluor- 647 labeled anti-human IgG (Catalog No.2049-31; Southern Biotech) as the detection reagent (Figure 6).

[0443] Brain uptake results are shown in Figures 5A-5B. Anti-CD98hc antibodies showed significant increase in brain uptake at all timepoints with the absolute values peaking at T=2 days and continuing to retain strong brain uptake at the day 4 timepoint (Figure 5A).CD98hc.04.048.WH1 had >40x increase in brain uptake at the day 4 timepoint as compared to the format-matched isotype control (Figure 5B). Example 17: In Vivo Brain Uptake of anti-CD98hc Antibodies in 2+1 Format

[0444] Brain uptake of anti-CD98hc antibodies is assayed in a 2+1 multi-specific binding protein format with an isotype control payload using the same methods as described above. Engineered binding domains are reformatted into scFvs and cloned into a 2+1 multi-specific binding protein format.2+1 antibodies are transiently transfected in Expi293 or ExpiCHO cells, purified on an AKTA system using Protein A column chromatography, and polished by various methods including size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC) and ion-exchange chromatography (IEX) to produce material with a high percentage of monomeric, heterodimeric 2+1 antibody, as determined by analytical SEC and mass spectrometry. Purified 2+1 anti-CD98hc antibodies are confirmed to retain binding to huCD98hc protein by SPR and for their ability to internalize into hCMEC / D3 cells, as described above.

[0445] Brain uptake of engineered anti-CD98hc 2+1 antibodies is confirmed in hCD98hc+ / + ECD KI mice as described above using an MSD assay. Example 18: Ability of anti-CD98 Antibodies to Modulate Amino Acid Transport

[0446] Since CD98hc forms heterodimers with CD98hc amino acid transporters, anti-CD98hc antibodies are tested to assess whether they interfere with the biological functions of amino acid transport. The amino acid transport level in the presence or absence of the anti-CD98hc antibodies is evaluated in wild-type HCMEC / D3 cells via a bioluminescent assay (Promega JE9400). The BCAA-Glo assay rapidly detects the branched chain amino acids (BCAA) leucine, valine and isoleucine using Leucine Dehydrogenase enzyme to produce NADH in the presence of BCAA, followed by NADH measurement using a bioluminescent NADH detection technology. HCMEC / D3 cells are seeded into clear tissue culture treated 96-well plates and allowed to adhere for an additional 24 hours. Anti-CD98hc antibodies are then incubated at varying dilutions in serum-free DMEM at 37 °C overnight. The next day, cells are processed according to assay manufacturer instructions and luminescence is recorded with an integration time of 1 second per well (Molecular Devices SpectraMax M5).Example 19: Ability of anti-CD98 Antibodies to Modulate Integrin-Dependent Signaling

[0447] CD98hc also forms heterodimers with integrins, which can trigger downstream signaling through kinases such as PI3k (Feral et al, 2004). Anti-CD98hc antibodies are tested to assess whether they modulate integrin-dependent signaling, which could impact cell adhesion or growth. One major signaling pathway impacted by CD98hc-dependent integrin signaling is the PI3k-AKT pathway, which terminates in AKT phosphorylation which can lead to many downstream effects. An ideal anti-CD98hc brain antibody for blood-brain barrier delivery would not constitutively activate or block native levels of signaling through this pathway.

[0448] The level of AKT phosphorylation in the presence or absence of the anti-CD98hc antibodies is evaluated in wild-type HCMEC / D3 cells via a commercial ELISA kit (Sigma RAB0012). The Phospho-AKT (pSer473) / pan-AKT ELISA is used to measure phospho-AKT (pSer473) relative to total AKT1 in lysates of treated cells upon incubation in pre-coated 96-well plates according to manufacturer’s instructions. Example 20: Ability of anti-CD98hc Antibodies to Affect CD98hc Levels or Localization

[0449] Binding of anti-CD98hc antibodies to their receptor on target cells could dysregulate its cell surface expression or recycling. To assess this, HCMEC / D3 cells treated with CD98hc antibodies are assessed for effects on cell surface and total cellular levels of CD98hc by flow cytometry (FACs) and quantitative western blot, respectively. HCMEC / D3 cells are seeded on appropriate tissue culture plates in culture medium (MV2, Sigma C22020). After 24 hours, cells are treated with anti-CD98hc antibodies for 24 hours at various concentrations before analysis by either flow cytometry (FACS) or western blot. Surface levels of CD98hc on dissociated cells are assessed by FACs using a non-competing anti-CD98hc antibody. Mean fluorescence intensities are obtained and analyzed using FlowJo software and Graphpad Prism. The total amount of CD98hc protein in cell lysatesis assessed using quantitative Western blot analysis and values normalized to a housekeeping protein such as GAPDH. Example 21: In Vivo Brain Uptake of anti-CD98hc Antibodies in 2+1 Format

[0450] Brain uptake of humanized anti-CD98hc antibody CD98hc.04.048.WH1 was assayed in a 2-week time course study in huCD98hc ECD+ / + knock-in homozygous mice, in the 2+1 antibody format with an isotype Fab payload. The CD98hc.04.048.WH1 antibody binding domains were cloned into the 2+1 format and produced as described above in Example 17. The animal study was conducted as described above in Example 12: animals were dosed with anti-CD98hc antibodies at 20 mg / kg with via intravenous injection of the tail vein. Animals were sacrificed for analysis at 1-, 4-, 7-, and 14-days post injection (3 animals per antibody per timepoint were used in these studies).

[0451] Antibody concentrations in brain samples were measured in duplicate using MSD methods as described above. Additional brain tissues were used for immunohistochemistry (IHC) analysis, and the results are described in Example 22. Whole blood samples were also collected at the terminal timepoint to evaluate hematological parameters, and the results are described in Example 23.

