Cd98 heavy chain transgenic models

Abstract

In some aspects, the present disclosure provides CD98hc polypeptides containing a partially or fully humanized ECD. In other aspects, this disclosure provides transgenic animal models expressing such polypeptides and methods of using the animal models to identify therapeutics that can cross the blood-brain barrier.

Description

CD98 HEAVY CHAIN TRANSGENIC MODELSCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 387,903, filed on December 16, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes.BACKGROUND

[0002] The blood-brain barrier (BBB) blocks the passage of most macromolecules from the periphery into the brain and thus limits the uses of large molecule therapeutics where brain exposure is required. CD98 heavy chain (CD98hc) is highly expressed at the BBB and can be used to transport such therapeutics across the BBB via receptor-mediated transcytosis. Transgenic animal models expressing forms of CD98hc that include human sequences are needed as tools to evaluate therapeutics that are capable of crossing the BBB to treat brain diseases. At least one prior attempt to make a mouse that expresses human (hCD98hc) resulted in no protein expression (Hattori et al., Cytologia 74: 65-70, 2009). There remains an unmet need for a transgenic animal model that expresses humanized CD98hc.BRIEF SUMMARY

[0003] Provided herein are CD98hc polypeptides comprising a partially or fully humanized ECD and transgenic animal models contain a partially or fully humanized ECD and methods of using the transgenic animal models.

[0004] In one aspect, this disclosure provides a non-human transgenic animal that expresses a chimeric CD98 heavy chain (CD98hc) polypeptide comprising a partially or fully humanized CD98hc extracellular domain (ECD), a transmembrane domain (TD), and an intracellular domain (ICD), wherein the animal comprises a nucleic acid sequence encoding the chimeric CD98hc polypeptide, and wherein: (1) a portion of the partially or fully humanized CD98hc ECD comprises an amino acid sequence having at least 90% identity to the amino acid sequence encoded by human exons 9-12 of a human CD98hc nucleic acid sequence; (2) the TD comprises an amino acid sequence having at least 90% identity to the amino acid sequenceof the TD endogenous to the animal; and (3) the ICD comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the ICD endogenous to the animal.

[0005] In some embodiments, the chimeric CD98hc polypeptide is expressed in the brain of the animal.

[0006] In some embodiments, the animal comprises a partially humanized CD98hc ECD. In certain embodiments, the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises four humanized exons, wherein the four humanized exons encode a portion of the partially humanized CD98hc ECD and replace a nucleic acid sequence encoding the corresponding portion of the CD98hc ECD endogenous to the animal. In some embodiments, the four humanized exons comprise human exons 9-12 of the human CD98hc nucleic acid sequence. In certain embodiments, the nucleic acid sequence encoding the chimeric CD98hc polypeptide, or a portion thereof, is codon optimized for expression in the animal. In some embodiments, the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises introns that are endogenous to the animal. In certain embodiments, the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises a sequence having at least 70% identity to the sequence of SEQ ID NO: 8. In certain embodiments, the partially humanized CD98hc ECD comprises an amino acid sequence having at least 95% identity to SEQ ID NO:9. In certain embodiments, the chimeric CD98hc polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:7.

[0007] In some embodiments, the animal comprises a fully humanized CD98hc ECD. In some embodiments, the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises six humanized exons, wherein the six humanized exons encode a portion of the fully humanized CD98hc ECD and replace a nucleic acid sequence encoding the corresponding portion of the CD98hc ECD endogenous to the animal. In certain embodiments, the six humanized exons comprise human exons 7-12 of the human CD98hc nucleic acid sequence. In certain embodiments, the nucleic acid sequence encoding the chimeric CD98hc polypeptide further comprises humanizing point mutations. In certain embodiments, the humanizing point mutations alter the encoded amino acid sequence to match that of the amino acid sequence encoded by human exons 4-6 of the human CD98hc nucleic acid sequence. In some embodiments, the nucleic acid sequence encoding the chimeric CD98hc polypeptide, or a portion thereof, is codon optimized for expression in the animal. In some embodiments, the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises introns that areendogenous to the animal. In some embodiments, the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises a sequence having at least 70% identity to the sequence of SEQ ID NO: 11. In some embodiments, the fully humanized CD98hc ECD comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 12. In some embodiments, the chimeric CD98hc polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 10.

[0008] In some embodiments, the animal is a rodent. In certain embodiments, the animal is a mouse. In certain embodiments, the animal is a rat.

[0009] In some embodiments, the animal is a mouse, and the mouse exons 7-10 of an endogenous mouse CD98hc nucleic acid sequence are replaced by human exons 9-12 of the human CD98hc nucleic acid sequence.

[0010] In some embodiments, the animal is a mouse, and the mouse exons 5-10 of an endogenous mouse CD98hc nucleic acid sequence are replaced by human exons 7-12 of the human CD98hc nucleic acid sequence.

[0011] In some embodiments, the animal is homozygous for the chimeric CD98hc polypeptide.

[0012] In some embodiments, the animal is heterozygous for the chimeric CD98hc polypeptide.

[0013] In another aspect, the disclosure features a genetically modified mouse comprising an amino acid sequence encoded by at least human exons 9-12 of a human CD98hc nucleic acid sequence, wherein the human exons 9-12 replace mouse exons 7-10 of an endogenous mouse CD98hc nucleic acid sequence, and wherein the mouse expresses a chimeric CD98hc polypeptide.

[0014] In some embodiments, the mouse comprises human exons 7-12 of the human CD98hc nucleic acid sequence, wherein the human exons 7-12 replace mouse exons 5-10 of an endogenous mouse CD98hc nucleic acid sequence.

[0015] In some embodiments, the mouse further comprises one or more humanizing point mutations in mouse exons 2-4. In some embodiments, the humanizing point mutations alter the amino acid sequence encoded by mouse exons 2-4 of the endogenous mouse CD98hcnucleic acid sequence to match that of the amino acid sequence encoded by human exons 4-6 of the human CD98hc nucleic acid sequence.

[0016] In some embodiments, the nucleic acid sequence encoding the chimeric CD98hc polypeptide, or a portion thereof, is codon optimized for expression in the mouse. In some embodiments, the nucleic acid encoding the chimeric CD98hc polypeptide is under the control of a promoter endogenous to the mouse.

[0017] In another aspect, the disclosure provides a genetically modified mouse comprising one or more humanizing point mutations introduced into an endogenous mouse CD98hc nucleic acid sequence, wherein the humanizing point mutations alter the encoded amino acids such that at least 70% of the amino acids selected from positions: 327, 295, 376, 383, 514, 478, 253, 214, 338, 384, 481, 491, 512, 507, 471, 220, 516, 135, 234, 235, 280, 182, 320, 139, 148,342, 346, 146, 527, 347, 186, 212, 136, 251, 173, 306, 224, 397, 524, 254, 400, 202, 500, 250,493, 145, 509, 172, 134, 396, 315, 528, 505, 256, 269, 177, 231, 308, 191, 501, 190, 289, 114,412, 309, 288, 170, 391, 328, 116, 313, 219, 257, 152, 420, 268, 506, 171, 518, 120, 401, 404,469, 473, and 476 of SEQ ID NO: 15 are humanized.