[0452] Brain uptake results from these studies are shown in FIG.7A and FIG.7B. Anti- CD98hc antibody CD98hc.04.048.WH1 showed a significant increase in brain uptake at all timepoints examined, with the absolute values peaking at T=7 days and remaining highly elevated even at 14 days post-antibody administration (FIG.7A). At the day 7 timepoint, brain uptake levels were 55 ng of antibody per mg of total protein for anti-CD98hc antibody CD98hc.04.048.WH1 compared to 2.7 ng of antibody per mg of total protein observed in isotype control antibody animals (FIG.7A). These results showed a 20-fold increase in brain uptake of anti-CD98hc antibody CD98hc.04.048.WH1 compared to that observed with isotype control antibody (FIG.7B). Example 22: Immunohistochemistry (IHC) Analysis of Brain Tissues from Mice Administered anti-CD98hc Antibody CD98hc.04.048.WH1 in 2+1 Format

[0453] Mouse brains from animals administered anti-CD98hc antibody CD98hc.04.048.WH1 and isotype control antibodies were drop-fixed in 4% PFA for 48 hours, then transferred to a 30% sucrose / PBS solution, and sectioned with a sliding microtome into 30 µm-thick sections. The sections were stained using the free-floating technique in 24-well plates. After washing in PBS, sections were permeabilized in 0.25% Triton X-100 / PBS at room temperature, then blocked in blocking buffer (2% goat serum, 2% BSA blocker, 0.05% Tween / PBS). The sections were then incubated at 4° C in secondary antibody solution. The following day, the sections were washed in 3 × 5 min PBS, then mounted onto slides. The stained sections were imaged at 20X with AxioScan.Z1 (Zeiss, Germany).

[0454] IHC analysis showed a substantial and broad increase in antibody staining present in the brains of animals administered anti-CD98hc antibody CD98hc.04.048.WH1 compared to that observed in animals administered isotype control antibody (FIG.8). As shown, the overall IHC staining pattern in mice administered anti-CD98hc antibody CD98hc.04.048.WH1 was diffuse inthese mouse brain samples. These results indicate that anti-CD98hc antibody CD98hc.04.048.WH1 without having a second binding region (i.e., without a second binding region directed to a specific brain target) was not directed towards a particular brain region but rather exhibited a more diffuse and widespread biodistribution in mice brains. Example 23: Evaluation of Hematological Parameters in Mice Administered anti-CD98hc Antibody CD98hc.04.048.WH1 in 2+1 Format

[0455] Whole blood samples from animals administered anti-CD98hc antibody CD98hc.04.048.WH1 and isotype control antibody were obtained from the animals prior to perfusion for hematology analysis (performed at Idexx Laboratories, Fremont CA) using a fully automated diagnostic instrument SYSMEX XT-V. The complete blood count (CBC) and reticulocyte count were measured by flow cytometry.

[0456] As shown in FIG.9, no major changes in red blood cell count (a key hematological parameter) were observed in animals administered anti-CD98hc antibody CD98hc.04.048.WH1. Hemoglobin levels also remained unchanged in animals administered anti-CD98hc antibody CD98hc.04.048.WH1 (data not shown). Example 24: Affinity Engineering of Anti-CD98hc Antibody CD98hc.04.048.WH1

[0457] Affinity variants of anti-CD98hc antibody CD98hc.04.048.WH1 were generated as follows. Amino acid substitutions within the variable heavy chains and variable light chains were introduced at various selected positions within or near the CDR regions of anti-CD98hc antibody CD98hc.04.048.WH1. The antibody variants were expressed in Expi293 or ExpiCHO cell cultures and either the supernatants or purified antibodies of these variants were subsequently used to evaluate binding and cell uptake.

[0458] Following binding and cell uptake evaluation, a subset of these variants were generated in monovalent scFv and Fab antibody formats for further characterization, as described below.

[0459] The antibody sequences of the CD98hc.04.048.WH1 affinity-engineered antibody variants are provided in Tables 19-21 below. Table 19. Heavy Chain CDR Sequences of CD98hc.04.048.WH1 Antibody VariantsTable 20. Light chain CDR Sequences of CD98hc.04.048.WH1 Antibody VariantsTable 21. VH and VL Sequences of CD98hc.04.048.WH1 Antibody VariantsExample 25: Binding Kinetics of CD98hc.04.048.WH1 Antibody Variants

[0460] Binding kinetics of CD98hc.04.048.WH1 affinity engineered antibody variants to human and cynomolgus CD98hc were evaluated using Biacore T200 (Cytiva). Binding domains were tested in both Fab and scFv monovalent formats. Briefly, CD98hc.04.048.WH1 variants were diluted and captured using a Cytiva Anti-Fab (Cytiva, # 28958325) surface on a CM4 chip that was prepared by amine coupling according to the instrument manufacturer’s recommendations. The captured binding domains were tested for their ability to bind human and cynomolgus CD98hc recombinant proteins as follows.

[0461] The human and cynomolgus CD98hc recombinant protein analytes were diluted in running buffer to a concentration of 1 µM, and then diluted 3-fold serially. Each sample injection was followed by three 30-second injections of 10 mM glycine pH 2.1 to regenerate the chip. Fresh binding domains were captured at the beginning of each cycle.

[0462] Data were analyzed using Biacore evaluation software to generate kinetic constants. The equilibrium dissociation constants (KD) were calculated from the fitted association and dissociation rate constants (k-on and k-off) for each of the CD98hc.04.048.WH1 antibody variants. The KDvalues for bindig domains in monovalent Fab format are summarized in Table 22, and the KD values for binding domains in monovalent scFv format are summarized in Table 23.Table 22. Kinetic Rate Constants for CD98hc.04.048.WH1 Affinity Variants in Monovalent Fab Format for Binding to Human and Cynomolgus CD98hcTable 23. Kinetic Rate Constants for CD98hc.04.048.WH1 Affinity Variants in Monovalent scFv Format for Binding to Human and Cynomolgus CD98hcExample 26: Brain Uptake of CD98hc.04.048.WH1 Affinity-Engineered Antibody Variants

[0463] To examine brain uptake of affinity-engineered CD98hc.04.048.WH1 antibody variants of the present disclosure, a subset of them were injected into huCD98hc ECD + / + knock-in homozygous mice in the C57Bl / 6 background at 7-9 weeks of age. Animals were administered CD98hc.04.048.WH1 antibody variants at 20 mg / kg via intravenous injection of the tail vein. Animals were sacrificed 48 hours following antibody administration, terminally bled, and perfused. Whole brains were harvested. Homogenized brain tissue samples were centrifuged to separate the vessel portion from the parenchyma portion and were frozen on dry ice and stored at −80 °C until analysis. Total protein concentration in the lysates was determined by BCA Assay (Pierce #23225). The validity of this method to remove blood vessels was confirmed using western blot analysis, in which markers of brain endothelial cells were absent in the parenchymal portions used in these studies.