[0018] In another aspect, the disclosure provides a genetically modified mouse comprising one or more humanizing point mutations introduced into an endogenous mouse CD98hc nucleic acid sequence, wherein the humanizing point mutations alter the encoded amino acids such that at least 70% of the amino acids selected from positions: 327, 295, 376, 383, 514, 478, 253, 214, 338, 384, 481, 491, 512, 507, 471, 220, 516, 135, 234, 235, 280, 182, 320, 139, 148,342, 346, 146, 527, 347, 186, 212, 136, 251, 173, 306, 224, 397, 524, 254, 400, 202, 500, 250,493, 145, 509, 172, 134, 396, 315, 528, 505, 256, 269, 177, 231, 308, 191, 501, 190, 289, 114,412, 309, 288, 170, 391, 328, 116, 313, 219, 257, 152, 268, 506, 171, 518, 120, 401, 404, 469,473, 476 of SEQ ID NO: 15 are humanized.

[0019] In another aspect, the disclosure features a genetically modified mouse comprising one or more humanizing point mutations introduced into an endogenous mouse CD98hc nucleic acid sequence, wherein the humanizing point mutations alter the encoded amino acids such that at least 70% of the amino acids selected from positions: 327, 295, 376, 383, 514, 478,253, 214, 338, 384, 481, 491, 512, 507, 471, 220, 516, 135, 234, 235, 280, 182, 320, 139, 148,342, 346, 146, 527, 347, 186, 212, 136, 251, 173, 306, 224, 397, 524, 254, 400, 202, 500, 250,493, 145, 509, 172, 134, 396, 315, 528, 505, 256, 269, 485, 177, 231, 308, 191, 501, 190, 289,114, 412, 180, 216, 309, 288, 170, 391, 328, 116, 430, 313, 219, 257, 152, 420, 268, 506, 258, 171, 518, 120, 387, 401, 404, 469, 473, and 476 of SEQ ID NO: 15 are humanized.

[0020] In some embodiments, the humanizing point mutations alter the amino acid sequence encoded by mouse exons 2-4 of the endogenous mouse CD98hc nucleic acid sequence to match that of the amino acid sequence encoded by human exons 4-6 of a human CD98hc nucleic acid sequence.

[0021] In some embodiments, the mouse comprises an amino acid sequence encoded by at least human exons 9-12 of a human CD98hc nucleic acid sequence, wherein the human exons 9-12 replace mouse exons 7-10 of the endogenous mouse CD98hc nucleic acid sequence.

[0022] In some embodiments, the mouse comprises an amino acid sequence encoded by human exons 7-12 of a human CD98hc nucleic acid sequence, wherein the human exons 7-12 replace mouse exons 5-10 of the endogenous mouse CD98hc nucleic acid sequence.

[0023] In another aspect, the disclosure provides a non-human embryonic stem (ES) cell that expresses a chimeric CD98hc polypeptide comprising a partially or fully humanized CD98hc extracellular domain (ECD), a transmembrane domain (TD), and an intracellular domain (ICD) in a non-human transgenic animal generated from the non-human ES cell, wherein: (1) a portion of the partially or fully humanized CD98hc ECD comprises an amino acid sequence having at least 90% identity to the amino acid sequence encoded by human exons 9-12 of a human CD98hc nucleic acid sequence; (2) the TD comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the TD endogenous to the animal; and (3) the ICD comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the ICD endogenous to the animal.

[0024] In some embodiments, the chimeric CD98hc polypeptide is expressed in the brain of the animal generated from the non-human ES cell.

[0025] In some embodiments, the partially humanized CD98hc ECD comprises an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO:9. In some embodiments, the chimeric CD98hc polypeptide comprises an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO:7. In some embodiments, the fully humanized CD98hc ECD comprises an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 12. In some embodiments, the CD98hc polypeptide comprises anamino acid sequence having at least 8O%% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 10.

[0026] In another aspect, the disclosure provides a polynucleotide encoding a chimeric CD98hc polypeptide, comprising a nucleic acid sequence encoding a partially or fully humanized CD98hc ECD and a nucleic acid sequence encoding a non-human CD98hc TD and / or non-human CD98hc ICD. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a partially humanized CD98hc ECD. In certain embodiments, the nucleic acid sequence comprises human exons 9-12 of a human CD98hc nucleic acid sequence. In certain embodiments, the polynucleotide comprises a nucleic acid sequence having at least 70% identity to the sequence of SEQ ID NO:8. In certain embodiments, the polynucleotide comprises a nucleic acid sequence encoding a fully humanized CD98hc ECD. In certain embodiments, the nucleic acid sequence comprises human exons 7-12 of a human CD98hc nucleic acid sequence.

[0027] In certain embodiments, the polynucleotide further comprises humanizing point mutations. In some embodiments, the humanizing point mutations alter the encoded amino acid sequence to match that of the amino acid sequence encoded by human exons 4-6 of the human CD98hc nucleic acid sequence.

[0028] In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 70% identity to the sequence of SEQ ID NO: 11.

[0029] In some embodiments, the polynucleotide, or a portion thereof, is codon optimized for expression in a non-human animal.

[0030] In some embodiments, the polynucleotide comprises introns that are endogenous to the non-human animal.

[0031] In some embodiments, the non-human animal is a mouse.

[0032] In another aspect, the disclosure provides a chimeric CD98 heavy chain (CD98hc) polypeptide or a portion thereof encoded by the polynucleotide described herein.

[0033] In another aspect, the disclosure provides an expression cassette for expressing a polynucleotide described herein in a non-human transgenic animal, comprising an endogenous promoter operably linked to the polynucleotide, wherein the introduction of the expression cassette into the animal results in the animal expressing a chimeric CD98hc polypeptide.

[0034] In another aspect, the disclosure provides an expression cassette for expressing a polynucleotide encoding a chimeric CD98hc polypeptide in a transgenic mouse, wherein the polynucleotide comprises a nucleic acid sequence having at least 70% identity to the sequence of SEQ ID NO:8 or 11.

[0035] In another aspect, the disclosure provides a host cell comprising the expression cassette described herein. In some embodiments, the host cell is a mouse cell. In some embodiments, the host cell is ex vivo. In some embodiments, the host cell is an embryonic stem cell.

[0036] In another aspect, the disclosure provides a non-human transgenic mouse comprising the host cell described herein.

[0037] In another aspect, the disclosure provides a method of screening for a chimeric CD98hc-binding polypeptide that crosses the blood-brain barrier, the method comprising: (a) administering a chimeric CD98hc-binding polypeptide to the animal described herein; and (b) measuring the presence or an activity of the chimeric CD98hc-binding polypeptide in the brain of the animal.

[0038] In some embodiments, the chimeric CD98hc-binding polypeptide is coupled to an effector molecule. In certain embodiments, the effector molecule is a small molecule, RNA, DNA, or polypeptide. In certain embodiments, the polypeptide is an antibody or an antigenbinding fragment thereof.

[0039] In some embodiments, the measuring step comprises contacting the brain or brain tissue of the animal with an agent that binds to the effector molecule and determining the level of the effector molecule present in the brain. In certain embodiments, the measuring step comprises measuring a pharmacodynamic (PD) effect of the effector molecule. In some embodiments, the effector molecule is an antibody or an antigen-binding fragment thereof that binds to a target in the brain.

[0040] In another aspect, the disclosure provides a method of screening for a CD98hc- binding polypeptide that binds to a chimeric CD98hc polypeptide comprising a partially or fully humanized CD98hc extracellular domain (ECD), the method comprising contacting a candidate CD98hc-binding polypeptide with a chimeric CD98hc polypeptide of claim 58; and determining the amount of the candidate CD98hc-binding polypeptide that binds to the chimeric CD98hc polypeptide.

[0041] In some embodiments, the step of contacting the candidate CD98hc-binding polypeptide with the chimeric CD98hc polypeptide comprises contacting the candidate CD98hc-binding polypeptide with a host cell that expresses the chimeric CD98hc polypeptide.

[0042] In some embodiments, the step of contacting the candidate CD98hc-binding polypeptide with the chimeric CD98hc polypeptide comprises contacting the candidate CD98hc-binding polypeptide with an endothelium that expresses the chimeric CD98hc polypeptide. In certain embodiments, the endothelium is a blood-brain barrier endothelium.

[0043] In some embodiments, the amount of the candidate CD98hc-binding polypeptide that binds the chimeric CD98hc polypeptide is determined by immunoassay.

[0044] In some embodiments, the amount of the candidate CD98hc-binding polypeptide that binds the chimeric CD98hc polypeptide is determined by surface plasmon resonance.

[0045] In some embodiments, the contacting step is performed in vivo.

[0046] In some embodiments, the candidate CD98hc-binding polypeptide is coupled to an effector molecule. In some embodiments, the effector molecule is a small molecule, RNA, DNA, or polypeptide. In some embodiments, the effector molecule is a polypeptide. In certain embodiments, the polypeptide is an antibody or an antigen-binding fragment thereof.

[0047] In another aspect, the disclosure provides a method of generating a transgenic nonhuman single cell embryo that expresses a chimeric CD98hc polypeptide comprising a partially or fully humanized CD98hc extracellular domain (ECD), the method comprises replacing at least a portion of the CD98hc ECD endogenous to the embryo with a corresponding heterologous portion having at least 95% identity to SEQ ID NO: 13 or 14.

[0048] In some embodiments, the replacing the portion of the CD98hc ECD is performed by homologous recombination.

[0049] In some embodiments, the method comprises contacting a Cas9 protein, at least one sgRNA, and a donor DNA comprising a nucleic acid sequence encoding the corresponding heterologous portion, wherein the corresponding heterologous portion is flanked by a left homology arm and a right homology arm, such that the corresponding heterologous portion coding sequence replaces the portion of the CD98hc ECD endogenous to the embryo.

[0050] In some embodiments, the corresponding heterologous portion is codon-optimized for expression in the embryo.

[0051] In another aspect, the disclosure provides a method of generating a transgenic nonhuman single cell embryo that expresses a chimeric CD98hc polypeptide comprising a partially or fully humanized CD98hc extracellular domain (ECD), the method comprises introducing one or more humanizing point mutations into the exons of the CD98hc ECD nucleic acid sequence endogenous to the embryo, wherein the humanizing point mutations alter the amino acid sequence encoded by the exons to match that of the amino acid sequence encoded by human exons of the human CD98hc ECD nucleic acid sequence. In some embodiments, the embryo is a mouse embryo.

[0052] In another aspect, the disclosure provides a method of generating a non-human transgenic animal comprising: (a) transferring the embryo of any one of claims 84 to 89 to a pseudo pregnant female of the same animal species as the embryo, and (b) selecting a transgenic animal from the progeny produced by the female, wherein the animal comprises a CD98hc polypeptide in which the CD98hc ECD endogenous to the embryo has been replaced with a heterologous ECD having an amino acid sequence of at least 95% identity to SEQ ID NO:9 or 12.

[0053] In another aspect, the disclosure provides a method of generating a transgenic animal comprising modifying the genome of a non-human animal such that the modified genome comprises a nucleic acid sequence encoding a chimeric CD98hc polypeptide, wherein the CD98hc ECD endogenous to the non-human animal has been replaced with a heterologous ECD having at least 95% identity to the amino acid sequence of SEQ ID NO:9 or 12.

[0054] In some embodiments, the transgenic animal expresses the chimeric CD98hc polypeptide comprising the heterologous ECD.

[0055] In some embodiments, the transgenic animal has functional sperm cells, functional ES cells, functional osteoclasts, functional keratinocytes, functional B cells, and functional T cells.

[0056] In some embodiments, the transgenic animal is a rodent, e.g., a mouse, a rat.

[0057] The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosure as claimed. Other objects, advantages and novel features will be readily apparent to those skilled in the art from the following detailed description of the disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG. 1A illustrates the construct for an unsucessful attempt at humanizing the extracellular domain (ECD) of CD98hc.

[0059] FIG. IB illustrates the construct design for full ECD knock-in CD98hc mouse.

[0060] FIGS. 2A-2D illustrate (A) msCD98hc protein was detected in cryosections of mouse (C57 / B6 mice) brain by IHC with an anti-mouse CD98hc antibody; (B) representative image of msCD98hc expression in C57 / B6 mice showed high expression in astrocytes and brain endothelial cells (identified by Glutl staining); (C) huCD98hc protein was detected in cryosections of huCD98hcKI / KImouse brain by IHC with an anti-humanCD98hc antibody; (D) representative image of human CD98hc expression in homozygous huCD98hcKI / KImouse brain was consistent with msCD98hc protein expression in C57 / B6 mice with high expression in astrocytes and brain endothelial cells (identified by anti-Glutl staining).

[0061] FIGS. 3A-3F illustrate msCD98hc protein was detected in wild-type mouse kidney, pancreas, and testis cryosections by IHC with an anti-mouse CD98hc antibody and huCD98hc protein was detected in cryosections of huCD98hcKI / KImouse kidney, pancreas, and testis by IHC with an anti -human CD98hc antibody: (A) representative image of msCD98hc expression in C57 / B6 mice in kidney showed high expression in a subset of kidney tubules; (B) representative image of human CD98hc expression in huCD98hcKI / KImouse kidney was consistent with msCD98hc protein expression in C57 / B6 mice with high expression in a subset of kidney tubules; (C) representative image of msCD98hc expression in C57 / B6 mice in pancreas showed high expression in lobules and lower expression in islets; (D) representative image of human CD98hc expression in huCD98hcKI / KImouse pancreas was consistent with msCD98hc protein expression in C57 / B6 mice with high expression in lobules and lower expression in islets; (E) representative image of msCD98hc expression in C57 / B6 mice in testis showed high expression in Leydig cells; and (F) representative image of human CD98hc expression in huCD98hcKI / KImouse testis was consistent with msCD98hc protein expression in C57 / B6 mice with high expression in Leydig cells.

[0062] FIGS. 4A and 4B illustrate (A) quantification of CD98hc protein levels in lysates from C57 / B6 control and huCD98hcKI / KImouse tissues: brain, small intestine, kidney, lung, liver, and heart (CD98hc protein concentrations were calculated from standard curves of mouse or human CD98hc recombinant protein run on the same western blots because a mouse / human cross-reactive detection antibody was not available); and (B) quantification of the proportionof wild-type and humanized CD98hc that were paired with light chains in brain tissue (nondenatured protein samples were run on Western blots and the proportion of paired and unpaired CD98hc was calculated based on total CD98hc).

[0063] FIGS. 5A-5C illustrate brain uptake of an CD98hc / BACEl bispecific antibody following the administration of the bispecific antibody in huCD98hcKI / KImice. (A) Plasma huIgGl concentration in huCD98hcKI / KImice 48 hours after the mice were administered with 50 mg / kg of anti-CD98hc / BACEl or anti-DNP02. CD98hc-mediated clearance of anti- CD98hc / BACEl was enhanced relative to that of anti-DNP02. (B) Mean brain uptake of anti- CD98hc / BACEl following a systemic dosing of the antibody. A roughly 2-fold increase in accumulation of anti-CD98hc / BACEl in mouse brains was seen compared to that of anti- DNP02. (C) There was a 3.5-fold increase in the ratio of anti-CD98hc / BACEl antibody concentration in brain to plasma compared to that of anti-DNP02.