[0464] Antibody concentrations in the brain and serum samples were measured using an MSD (Meso Scale Discovery) assay as described above. Antibody concentrations were normalized to the total protein concentration in the lysate and then graphed as fold-change over the format- matched control antibody.

[0465] Brain uptake measurements obtained for the CD98hc.04.048.WH1 affinity-engineered antibody variants are shown in FIG.10A (ng binding domain / mg total protein) and FIG.10B (fold-change over control). In these studies, all CD98hc.04.048.WH1 affinity-engineered antibody variants tested showed increased brain uptake at the 48 hour timepoint and showed a 3.5-fold to 8-fold enrichment in brain uptake as compared to a format-matched antibody isotype control.Example 27: Effect of CD98hc.04.048.WH1 Antibody Affinity Variants on Amino Acid Uptake

[0466] The ability of CD98hc.04.048.WH1 affinity-engineered antibody variants to affect amino acid uptake was evaluated using a commercially available bioluminescent assay (Promega JE9400) as described in Example 18. Briefly, hCMEC / D3 cells were seeded into clear tissue culture treated 96-well plates and allowed to adhere for an additional 24 hours. Anti-CD98hc antibodies were then added at varying dilutions in serum-free DMEM, and the cells were incubated at 37°C overnight. The following day, the cells were processed according to assay manufacturer instructions, and luminescence was recorded with an integration time of 1 second per well (Molecular Devices SpectraMax M5).

[0467] As shown in FIG.11, none of the CD98hc.04.048.WH1 antibody variants tested in this assay decreased amino acid uptake. These results indicated that use of CD98hc.04.048.WH1 antibody variants of the present disclosure to transport molecules across the blood brain barrier would not interfere with amino acid transport. Example 28: Anti-CD98hc / multi-specific binding proteins show significant enhancement of brain penetration

[0468] Mice were injected intravenously with a single 20 mg / kg dose of the following antibodies: anti-CD98hc.04.048.WH1, the isotype IgG control, anti-GPNMB antibody, or anti- GPNMB / CD98hc.04.048.WH1 multi-specific antibody. After 72 hours, antibody levels in vessel- depleted mouse brain fractions were determined. As shown in FIGs.12A-12B, inclusion of a CD98hc.04.048.WH1 binding moiety greatly increased antibody brain penetration compared to that observed with isotype control antibody and compared to that observed with anti-GPNMB antibody without the CD98hc binding moiety. Taken together, these results demonstrated that significantly higher brain parenchyma concentrations of anti-GPNMB antibodies were achieved with an anti-GPNMB antibody / anti-CD98hc multi-specific binding protein. Example 29: Engineering of a Rituximab – Anti-CD98hc Bispecific Protein

[0469] A multi-specific protein comprising an anti-CD98hc antigen-binding domain and rituximab with an effector-function silencing mutation (LALA-PS) was created in silico. The multi-specific protein comprising an anti-CD98hc antigen-binding domain and rituximab could be designed in one of two exemplary formats as shown in FIG.13. Table 24 shows the sequencesfor a multi-specific protein comprising an anti-CD98hc antigen-binding domain and rituximab with a LALA-PS sequence. Table 24. Sequences to be used in a Multi-specific Protein Comprising an Anti-CD98hc Antigen-Binding Domain and LALA-P331S Rituximab

[0470] Table 25 provides the sequences to create a multi-specific protein comprising an anti- CD98hc antigen-binding domain and a LALA-P331S rituximab. In some aspects, the constant domain of the heavy chain contains LALA-P329S. Figure 13 provides exemplary formats forthis multi-specific protein with the sequences in Table 25 forming the multi-specific protein in a 2+1 format. Table 25. Sequences of a Multi-specific Protein Comprising an Anti-CD98hc Antigen- Binding Domain and LALA-PS Rituximab

[0471] The multi-specific protein comprising an anti-CD98hc antigen-binding domain and a LALA-PS rituximab will be produced in cells. The produced multi-specific protein will be administered in in vitro and in vivo models to demonstrate their anti-cancer effect on CD20- expressing cancers.Example 30: Brain Uptake of Affinity-Engineered Anti-CD98hc.04.048.WH1 Monovalent Fab Antibody Variants

[0472] Brain uptake of various a...

Claims

CLAIMS What is claimed is:

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 comprising the amino acid sequences of: (i) SEQ ID NOs: 70, 31, 12, 93, 17, and 18, respectively; (ii) SEQ ID NOs: 70, 31, 12, 16, 17, and 18, respectively; (iii) SEQ ID NOs: 70, 75, 12, 16, 17, and 18, respectively; (iv) SEQ ID NOs: 70, 80, 12, 16, 17 and 18, respectively; (v) SEQ ID NOs:70, 35, 12, 16, 17 and 18, respectively; (vi) SEQ ID NOs: 70, 7412, 16, 17, and 18, respectively; (vii) SEQ ID NOs: 70, 33, 31, 16, 17, and 18, respectively; (viii) SEQ ID NOs: 70, 7612, 16, 17, and 18, respectively; (ix) SEQ ID NOs: 70, 77, 12, 16, 17, and 18, respectively; (x) SEQ ID NOs: 70, 78, 12, 16, 17, and 18, respectively; (xi) SEQ ID NOs:70, 79, 12, 16, 17, and 18, respectively; (xii) SEQ ID NOs:70, 34, 12, 16, 17, and 18, respectively; (xiii) SEQ ID NOs:70, 31, 12, 91, 17, and 18, respectively; (xiv) SEQ ID NOs:70, 31, 12, 92, 17, and 18, respectively; (xv) SEQ ID NOs:70, 71, 12, 16, 17, and 18, respectively; (xvi) SEQ ID NOs:70, 31, 12, 94, 17, and 18, respectively; (xvii) SEQ ID NOs:70, 31, 12, 95, 17, and 18, respectively; (xviii) SEQ ID NOs:70, 31, 12, 96, 17, and 18, respectively; (xix) SEQ ID NOs:70, 31, 12, 97, 17, and 18, respectively; (xx) SEQ ID NOs:70, 76, 12, 93, 17, and 18, respectively; (xxi) SEQ ID NOs:70, 90, 12, 16, 17, and 18, respectively; (xxii) SEQ ID NOs:70, 31, 81, 16, 17, and 18, respectively; (xxiii) SEQ ID NOs:70, 31, 82, 16, 17, and 18, respectively; (xxiv) SEQ ID NOs:70, 31, 83, 16, 17, and 18, respectively; (xxv) SEQ ID NOs:70, 31, 84, 16, 17, and 18, respectively(xxvi) SEQ ID NOs:70, 31, 85, 16, 17, and 18, respectively; (xxvii) SEQ ID NOs:70, 31, 86, 16, 17, and 18, respectively; (xxviii) SEQ ID NOs:70, 31, 87, 16, 17, and 18, respectively; (xxix) SEQ ID NOs:70, 31, 88, 16, 17, and 18, respectively; (xxx) SEQ ID NOs:70, 31, 89, 16, 17, and 18, respectively; (xxxi) SEQ ID NOs:70, 31, 12, 98, 17, and 18, respectively; (xxxii) SEQ ID NOs:70, 31, 12, 99, 17, and 18, respectively; (xxxiii) SEQ ID NOs:70, 31, 12, 100, 17, and 18, respectively; (xxxiv) SEQ ID NOs:70, 31, 12, 101, 17, and 18, respectively; (xxxv) SEQ ID NOs:70, 31, 12, 102, 17, and 18, respectively; (xxxvi) SEQ ID NOs:70, 31, 12, 103, 17, and 18, respectively; (xxxvii) SEQ ID NOs:70, 31, 12, 16, 17, and 104, respectively; (xxxviii) SEQ ID NOs:70, 31, 12, 16, 17, and 105, respectively; (xxxix) SEQ ID NOs:70, 31, 12, 16, 17, and 106, respectively; (xl) SEQ ID NOs:70, 31, 12, 16, 17, and 107, respectively; (xli) SEQ ID NOs:70, 31, 12, 16, 17, and 108, respectively; (xlii) SEQ ID NOs:70, 31, 12, 16, 17, and 109, respectively; (xliii) SEQ ID NOs:70, 31, 12, 16, 17, and 110, respectively; (xliv) SEQ ID NOs:70, 31, 12, 16, 17, and 111, respectively; (xlv) SEQ ID NOs:70, 31, 12, 16, 17, and 112, respectively; (xlvi) SEQ ID NOs:70, 31, 12, 16, 17, and 113, respectively; (xlvii) SEQ ID NOs:73, 31, 12, 16, 17, and 18, respectively; or (xlviii) SEQ ID NOs:73, 31, 12, 16, 17, and 113, 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 amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequences of: (i) SEQ ID NOs: 22 and 123, respectively; (ii) SEQ ID NOs: 22 and 23, respectively; (iii) SEQ ID NOs: 115 and 23, respectively; (iv) SEQ ID NOs: 120 and 23, respectively; (v) SEQ ID NOs: 28 and 23, respectively;(vi) SEQ ID NOs: 114 and 23, respectively; (vii) SEQ ID NOs: 24 and 23, respectively; (viii) SEQ ID NOs: 116 and 23, respectively; (ix) SEQ ID NOs: 117 and 23, respectively; (x) SEQ ID NOs: 118 and 23, respectively; (xi) SEQ ID NOs:119 and 23, respectively; (xii) SEQ ID NOs:26 and 23, respectively; (xiii) SEQ ID NOs:22 and 121, respectively; (xiv) SEQ ID NOs:22 and 122, respectively; (xv) SEQ ID NOs:5 and 6, respectively; (xvi) SEQ ID NOs:22 and 124, respectively; (xvii) SEQ ID NOs:22 and 125, respectively; (xviii) SEQ ID NOs:22 and 126, respectively; (xix) SEQ ID NOs:22 and 127, respectively; (xx) SEQ ID NOs:116 and 123, respectively; (xxi) SEQ ID NOs:128 and 23, respectively; (xxii) SEQ ID NOs:129 and 23, respectively; (xxiii) SEQ ID NOs:130 and 23, respectively; (xxiv) SEQ ID NOs:131 and 23, respectively; (xxv) SEQ ID NOs:132 and 23, respectively; (xxvi) SEQ ID NOs:133 and 23, respectively; (xxvii) SEQ ID NOs:134 and 23, respectively; (xxviii)SEQ ID NOs:135 and 23, respectively; (xxix) SEQ ID NOs:136 and 23, respectively; (xxx) SEQ ID NOs:137 and 23, respectively; (xxxi) SEQ ID NOs:22 and 138, respectively; (xxxii) SEQ ID NOs:22 and 139, respectively; (xxxiii) SEQ ID NOs:22 and 140, respectively; (xxxiv) SEQ ID NOs:22 and 141, respectively; (xxxv) SEQ ID NOs:22 and 142, respectively; (xxxvi) SEQ ID NOs:22 and 143, respectively; (xxxvii) SEQ ID NOs:22 and 144, respectively; (xxxviii) SEQ ID NOs:22 and 145, respectively;(xxxix) SEQ ID NOs:22 and 146, respectively; (xl) SEQ ID NOs:22 and 147, respectively; (xli) SEQ ID NOs:22 and 148, respectively; (xlii) SEQ ID NOs:22 and 149, respectively; (xliii) SEQ ID NOs:22 and 150, respectively; (xliv) SEQ ID NOs:22 and 151, respectively; (xlv) SEQ ID NOs:22 and 152, respectively; (xlvi) SEQ ID NOs:22 and 153, respectively; (xlvii) SEQ ID NOs:154 and 6, respectively; or (xlviii) SEQ ID NOs:154 and 153, respectively.

3. An antigen-binding domain that specifically binds to human CD98hc, wherein the antigen-binding domain comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 5, 22, 24, 2628, 114, 115, 116, 117, 118, 119, 120, 22, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, or 154.

4. An antigen-binding domain that specifically binds to human CD98hc, wherein the antigen-binding domain comprises a VH and a VL, wherein the VL comprises the amino acid sequence of SEQ ID NO: 6, 23, 121, 122, 123, 124, 125, 126, 127, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, or 153.

5. The antigen-binding domain of any one of claims 1-4, wherein the antigen-binding domain comprises a VH and VL comprising the amino acid sequences of: (i) SEQ ID NOs: 22 and 123, respectively; (ii) SEQ ID NOs: 22 and 23, respectively; (iii) SEQ ID NOs: 115 and 23, respectively; (iv) SEQ ID NOs: 120 and 23, respectively; (v) SEQ ID NOs: 28 and 23, respectively; (vi) SEQ ID NOs: 114 and 23, respectively; (vii) SEQ ID NOs: 24 and 23, respectively; (viii) SEQ ID NOs: 116 and 23, respectively; (ix) SEQ ID NOs: 117 and 23, respectively; (x) SEQ ID NOs: 118 and 23, respectively; (xi) SEQ ID NOs:119 and 23, respectively;(xii) SEQ ID NOs:26 and 23, respectively; (xiii) SEQ ID NOs:22 and 121, respectively; (xiv) SEQ ID NOs:22 and 122, respectively; (xv) SEQ ID NOs:5 and 6, respectively; (xvi) SEQ ID NOs:22 and 124, respectively; (xvii) SEQ ID NOs:22 and 125, respectively; (xviii) SEQ ID NOs:22 and 126, respectively; (xix) SEQ ID NOs:22 and 127, respectively; (xx) SEQ ID NOs:116 and 123, respectively; (xxi) SEQ ID NOs:128 and 23, respectively; (xxii) SEQ ID NOs:129 and 23, respectively; (xxiii) SEQ ID NOs:130 and 23, respectively; (xxiv) SEQ ID NOs:131 and 23, respectively; (xxv) SEQ ID NOs:132 and 23, respectively; (xxvi) SEQ ID NOs:133 and 23, respectively; (xxvii) SEQ ID NOs:134 and 23, respectively; (xxviii)SEQ ID NOs:135 and 23, respectively; (xxix) SEQ ID NOs:136 and 23, respectively; (xxx) SEQ ID NOs:137 and 23, respectively; (xxxi) SEQ ID NOs:22 and 138, respectively; (xxxii) SEQ ID NOs:22 and 139, respectively; (xxxiii) SEQ ID NOs:22 and 140, respectively; (xxxiv) SEQ ID NOs:22 and 141, respectively; (xxxv) SEQ ID NOs:22 and 142, respectively; (xxxvi) SEQ ID NOs:22 and 143, respectively; (xxxvii) SEQ ID NOs:22 and 144, respectively; (xxxviii) SEQ ID NOs:22 and 145, respectively; (xxxix) SEQ ID NOs:22 and 146, respectively; (xl) SEQ ID NOs:22 and 147, respectively; (xli) SEQ ID NOs:22 and 148, respectively; (xlii) SEQ ID NOs:22 and 149, respectively; (xliii) SEQ ID NOs:22 and 150, respectively; (xliv) SEQ ID NOs:22 and 151, respectively;(xlv) SEQ ID NOs:22 and 152, respectively; (xlvi) SEQ ID NOs:22 and 153, respectively; (xlvii) SEQ ID NOs:154 and 6, respectively; or (xlviii) SEQ ID NOs:154 and 153, respectively.

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

7. The antigen binding domain of any one of claims 1-6, wherein the antigen-binding domain binds human CD98hc with an affinity of 10 nM to 100 nM.

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

9. The antigen binding domain of any one of claims 1-6, wherein the antigen-binding domain binds human CD98hc with an affinity of 10 nM to 1000 nM.

10. The antigen-binding domain of any one of claims 1-6, wherein the antigen-binding domain binds to cynomolgus monkey CD98hc.

11. The antigen-binding domain of claim 10, wherein the antigen-binding domain binds cynomolgus monkey CD98hc with an affinity of 10 nM to 1000 nM, optionally wherein the affinity is measured by high throughput surface plasmon resonance (SPR) detection.

12. The antigen-binding domain of claim 10, wherein the antigen-binding domain binds cynomolgus monkey CD98hc with an affinity of 10 nM to 500 nM, optionally wherein the affinity is measured by high throughput surface plasmon resonance (SPR) detection.

13. The antigen-binding domain of claim 10, wherein the antigen-binding domain binds cynomolgus monkey CD98hc with an affinity of 10 nM to 100 nM, optionally wherein the affinity is measured by high throughput surface plasmon resonance (SPR) detection.

14. The antigen-binding domain of any one of claims 1-13, wherein 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.

15. The antigen-binding domain of any one of claims 1-14, wherein the antigen-binding domain is internalized in blood-brain barrier epithelial cells, optionally wherein the blood-brain barrier epithelial cells are HCMEC / D3 cells.

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

17. The antigen-binding domain of any one of claims 1-15, wherein the antigen-binding domain does not increase cell-surface expression of CD98hc on HCMEC / D3 cells by more than 50% relative to cell-surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control.

18. The antigen-binding domain of any one of claims 1-17, wherein the antigen-binding domain accumulates at least 3-fold more than an isotype control in vessel-depleted mouse brain.

19. The antigen-binding domain of any one of claims 1-17, wherein the antigen-binding domain accumulates at least 5-fold more than an isotype control in vessel-depleted mouse brain.

20. The antigen-binding domain of any one of claims 1-17, wherein the antigen-binding domain accumulates at least 10-fold or at least 20-fold more than an isotype control in vessel-depleted mouse brain.

21. The antigen-binding domain of any one of claims 1-20, wherein the antigen-binding domain has at least a 5-fold increase in brain concentration ratio over an isotype control 24 hours after administration to a mouse.