[0064] FIG. 6A illustrates the construct design for partial ECD knock-in CD98hc mouse.

[0065] FIG. 6B illustrates the the portion of the CD98hc that is humanized with the construct in FIG. 6A.

[0066] FIGS. 7A and 7B illustrate the normalized concentration of the antibody (50 mg / kg, 48hrs post IV dose) in the (A) brain vasculature and (B) isolated brain parenchymal cell lysates obtained from fresh brain tissue according to the capillary depletion method. The normalized concentration anti-CD98hc / BACEl antibody was 8-fold higher than the non-binding anti-DNP02 antibody in the parenchymal fraction. There was also more anti-CD98hc / BACEl antibody in vascular fraction, consistent with anti-CD98hc / BACEl binding brain endothelial cells in huCD98hcKI / KImice.

[0067] FIGS. 8A and 8B illustrate (A) the brain parenchymal distribution of systemically administered anti-CD98hc / BACEl antibody in huCD98hcKI / KImice. A representative image of hippocampal brain sections from huCD98hcKI / KImice showed broad parenchymal distribution of intravenously-injected anti-CD98hc / BACEl (50 mg / kg, 48hrs post-dose). In contrast, (B) a representative image of brain sections from huCD98hcKI / KImice injected with anti-DNP02 showed minimal staining.

[0068] FIGS. 9A and 9B illustrate PK of an anti-human CD98hc / DNP02 bispecific antibody in brain and plasma of full extracellular domain (ECD) huCD98hc KI mice. (A) Plasma huIgGl PK in huCD98hcKI / KImice out to 21 days after the mice were administered with 50 mg / kg ofanti-CD98hc / DNP02 or 15 mg / kg of anti-DNP02 as the control mAB. CD98hc-mediated clearance of anti-CD98hc / DNP02 was enhanced relative to that of anti-DNP02. (B) Brain PK of anti-CD98hc / DNP02 out to 21 days following a systemic dosing of the antibody. An accumulation of anti-CD98hc / DNP02 was seen in mouse brains compared to that of anti- DNP02.

[0069] FIG. 10 illustrates quantification of CD98hc protein levels in lysates from wild-type C57 / B6 control and the homozygous 9-12 CD98hc KI (SLC3A2hu9~12 / hu9~12) mouse tissues: brain, lung, small intestine, kidney, heart, and testis.

[0070] FIGS. 11 A-l ID illustrate spatial expression msCD98hc in wild-type C57 / B6 control mice and 9-12 hu / msCD98hc chimeric protein in brain of homozygous 9-12 CD98hc KI (SLC3A2hu9~12 / hu9~12') mice. The expression pattern of CD98hc was conserved in SLC3A2hu9~ i2 / hu9-i2micejnbrains

[0071] FIGS. 12A-12D illustrate spatial expression msCD98hc in wild-type C57 / B6 control mice and the 9-12 hu / msCD98hc chimeric protein in kidney of homozygous 9-12 CD98hc KI (SLC3A2hu9~12 / hu9~12)' mice. Chimeric hu / msCD98hc expression in kidney of SLC3A2hu9~12 / hu9~12mice was consistent with msCD98hc protein expression in C57 / B6 mice with high expression in a subset of kidney tubules.

[0072] FIGS. 13A-13D illustrate spatial expression msCD98hc in wild-type C57 / B6 control mice and the 9-12 hu / msCD98hc chimeric protein in testis of homozygous 9-12 CD98hc KI SLC3A2hu9~12 / hu9~12') mice. Chimeric hu / msCD98hc expression in testis of SLC3A2hu9~12 / hu9~12mice was consistent with msCD98hc protein expression in C57 / B6 mice with high expression in Leydig cells.

[0073] FIGS. 14A-14D illustrate spatial expression msCD98hc in wild-type C57 / B6 control mice and the 9-12 hu / msCD98hc chimeric protein in pancreas of homozygous 9-12 CD98hc KI (SLC 3A2hu9~12 / hu9~12) mice. These data showed that chimeric hu / msCD98hc expression in in pancreas of SLC3A2hu9~12 / hu9~12mice was consistent with msCD98hc protein expression in C57 / B6 mice with high expression in lobules and lower expression in islets.

[0074] FIGS. 15A-15C illustrate: (A) Biacore binding affinity of anti-msCD98hc / BACEl and anti-huCD98hc / BACEl bispecific antibody to the 9-12 hu / msCD98hc chimeric protein; (B) plasma huIgG levels in SLC3A2hu9~12 / hu9~12mice; and (C) brain huIgG levels in SLC3A2hu9~ 12 / hu9-121111 C.

[0075] FIGS. 16A-16C illustrate amino acid residues in the hCD98hc extracellular domain that are solvent exposed at exemplary solvent exposure thresholds: (A) solvent exposure threshold of 20%, (B) solvent exposure threshold of 10%, and (C) solvent exposure threshold of 9%. Residues that are at or above the solvent exposure threshold in mouse or human are shown in black.

[0076] FIGS. 17A and 17B illustrate amino acid residues in the hCD98hc extracellular domain that are solvent exposed at exemplary solvent exposure thresholds: (A) solvent exposure threshold of 5% and (B) solvent exposure threshold of 0.01%. Residues that are at or above the solvent exposure threshold in mouse or human are shown in black.

[0077] FIG. 18 shows the the alignment of human CD98hc sequence (SEQ ID NO: 1) and mouse CD98hc sequence (SEQ ID NO:4).DETAILED DESCRIPTION

[0078] We have developed non-human transgenic animals that express CD98 heavy chain (CD98hc) polypeptides that include a partially or fully humanized CD98hc extracellular domain (ECD), as well as an intracellular domain (ICD) and transmembrane domain (TD) that are or are derived from the ICD and TD endogenous to the animal. The present disclosure therefore provides non-human transgenic animals that express CD98hc polypeptides with a partially or fully humanized CD98hc ECD. In some embodiments, the partially or fully humanized CD98hc ECD comprises an amino acid sequence having at least 80% identity to SEQ ID NO:2. In some embodiments, the non-human transgenic animal contains a nucleic acid sequence encoding the CD98hc polypeptide with the partially or fully humanized CD98hc ECD. In some embodiments, the nucleic acid sequence comprises humanized exons encoding at least a portion of the humanized CD98hc ECD, where the humanized exons replace the nucleic acid sequence encoding the corresponding portion of the CD98hc ECD endogenous to the non-human transgenic animal.

[0079] The non-human transgenic animals provided herein are healthy and suitable for use in discovery and development of therapeutics for treating brain diseases. The disclosure also provides the use of the non-human transgenic animal to screen for CD98hc-binding polypeptides that can cross the BBB by binding to these CD98hc polypeptides with humanized CD98hc ECD in vivo, as well as non-human embryonic stem (ES) cells that express these CD98hc polypeptides, methods of generating transgenic single-cell embryos expressing the CD98hc polypeptide, and methods of generating the transgenic animal.I. TERMINOLOGY

[0080] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an antibody” optionally includes a combination of two or more such molecules, and the like.