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

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

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

28. The antigen-binding domain of claim 24 or 25, wherein the linker is about 15 to about 25 amino acids.

29. The antigen-binding domain of any one of claims 26-28, wherein the linker comprises the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 48) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 49).

30. The antigen-binding domain of claim 23, wherein the scFv comprises the amino acid sequence of any one of SEQ ID NOs: 21, 39, 40, 41 or 42.

31. The antigen-binding domain of any one of claims 1-22, wherein the antigen-binding domain comprises a VH on a first polypeptide and a VL on a second polypeptide.

32. The antigen-binding domain of any one of claims 1-31, 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.

33. An antigen-binding domain that specifically binds to human CD98hc, wherein the antigen-binding domain is a VHH comprising (i) the VH CDR1, VH CDR2, and VH CDR3 of the antigen-binding domain of any one of claims 1-3, 5-19, and 30 or (ii) the VH of the antigen-binding domain of any one of claims 1-3, 5-19, and 30, optionally wherein the VHH is capable of crossing the blood brain barrier (BBB).

34. A complex or fusion protein comprising the antigen-binding domain of any one of claims 1-33 and a heterologous protein or peptide.

35. The complex or fusion protein of claim 34, wherein the heterologous protein or peptide (a) comprises the amino acid sequence of β-glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), clusterin (APOJ), Reelin, Tripeptidyl Peptidase 1 (CLN2 / TPP1), Alpha-L-Iduronidase (IDUA), Iduronate 2-Sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), N-sulfoglucosamine sulfohydrolase (SGSH), ubiquitin protein ligase E3A (UBE3A), or a variant or portion thereof or (b) specifically binds to beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, 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 expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4- Domains A 6A (MS4A6A), or Transmembrane Protein 106B (TMEM106b).

36. An antibody comprising the antigen-binding domain of any one of claims 1-33.

37. An antibody or antigen-binding fragment thereof that binds to the same human CD98hc epitope as the antigen-binding domain of any one of claims 1-33.

38. An antibody or antigen-binding fragment thereof that competitively inhibits binding of the antigen-binding domain of any one of claims 1-33 to human CD98hc.

39. A multi-specific protein comprising a first antigen-binding domain that is the antigen- binding domain of any one of claims 1-33 linked to a second antigen-binding domain, optionally wherein the second antigen-binding domain specifically binds to a CNS antigen.

40. A multi-specific protein comprising a first antigen-binding domain that is the antigen- binding domain of any one of claims 1-33 linked to an antibody or antigen-binding domain that binds to a cancer antigen.

41. The multi-specific protein of claim 40, wherein the antibody or antigen-binding domain that binds to a cancer antigen is selected from the group consisting of rituximab, cetuximab, trastuzumab, pertuzumab, bevacizumab, nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab.

42. The multi-specific protein of claim 40, wherein the antibody or antigen-binding domain that binds to a cancer antigen is selected from the group consisting of a version of rituximab comprising reduced effector function, a version of cetuximab comprising reduced effector function, a version of trastuzumab comprising reduced effector function, a version of pertuzumab comprising reduced effector function, a version of bevacizumab comprising reduced effector function, a version of nivolumab comprising reduced effector function, a version of pembrolizumab comprising reduced effector function, a verison of atezolizumab comprising reduced effector function, a verison of avelumab comprising reduced effector function, and a verison of durvalumab comprising reduced effector function.

43. The multi-specific protein of claim 42, wherein the reduced effector function comprises a mutation of LALA-P331S and / or LALA-P329S.

44. The multi-specific protein of claim 41, wherein the antibody or antigen-binding domain that binds to a cancer antigen is a verison of rituximab comprising reduced effector function.

45. The multi-specific protein of claim 41, wherein the antibody or antigen-binding domain that binds to a cancer antigen is rituximab.

46. The multi-specific protein of claim 42, wherein the multi-specific protein comprises the amino acid sequences of SEQ ID NOs:161-163.

47. A multi-specific protein comprising the antigen-binding domain of any one of claims 1- 33 linked to an antibody or antigen-binding fragment thereof, optionally wherein the antibody or antigen-binding fragment thereof specifically binds to a CNS antigen.

48. The multi-specific protein of claim 47, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region.

49. The multi-specific protein of claim 48, wherein the antigen-binding domain of any one of claims 1-31 is linked, optionally via an amino acid linker, to the C-terminus of the heavy chain constant region.

50. The multi-specific protein of any one of claims 39-49, wherein the multi-specific protein is bispecific.

51. The multi-specific protein of any one of claims 39-50, wherein the multi-specific protein is bivalent, trivalent, or tetravalent.

52. The multi-specific protein of claim 51, wherein the multi-specific protein is bivalent.

53. The multi-specific protein of claim 51, wherein the multi-specific protein is trivalent, optionally wherein the trivalent protein comprises one of the antigen-binding domain that binds to human CD98hc and two antigen-binding domains that bind to a CNS antigen.

54. The multi-specific protein of claim 51, wherein the multi-specific 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 a CNS antigen.

55. A multi-specific protein that is trivalent and bi-specific and comprises the antigen-binding domain of any one of claims 1-31 and 33 linked to an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, and wherein the antigen-binding domain is an scFv linked, optionally via an amino acid linker, to the C-terminus of one of the two antibody heavy chains.

56. A multi-specific protein that is tetravalent and bi-specific and comprises two antigen- binding domains of any one of claims 1-33, and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, wherein each ofthe two antigen-binding domains is an scFv, Fab, or VHH, wherein one of the two antigen-binding domains is linked, optionally via an amino acid linker, to the C-terminus of one of the antibody heavy chains, and wherein the other antigen-binding domain is linked, optionally via an amino acid linker, to the C-terminus of the other antibody heavy chain.

57. The multi-specific protein of any one of claims 47-56, wherein the antibody or antigen- binding fragment thereof comprises a constant region comprising a knob mutation and a constant region comprising a hole mutation.

58. The multi-specific protein of claim 57, wherein the antigen-binding domain is linked, optionally via an amino acid linker, to the constant region comprising a hole mutation.

59. The multi-specific protein of claim 57, wherein the antigen-binding domain is linked, optionally via an amino acid linker, to the constant region comprising a knob mutation.

60. The multi-specific protein of claim 55-59, wherein the amino acid linker is a glycine- serine linker, optionally wherein the glycine-serine linker comprises the amino acid sequence (GGGGS)x3 (SEQ ID NO: 50).

61. The multi-specific protein of claim 55-59, 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: 51).

62. The multi-specific protein of any one of claims 39 and 47-61, wherein the CNS antigen is a brain antigen.

63. The multi-specific protein of any one of claims 39 and 47-62, wherein the CNS antigen is not CD98hc.

64. The multi-specific protein of any one of claims 47-63, 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.

65. The multi-specific protein of any one of claims 47-64, 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.

66. The multi-specific protein of any one of claims 47-65, 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.

67. The multi-specific protein of any one of claims 47-66, wherein the antibody or antigen- binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

68. The multi-specific protein of claim 67, 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.

69. The multi-specific protein of any one of claims 39-68, wherein the multi-specific 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 KDof about 3 nM to about 225 nM.

70. The multi-specific protein of any one of claims 39-68, wherein the multi-specific protein binds cynomolgus monkey CD98hc with a KDof about 27 nM to about 1430 nM.

71. The multi-specific protein of any one of claims 39-68, wherein the multi-specific protein binds cynomolgus monkey CD98hc with a KDof about 28 nM to about 651 nM.

72. The multi-specific protein of any one of claims 39-68, wherein the multi-specific protein binds cynomolgus monkey CD98hc with a KD of about 53 nM to about 859 nM.

73. The multi-specific protein of any one of claims 39-72, wherein the multi-specific protein is internalized in blood-brain barrier epithelial cells greater than 10-fold as compared tointernalization by an isotype control, optionally wherein the blood-brain barrier epithelial cells are HCMEC / D3 cells.

74. The multi-specific protein of any one of claims 39-73, wherein the multi-specific protein does not reduce cell-surface expression of CD98hc on HCMEC / D3 cells by more than 20% relative to cell-surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control.

75. The multi-specific protein of any one of claims 39-74, wherein the multi-specific protein does not increase cell-surface expression of CD98hc on HCMEC / D3 cells by more than 50% relative to cell-surface expression of CD98hc on HCMEC / D3 cells treated with an isotype control.

76. The multi-specific protein of any one of claims 39-75, wherein the multi-specific protein accumulates at least 3-fold, at least 5-fold, at least 10-fold, or at least 20-fold more than an isotype control in vessel-depleted mouse brain.

77. The multi-specific protein of any one of claims 39-76, wherein the multi-specific protein has at least a 5-fold increase in brain concentration ratio over an isotype control 24 hours after administration to a mouse.

78. The multi-specific protein of claim 39-77, wherein the CNS antigen is beta-secretase 1 (BACE1), amyloid beta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, β- glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), sialic acid binding Ig-like lectin 11 (Siglec11), glycoprotein nonmetastatic melanoma protein B (GPNMB), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), MS4A4E Transmembrane Protein106B (TMEM106b), ubiquitin protein ligase E3A (UBE3A), CR1, ABCA1, ABCA7, HLA-DR1, HLA-DR5, IL1RAP, TREML2, IL-34, SORL1, reelin, very low density lipoprotein receptor (VLDLR), apolipoprotein E receptor 2 (APOER2 / low-density lipoprotein receptor-related protein 8 (LRP8)), or ADAM1.

79. The complex or fusion protein of claim 34 or 35, antibody or antigen-binding fragment thereof of any one of claims 36-38, or the multi-specific protein of any one of claims 39- 78, that is capable of crossing the BBB.

80. The complex, fusion protein, antibody or antigen-binding fragment thereof, or multi- specific protein of any one of claims 34-79, wherein the complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein is linked to an imaging agent.

81. A composition comprising a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode the multi- specific protein of any one of claims 39-54 and 57-80, 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.

82. A composition comprising a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode the multi- specific protein of any one of claims 39-53 and 56-80, 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.

83. The composition of claim 81 or 82, wherein the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation.

84. The composition of any one of claims 81-83, wherein the ratio of the first, second, and third polynucleotides is about 1:3:6.

85. The composition of claim 81 or 82, wherein the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

86. A composition comprising a first polynucleotide and a second polynucleotide, wherein the first and second polynucleotides encode the multi-specific protein of any one of claims 39-53 and 56-80, wherein the first polynucleotide encodes a heavy chain and the antigen-binding domain that bind to human CD98hc, and wherein the second polynucleotide encodes a light chain.

87. A host cell comprising the composition of any one of claims 81-86.

88. An isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain of the antigen-binding domain of any one of claims 1-33.

89. An isolated polynucleotide comprising a nucleic acid molecule encoding the light chain variable region of the antigen-binding domain of any one of claims 1-33.

90. An isolated vector comprising the polynucleotide of claim 82 and / or the polynucleotide of claim 89.

91. An isolated vector comprising a nucleic acid molecule encoding the heavy chain variable region of the antigen-binding domain of any one of claims 1-33 and a nucleic acid molecule encoding the light chain variable region of the antigen-binding domain.

92. A host cell comprising the polynucleotide of claim 88 or 89 or the vector of claim 90 or 91.

93. The host cell of claim 87 or 92, wherein the host cell is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, BMT10 cell, plant cell, insect cell, and human cell in tissue culture.

94. A method of producing an antigen-binding domain or multi-specific protein comprising culturing the host cell of any one of claims 87, 92, and 93 so that the antigen-binding domain or multi-specific protein is produced, optionally wherein the method further comprises isolating the antigen-binding domain or multi-specific protein from the culture.

95. An isolated antigen-binding domain or multi-specific protein thereof produced by the method of claim 94.

96. The antigen-binding domain of any one of claims 1-33, the antibody or antigen-binding fragment thereof of claim 36, the complex or fusion protein of claim 34 or 35, or the multi-specific protein of any one of claims 39-75 further comprising a cytotoxic drug.