[0081] As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art, for example, ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value.

[0082] As used herein, the term “CD98 heavy chain” or “CD98hc” refers to 4F2 cell-surface antigen heavy chain and is encoded by the SLC3A2 gene. CD98hc is also known as 4F2 heavy chain. The human CD98hc sequence is set forth in SEQ ID NO: 1 and UNIPROT Accession No. P08195. CD98hc sequences from other species are also known (e.g., mouse, UNIPROT Accession No. P10852 and cynomolgus monkey, UNIPROT Accession No. G8F3Z0).

[0083] As used herein, the term “non-human CD98hc polypeptide” refers to a CD98hc polypeptide that is from or is derived from of a non-human mammal. In some embodiments, the non-human CD98hc polypeptide comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to sequence of the native the CD98hc polypeptide of the non-human mammal. Examples of the non-human mammals include mouse, rat, rabbit, bovine, ovine, canine, feline, equine, porcine, non-human primates, and the like.

[0084] As used herein, the term “humanized” is used to describe a non-human nucleic acid, polypeptide, or protein whose sequence is partially or fully modified to match a portion of or the entire sequence of the corresponding nucleic acid, polypeptide, or protein found in humans. In certain embodiments, a humanized nucleic acid, polypeptide, or protein can be a partially humanized nucleic acid, polypeptide, or protein, which means that a portion of the non-human nucleic acid, polypeptide, or protein sequence is modified to match the sequence of the corresponding portion found in humans. In certain embodiments, a humanized nucleic acid, polypeptide, or protein can be a fully humanized nucleic acid, polypeptide, or protein, which means that the entire sequence of the non-human nucleic acid, polypeptide, or protein is modified to match the corresponding sequence found in humans. For example, a transgenic mouse with a fully humanized CD98hc ECD means that the entire mouse CD98hc ECD has been modified to match the amino acid sequence of the human CD98hc ECD. Techniques to humanize a non-human nucleic acid, polypeptide, or protein sequence are available in the art.For example, at least a portion of a non-human nucleic acid sequence can be replaced by a corresponding human complementary DNA (cDNA) sequence. In another example, one or more point mutations in a non-human nucleic acid sequence can be made such that the resulting codons encode for the corresponding amino acid found in the human sequence. The one or more point mutations can be made such that the resulting codons are codon-optimized for expression in the non-human animal.

[0085] As used herein, the term "solvent exposed residue" is an amino acid residue that meets or is above a solvent exposure threshold. Solvent exposed residues may differ between species, e.g., between humans and non-human animals or between human and mouse.

[0086] As used herein, the term "solvent exposure threshold" defines the precent of total surface area of a residue that is exposed. For example, an about 9% solvent exposure threshold means at least 9% of the total surface area of an amino acid residue is exposed. In one embodiment, the solvent exposure threshold is about 9%, 10%, 15%, 20%, 25%, 30%, 35%, or 36%. In one embodiment, the solvent threshold is between about 9% and about 36%, between about 9% and about 20%, between about 10% and about 20%, between about 9% and about 25%, or between about 10% and about 25%.

[0087] As used herein, the term “humanized CD98hc extracellular domain (ECD)” refers to a non-human CD98hc ECD whose coding sequence has been partially or fully modified, such that at least a portion of or the entire amino acid sequence of the CD98hc ECD match that of the amino acid sequence of the human CD98hc ECD.

[0088] As used herein, the term “humanized exon” can refer to an exon in a non-human animal whose sequence has been modified to match the sequence of the corresponding exon found in humans. For example, mouse exons 5-10 in a mouse CD98hc nucleic acid sequence correspond to human exons 7-12 in a human CD98hc nucleic acid sequence. A comparison of mouse and human sequences and exons is provided in FIG. 18. A humanized exon can also refer to an exon whose sequence has been modified, such that the modifed exon encodes for an amino acid sequence that matches the corresponding amino acid sequence found in humans.

[0089] As used herein, a “CD98hcmu / huKI,” “SLC3A2huECD / huECDmouse,” or “huCD98hcKI / KImouse” refers to a transgenic mouse in which the ECD of the mouse CD98hc has been fully humanized to match the amino acid sequence of the ECD of human CD98hc.

[0090] As used herein, a “SLC3A2hu9~12 / hu9~12mouse” or “SLC3 A29'12 KI / KImouse” refers to a transgenic mouse in which the exons 7-10 encoding a portion of the ECD of the mouse CD98hc have been humanized to match the nucleic acid sequence of exons 9-12 encoding a portion of the ECD of human CD98hc.

[0091] As used herein, the terms “wild-type,” “native,” and “naturally occurring” with respect to a CD98hc or a domain thereof, refer to a CD98hc or a domain thereof that has a sequence that occurs in nature.

[0092] As used herein, an “endogenous” CD98hc or a domain thereof refers to a CD98hc or a domain thereof that naturally occurs in a cell or non-human animal, i.e., in the absence of genetic modification to the cell or animal.

[0093] As used herein, the term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y- carboxyglutamate and O-phosphoserine. Naturally occurring a-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (He), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Vai), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of a naturally occurring a-amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D- phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D- lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D- tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof. “Amino acid analogs” refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring aminoacid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0094] As used herein, the terms “polypeptide” and “peptide” are used interchangeably to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L and D amino acids.

[0095] As used herein, the terms “polynucleotide,” “nucleic acid,” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double and single stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double stranded form and each of two complementary single stranded forms known or predicted to make up the double stranded form.

[0096] As used herein, the term “non-targeting Fab fragment” refers to a Fab fragment that does not specifically bind to an antigen via its heavy or light chain variable domains or does not specifically bind to an antigen expressed in a given mammal, such as a primates, e.g.,human and non-human primates, or rodents, e.g., mouse, or in a particular tissue within such a mammal via its heavy or light chain variable domains. Non-limiting examples of non-targeting Fab fragments include (a) RSV (palivizumab) Fab fragments, which are non-targeting in mice and non-human primates, and (b) Fab fragments to dinitrophenyl hapten (DNP) (See, e.g., Leahy, PNAS 3661-3665, 1988). In one embodiment, the Fab fragments to dinitrophenyl hapten is DNP02.

[0097] As used herein, the terms “identical” or percent “identity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues, e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater, that are identical over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0098] For sequence comparison of polypeptides, typically one amino acid sequence acts as a reference sequence, to which a candidate sequence is compared. Alignment can be performed using various methods available to one of skill in the art, e.g., visual alignment or using publicly available software using known algorithms to achieve maximal alignment. Such programs include the BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters employed for an alignment to achieve maximal alignment can be determined by one of skill in the art. For sequence comparison of polypeptide sequences for purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two proteins sequence with the default parameters is used.

[0099] As used herein, the term “knock-in” refers to a one-for-one substitution of DNA sequence information in a predetermined genetic locus or the insertion of sequence information not found within the locus. Those skilled in the art will readily appreciate how to use various genetic approaches, e.g., CRISPR / Cas9 systems, ZFN, TALEN, transposon-mediated insertion, to knock in a target polynucleotide sequence in a specific locus of the genome.

[0100] As used herein, the term “point mutation” refers to a single nucleotide change in a genetic sequence. A point mutation can result from a direct substitution of one nucleotide for another nucleotide. A point mutation can also result from an addition or deletion of one or more nucleotides at other locations in the genetic sequence. In some cases, a point mutation ina codon can change the amino acid encoded by the codon. In other cases, a point mutation in a codon does not change the amino acid encoded by the codon, i.e., a silent mutation.

[0101] As used herein, the term “blood-brain barrier” or “BBB” refers to a highly selective semipermeable membrane barrier that separates the circulating blood from the brain extracellular fluid in the central nervous system (CNS). The blood-brain barrier is formed by brain endothelial cells, which are connected by tight junctions.II. CD98 HEAVY CHAIN

[0102] CD98 is a heterodimer composed of CD98 heavy chain (CD98hc) (also referred to as 4F2hc) encoded by the SLC3A2 gene and CD98 light chain encoded by the SLC7A5 gene. CD98 is highly expressed in brain capillaries. To date six CD98 light chains have been identified, i.e., LAT1 (SLC7A5, 4F2 light chain), LAT2 (SLC7A8), y+LATl (SLC7A7), y+LAT2 (SLC7A6), Asc-1 (SLC7A10), or xCT (SLC7A11). In complex, CD98 heavy chain transports the light chain to the cell surface where it functions as a large neutral amino acid transporter which preferentially transports branched-chain (valine, leucine, isoleucine) and aromatic (tryptophan, tyrosine, phenylalanine) amino acids.

[0103] CD98hc is present both in human and non-human species, such as non-human primates and rodents. The native human CD98hc, UNIPROT Accession No. P08195, SEQ ID NO: 1, is a transmembrane protein; it has an extracellular domain (ECD), a transmembrane domain (TD), and an intracellular domain (ICD). The ECD of human CD98hc, which has the sequence of SEQ ID NO:2 (encoded by the nucleic acid sequence of SEQ ID NO:3), is essential for mediating the transport of amino acids. The native cynomolgous monkey, native rhesus monkey, and native chimpanzee CD98hcs are also known, as represented, for example, by accession numbers G8F3Z0, F6W941, and G2HIE1, respectively. For example, the ECDs of the native rhesus monkey and native chimpanzee CD98hcs share about 95% sequence identity, respectively, with the ECD of the native human CD98hc of SEQ ID NO:2.

[0104] The native mouse CD98hc, UNIPROT Accession No. P10852, SEQ ID NO:4, has about 75% amino acid sequence identity with human CD98hc. The ECD of the native mouse CD98hc (e.g., SEQ ID NO:5, encoded by the nucleic acid sequence of SEQ ID NO:6) is about 71% identical to the ECD of the native human CD98hc (e.g., SEQ ID NO:2). The complete gene sequence for mouse CD98hc, with annotated exons and introns, can be found from the NCBI database (Gene ID: 17254). Mouse CD98hc is found on chromosome 19 (NCBI reference sequence NC_000085.7).

[0105] The alignment in FIG. 18 shows the comparison of human CD98hc sequence (SEQ ID NO: 1) and mouse CD98hc sequence (SEQ ID NON). In FIG. 18, the alternating, with and without squiggly underlining indicates the alternating exons. Bold residues indicate residual overlap splice site. The ECD of human CD98hc is in SEQ ID NO:2. The ECD of mouse CD98hc is in SEQ ID NO:5. The table below shows the exon by exon comparison and their percent identity.Table 1III. NON-HUMAN TRANSGENIC ANIMALS

[0106] The disclosure provides a non-human transgenic animal that expresses a chimeric CD98 heavy chain (CD98hc) polypeptide comprising a partially or fully humanized CD98hc extracellular domain (ECD), a transmembrane domain (TD), and an intracellular domain (ICD). The non-human transgenic animal contains a nucleic acid sequence encoding the chimeric CD98hc polypeptide. In some embodiments, the partially or fully humanized CD98hc ECD comprises a sequence having at least 85% (e.g., 85%, 90%, 95%, 97%, 99%, or 100%) identity to the sequence of SEQ ID NO:2; the TD comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the TD endogenous to the animal; and the ICD comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the ICD endogenous to the animal. In some embodiments, the nucleic acid sequence encoding the chimeric CD98hc polypeptide, or a portion thereof, is codon optimized for expression in the non-human transgenic animal. In some embodiments, the nucleic acidsequence encoding the chimeric CD98hc polypeptide containing a partially or fully humanized CD98hc ECD in the non-human transgenic animal comprises introns that are endogenous to the non-human transgenic animal.

[0107] In some embodiments, residues that are solvent exposed in the mouse and / or human CD98hc ECD can be humanized by making point mutations to an endogenous mouse CD98hc nucleic acid encoding the CD98hc ECD. In one embodiment, residues that are solvent exposed in the human CD98hc ECD are humanized by making point mutations to an endogenous mouse CD98hc nucleic acid encoding the CD98hc ECD. Additionally, residues that are solvent exposed in one species but not in another species may be important positions to humanize. For example, a residue that is solvent exposed in the mouse CD98hc ECD but not in the human CD98hc ECD should also be considered for humanization because this represents a substantial difference in the mouse CD98hc ECD and human CD98hc ECD.

[0108] In certain embodiments, the solvent exposure threshold is about 0.01%, 5%, 9%, 10%, or 20%. Examples of the total solvent exposed residues in the mouse and human CD98hc ECD as shown on the human CD98hc ECD protein crystal structure are shown in FIGS. 14A-15C and 15A-15B. In some embodiments, the solvent exposure threshold is 0.01% and 108 residues are considred solvent exposed in the mouse and human protein. In some embodiments, the solvent exposure threshold is 5% and 91 residues are considered solvent exposed in the mouse and human protein. In some embodiments, the solvent exposure threshold is 9% and 85 residues are considered solvent exposed in the mouse and human protein. In some embodiments, the solvent exposure threshold is 10% and 84 residues are considered solvent exposed in the mouse and human protein. In some embodiments, the solvent exposure threshold is 5% and 67 residues are considered solvent exposed in the mouse and human protein. In some embodiments, at least 70%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, or 99% of solvent exposed residues are altered by point mutation to an endogenous mouse CD98hc nucleic acid encoding the CD98hc ECD to match the human amino acid sequence, wherein the solvent exposure threshold is 9%. In some embodiments, at least 70%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, or 99% of solvent exposed residues are changed are altered by point mutation to an endogenous mouse CD98hc nucleic acid to match the human amino acid sequence, wherein the solvent exposure threshold is 10%. In some embodiments, at least 70%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, or 99% of solvent exposed residues are changed are altered by point mutation to an endogenous mouse CD98hc nucleic acid to match the human amino acid sequence, wherein the solvent exposure threshold is 20%.

[0109] In one embodiment, at least 70%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% of residues selected from: 327, 295, 376, 383, 514, 478, 253, 214, 338, 384, 481, 491, 512, 507, 471, 220, 516, 135, 234, 235, 280, 182, 320, 139, 148, 342, 346, 527, 347, 212, 136, 251, 173, 224, 397, 254, 400, 500, 145, 172, 396, 528, 505, 269, 177, 231, 308, 501, 190, 289, 114, 412, 309, 170, 391, 313, 219, 257, 152, 268, 506, 171, 120, 401, 404, 469, and 476 of SEQ ID NO: 15 are humanized via point mutations to an endogenous mouse CD98hc nucleic acid encoding the CD98hc ECD.