97. The antign-binding domain, antibody or antigen-binding fragment thereof, the complex or fusion protein, or the multispecific protein of claim 96, wherein the cytotoxic drug is a microtubule disrupting agent, optionally wherein the microtubule disrupting agent is monomethyl auristatin E (MMAE).

98. An antibody drug conjugate (ADC) comprising a drug and (i) the antigen-binding domain of any one of claims 1-33, (ii) the antibody or antigen-binding fragment thereof of claim 36, (iii) the complex or fusion protein of claim 34 or 35, or (iv) the multi-specific protein of any one of claims 39-75.

99. The ADC of claim 98, wherein the ADC binds to tissue factor (TF), human epidermal growth factor receptor (HER2), B7-H4, or Nectin-4.

100. The ADC of claim 98 or 99, wherein the ADC comprises a microtubule disrupting agent, optionally wherein the microtubule disrupting agent is monomethyl auristatin E (MMAE).

101. The ADC of any one of claims 98-100, wherein the ADC comprises tisotumab vedotin, disitamab vedotin, felmetatug vedotin, enfortumab vedotin or trastuzumab deruxtecan with reduced effector function.

102. A pharmaceutical composition comprising (i) the antigen-binding domain, complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein of any one of claims 1-80 and (ii) a pharmaceutically acceptable carrier.

103. The pharmaceutical composition of claim 102, wherein the concentration of the complex, fusion protein, antibody or an antigen-binding fragment thereof, or multi-specific protein is increased in the brain following administration to a subject as compared to an isotype control.

104. The pharmaceutical composition of claim 102 or 103, wherein administration increases delivery of the complex, fusion protein, antibody or antigen-binding fragment thereof, multi-specific protein, or pharmaceutical composition into the brain by at least 50%, at least 100%, at least 200%, at least 500% or at least 1000% as compared to an isotype control.

105. A method of treating a neurological disease or disorder in a subject comprising administering the complex, fusion protein, antibody or antigen-binding fragment thereof, multi-specific protein, or pharmaceutical composition of any one of claims 34-39, 47-80, 102, and 103 to the subject.

106. The method of claim 105, wherein administration increases delivery of the complex, fusion protein, antibody or antigen-binding fragment thereof, multi-specific protein, or pharmaceutical composition into the brain by at least 50%, at least 100%, at least 200%, at least 500% or at least 1000% as compared to an isotype control.

107. The method of claim 105 or 106, wherein administration increases delivery of the complex, fusion protein, antibody or antigen-binding fragment thereof, multi-specific protein, or pharmaceutical composition into the frontal cortex, the entorhinal cortex and / or the hippocampus.

108. The method of any one of claims 105-107, wherein the neurological disease or disorder is selected from a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation.

109. The method of claim 108, wherein 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).

110. The method of claim 109, wherein the dementia is frontotemporal dementia (FTD).

111. The method of claim 109, wherein the neurological disease or disorder is Alzheimer’s disease.

112. The method of claim 111, 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.

113. The method of claim 109, wherein the neurological disease or disorder is Parkinson’s disease.

114. The method of claim 105, wherein the neurological disease or disorder is frontal temporal epilepsy.

115. The method of claim 105, wherein the neurological disease or disorder is autism.

116. The method of claim 105, wherein the neurological disease or disorder is lissencephaly.

117. A method of treating a lysosomal storage disease in a subject, comprising administering the complex or fusion protein of claim 34 or 35 to the subject.

118. The method of claim 117, wherein the lysosomal storage disease is selected from Gaucher disease, Ceroid lipofuscinosis (Batten disease), Mucopolysaccharidosis (MPS) Type I, MPS Type II and MPS Type III.

119. A method of transporting a complex, fusion protein, antibody or an antigen-binding fragment thereof, or multi-specific protein across the BBB of a subject, comprising administering to the subject the complex or fusion protein of any one of claims 34, 35, 79, or 80, the antibody or antigen-binding fragment thereof of any one of claims 36 or 37, the multi-specific protein of any one of claims 38-78, or the pharmaceutical composition of any one of claims 102-104 to the subject.

120. The method of claim 119, wherein the concentration of the complex, fusion protein, antibody or an antigen-binding fragment thereof, or multi-specific protein is increased in the brain following administration as compared to an isotype control.

121. The method of claim 119 or 120, wherein the concentration of the complex, fusion protein, antibody or antigen-binding fragment thereof, multi-specific protein, orpharmaceutical composition in the brain is increased by at least 50%, at least 100%, at least 200%, at least 500% or at least 1000% as compared to an isotype control.

122. The method of claims 119-121, wherein administration of the complex, fusion protein, antibody or an antigen-binding fragment thereof, or multi-specific protein does not result in reticulocyte count reduced in the subject by more than 10%, as compared to administration of an isotype control.

123. The method of claim 122, wherein administration of the complex, fusion protein, antibody or an antigen-binding fragment thereof, or multi-specific protein does not result in reticulocyte count reduction in the subject, as compared to an isotype control.

124. A method of increasing the concentration of a CNS binding antigen in the CSF of a subject, comprising administering the multi-specific protein of any one of claims 39-80 to the subject, wherein the concentration of the CNS binding antigen is increased as compared to administering the CNS binding antigen alone to the subject.

125. A method of imaging a CNS antigen within a subject, comprising administering to the subject the complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein of claim 77 and locating the imaging agent within the subject.

126. A method of detecting a CNS antigen in vitro, comprising contacting an in vitro sample with the complex, fusion protein, antibody or antigen-binding fragment thereof, or multi- specific protein of claim 80 and locating the imaging agent within the sample.

127. Use of the complex or fusion protein of any one of claims 34, 35, 79, or 80, the antibody or antigen-binding fragment thereof of any one of claims 36 or 37, the multi-specific protein of any one of claims 38-78, or the pharmaceutical composition according to any one of claims 102-104 in the method of any one of claims 105-126.

128. The complex or fusion protein of any one of claims 34, 35, 79, or 80, the antibody or antigen-binding fragment thereof of any one of claims 36 or 37, the multi-specific protein of any one of claims 38-78, or the pharmaceutical composition of any one of claims 102- 104 for use in the method of any one of claims 105-126.

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