[0110] In one embodiment, at least 70%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% of residues selected from: 327, 295, 376, 383, 514, 478, 253, 214, 338, 384, 481, 491, 512, 507,471, 220, 516, 135, 234, 235, 280, 182, 320, 139, 148, 342, 346, 146, 527, 347, 186, 212, 136,251, 173, 306, 224, 397, 524, 254, 400, 202, 500, 250, 493, 145, 509, 172, 134, 396, 315, 528,505, 256, 269, 177, 231, 308, 191, 501, 190, 289, 114, 412, 309, 288, 170, 391, 328, 116, 313,219, 257, 152, 268, 506, 171, 518, 120, 401, 404, 469, 473, 476 of SEQ ID NO: 15 are humanized via point mutations to an endogenous mouse CD98hc nucleic acid encoding the CD98hc ECD.

[0111] In one embodiment, at least 70%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% of residues selected from: 327, 295, 376, 383, 514, 478, 253, 214, 338, 384, 481, 491, 512, 507,471, 220, 516, 135, 234, 235, 280, 182, 320, 139, 148, 342, 346, 146, 527, 347, 186, 212, 136,251, 173, 306, 224, 397, 524, 254, 400, 202, 500, 250, 493, 145, 509, 172, 134, 396, 315, 528,505, 256, 269, 177, 231, 308, 191, 501, 190, 289, 114, 412, 309, 288, 170, 391, 328, 116, 313,219, 257, 152, 420, 268, 506, 171, 518, 120, 401, 404, 469, 473, and 476 of SEQ ID NO: 15 are humanized via point mutations to an endogenous mouse CD98hc nucleic acid encoding the CD98hc ECD.

[0112] In one embodiment, at least 70%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% of residues selected from: 327, 295, 376, 383, 514, 478, 253, 214, 338, 384, 481, 491, 512, 507, 471, 220, 516, 135, 234, 235, 280, 182, 320, 139, 148, 342, 346, 146, 527, 347, 186, 212, 136,251, 173, 306, 224, 397, 524, 254, 400, 202, 500, 250, 493, 145, 509, 172, 134, 396, 315, 528,505, 256, 269, 485, 177, 231, 308, 191, 501, 190, 289, 114, 412, 180, 216, 309, 288, 170, 391,328, 116, 430, 313, 219, 257, 152, 420, 268, 506, 258, 171, 518, 120, 387, 401, 404, 469, 473, and 476 of SEQ ID NO: 15 are humanized via point mutations to an endogenous mouse CD98hc nucleic acid encoding the CD98hc ECD.

[0113] In one embodiment, at least 70%, 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% of residues selected from: 327, 295, 376, 383, 514, 478, 253, 214, 338, 384, 481, 491, 512, 507, 471, 220, 516, 135, 234, 235, 280, 182, 320, 139, 148, 342, 346, 146, 527, 347, 186, 212, 136,251, 173, 306, 224, 397, 524, 254, 400, 202, 500, 250, 493, 145, 509, 172, 134, 396, 315, 528,505, 256, 269, 485, 177, 231, 308, 191, 501, 190, 289, 114, 412, 180, 216, 309, 288, 170, 391,290, 328, 374, 428, 116, 430, 341, 313, 219, 239, 375, 257, 445, 152, 133, 420, 183, 268, 421,506, 258, 443, 344, 294, 171, 518, 321, 371, 120, 387, 401, 404, 446, 448, 469, 473, 476 of SEQ ID NO: 15 are humanized via point mutations to an endogenous mouse CD98hc nucleic acid encoding the CD98hc ECD.

[0114] The non-human transgenic animal can be a rodent (e.g., a mouse or a rat). In certain embodiments, the chimeric CD98hc polypeptide with the partially or fully humanized CD98hc ECD is expressed in the brain of the non-human transgenic animal. The transgenic animal can be heterozygous or homozygous for the chimeric CD98hc polypeptide. In some embodiments, the non-human transgenic animal expresses a level of the chimeric CD98hc polypeptide described herein in the brain, small intestine, kidney, lung, liver, or heart within about 20% (e.g., within about 15%, within about 10%, within about 5%, or within about 1%) of the level of expression of the endogenous CD98hc polypeptide in the same tissue of a corresponding wild-type non-human animal.Partially humanized CD98hc ECD

[0115] In some embodiments, the non-human transgenic animal expresses a chimeric CD98hc polypeptide that comprises a partially humanized CD98hc ECD. A nucleic acid sequence encoding a portion of the CD98hc ECD endogenous to the non-human transgenic animal can be replaced by at least 2 exons encoding the corresponding portion of the CD98hc ECD found in humans. In some embodiments, 4 exons from a human CD98hc ECD nucleic acid sequence (e.g., exons 9-12 of a human CD98hc nucleic acid sequence) replace a nucleic acid sequence encoding the corresponding portion of the CD98hc ECD endogenous to the non- human transgenic animal.

[0116] In certain embodiments, the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises four humanized exons, in which the four humanized exons encode a portion of the partially humanized CD98hc ECD and replace a nucleic acid sequence encoding the corresponding portion of the CD98hc ECD endogenous to the animal. In particularembodiments, the four humanized exons comprise human exons 9-12 of the human CD98hc nucleic acid sequence.

[0117] In some embodiments, the partially humanized CD98hc ECD in the chimeric CD98hc polypeptide comprises a sequence having at least 85% (e.g., 85%, 90%, 95%, 97%, or 99%) identity to the sequence of SEQ ID NO:2. In some embodiments, the partially humanized CD98hc ECD comprises at least a portion having an amino acid sequence with at least 95% (e.g., 95%, 97%, 99%, or 100%) identity to the amino acid sequence of SEQ ID NO: 13 or 14. In certain embodiments, the chimeric CD98hc polypeptide containing a partially humanized CD98hc ECD in the non-human transgenic animal comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) identity to SEQ ID NO:7. A nucleic acid sequence encoding the chimeric CD98hc polypeptide containing the partially humanized CD98hc ECD (i.e., a nucleic acid sequence containing exons 9-12 from a human CD98hc nucleic acid sequence) can have a sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) identity to the sequence of SEQ ID NO:8. In certain embodiments, the partially humanized CD98hc ECD in the non-human transgenic animal comprises an amino acid sequence having at least 95% (e.g., 95%, 97%, 99%, or 100%) identity to SEQ ID NO: 9.

[0118] Various techniques are available to replace a nucleic acid sequence encoding a portion of the CD98hc ECD endogenous to the non-human transgenic animal by exons encoding the corresponding portion of the human CD98hc ECD (e.g., exons 9-12 from a human CD98hc nucleic acid sequence). For example, the nucleic acid sequence encoding a portion of the CD98hc ECD endogenous to the non-human transgenic animal can be replaced via genetic knock-in by a human complementary DNA (cDNA) sequence encoding exons 9-12 in the human CD98hc nucleic acid sequence.Fully humanized CD98hc ECD

[0119] In some embodiments, the non-human transgenic animal expresses a chimeric CD98hc polypeptide that comprises a fully humanized CD98hc ECD. A nucleic acid sequence encoding the CD98hc ECD in the non-human transgenic animal can be modified in several ways such that the resulting nucleic acid sequence encodes for the CD98hc ECD found in humans. First, a portion of the CD98hc ECD endogenous to the non-human transgenic animal can be replaced by the corresponding portion of the human CD98hc ECD. In some embodiments, six exons from a human CD98hc ECD nucleic acid sequence (e.g., exons 7-12of a a human CD98hc nucleic acid sequence) can replace encoding the corresponding portion of the CD98hc ECD endogenous to the non-human transgenic animal. In particular embodiments, exons 7-12 of the human CD98hc nucleic acid sequence replace the corresponding exons of the CD98hc nucleic acid sequence in the non-human transgenic animal.

[0120] Moreover, the remaining exons in the nucleic acid sequence encoding the CD98hc ECD in the non-human transgenic animal can be further modified by point mutations. The point mutations in each of the remaining exons (i.e., the exons of the CD98hc nucleic acid sequence in the non-human transgenic animal that are not replaced by exons 7-12 of the human CD98hc nucleic acid sequence) can alter the encoded amino acid sequence to match that of the human amino acid sequence encoded by each of exons 4-6 of a human CD98hc nucleic acid sequence. The point mutations can be made such that sequences in the remaining exons in the nucleic acid sequence encoding the CD98hc ECD in the non-human transgenic animal are codon-optimized for expression in the non-human transgenic animal. Methods for codon optimization are readily available, for example, optimizer, which is accessible at http: / / genomes.urv.es / OPTIMIZER, and GeneGPS® Expression Optimization Technology from DNA 2.0 (Newark, California). In a preferred embodiment, the coding sequence is codon-optimized for expression in the non-human transgenic animal using the OptimumGene™ algorithm from GenScript (Piscataway, New Jersey). In certain embodiments, the nucleic acid sequence encoding the chimeric CD98hc polypeptide having a fully humanized CD98hc ECD can include introns that are endogenous to the non-human transgenic animal.

[0121] In some embodiments, the fully humanized CD98hc ECD in the chimeric CD98hc polypeptide comprises a sequence having at least 95% (e.g., 95%, 97%, 99%, Or 100%) identity to the sequence of SEQ ID NO:2. In certain embodiments, the chimeric CD98hc polypeptide containing a fully humanized CD98hc ECD in the non-human transgenic animal comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) identity to SEQ ID NO: 10. A nucleic acid sequence encoding the chimeric CD98hc polypeptide containing the full humanized CD98hc ECD can have a sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) identity to the sequence of SEQ ID NO: 11. In certain embodiments, the fully humanized CD98hc ECD in the non-human transgenic animal comprises an amino acid sequence having at least 95% (e.g., 95%, 97%, 99%, or 100%) identity to SEQ ID NO: 12.

[0122] The non-human transgenic animals described herein are generally healthy and demonstrate physiological conditions that are similar to those of wild-type non-human animals of the same species. For example, the CD98hc polypeptide expression levels are similar to a wild-type animal of the same species; the expression level in the transgenic animal is no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (e.g, 5%, 10%, 20%, or 30%) less than that of the wild-type animal or is no more than 5%, 10%, 20%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 300%, or 500% (e.g., 5%, 10%, 20%, or 30%) greater than that of the wild-type animal. In typical embodiments, transgenic animals in accordance with the disclosure retain selective BBB transport that enables import of nutrients and proteins and retain the ability to protect CNS from toxins. Typically, CD98-mediated cellular trafficking in the transgenic animal is also similar to those wild-type animals. The transgenic animals in accordance with the disclosure are more relevant as a model for pharmacokinetic or pharmacodynamic studies of human BBB -penetrating drugs than the wild-type mice that lack the partially or fully humanized CD98hc ECD entirely.

[0123] In some embodiments, the transgenic animals in accordance with the disclosure have functional cells, tissues, and / or organs. In certain embodiments, the transgenic animals in accordance with the disclosure have functional cells, tissues, and / or organs that are substantially similar to those of the wild-type animal. In certain embodiments, the the transgenic animals in accordance with the disclosure have functional sperm cells, functional ES cells, functional osteoclasts, functional keratinocytes, functional B cells, and / or functional T cells. In certain embodiments, cells that proliferate and / or expand, are functional in the transgenic animals in accordance with the disclosure. In one example, B cells and T cells in the transgenic animals can undergo expansion. In another examples, cancer cells are also able to proliferate in the transgenic animals in accordance with the disclosure.

[0124] Although the present disclosure is illustrated in mouse as shown in the examples, one of ordinary skill in the art would understand that other non-human mammals, for example, rodent (e.g., rat, mouse), rabbit, bovine, ovine, canine, feline, equine, porcine, camelid, non- human primate, and other mammals, can also be engineered to express the CD98hc polypeptide with a partially or fully humanized CD98hc ECD in a similar fashion, and these transgenic animals can also be used for applications as disclosed herein.IV. TRANSGENIC MOUSE MODELS

[0125] The disclosure provides a transgenic mouse that expresses a chimeric CD98 heavy chain (CD98hc) polypeptide containing a partially or humanized CD98hc ECD. The chimeric CD98hc polypeptide also comprises a transmembrane domain (TD) and an intracellular domain (ICD). The transgenic mouse contains a nucleic acid sequence encoding the chimeric CD98hc polypeptide. The partially or fully humanized CD98hc ECD comprises at least a portion having an amino acid sequence with at least 90% identity to the amino acid sequence encoded by human exons 9-12 of a human CD98hc nucleic acid sequence; the TD comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the TD endogenous to the mouse; and the ICD comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the ICD endogenous to the mouse. In some embodiments, the nucleic acid sequence encoding the chimeric CD98hc polypeptide, or a portion thereof, is codon optimized for expression in the transgenic mouse. In some embodiments, the nucleic acid sequence encoding the chimeric CD98hc polypeptide containing a partially or fully humanized CD98hc ECD in the transgenic mouse comprises introns that are endogenous to the transgenic mouse.

[0126] In certain embodiments, the chimeric CD98hc polypeptide with the partially or fully humanized CD98hc ECD is expressed in the brain of the transgenic mouse. The transgenic mouse can be homozygous or heterozygous for the nucleic acid encoding the chimeric CD98hc polypeptide. In some embodiments, the transgenic mouse expresses a level of the chimeric CD98hc polypeptide described herein in the brain, small intestine, kidney, lung, liver, or heart within about 20% (e.g., within about 15%, within about 10%, within about 5%, or within about 1%) of the level of expression of the endogenous CD98hc polypeptide in the same tissue of a corresponding wild-type mouse.

[0127] In some embodiments, the transgenic mouse expresses a chimeric CD98hc polypeptide (e.g., a chimeric CD98hc polypeptide having at least 95% identity to the sequence of SEQ ID NO:7 or 10) at a level capable of mediating transport (i.e., transport of a therapeutic agent) across the BBB. Transgenic mice expressing the chimeric CD98hc polypeptide (e.g., a chimeric CD98hc polypeptide having at least 95% identity to the sequence of SEQ ID NO: 7 or 10) are useful as a model for in vivo functional studies of CD98hc polypeptides having at least 95% identity to the sequence of SEQ ID NO: 1. Further, transgenic mice expressing the chimeric CD98hc polypeptide (e.g., a chimeric CD98hc polypeptide having at least 95%identity to the sequence of SEQ ID NO:7 or 10) are also useful as a model for in vivo screening for CD98hc-binding polypeptides.Partially humanized CD98hc ECD

[0128] In some embodiments, the transgenic mouse expresses a chimeric CD98hc polypeptide comprising a partially humanized CD98hc ECD. In some embodiments, at least four exons encoding a portion of the mouse CD98hc ECD are replaced by the corresponding exons of a human CD98hc ECD. In particular embodiments, mouse exons 7-10 of an endogenous mouse CD98hc nucleic acid sequence are replaced by human exons 9-12 of the human CD98hc nucleic acid sequence. In particular embodiments, mouse exons 5-10 of an endogenous mouse CD98hc nucleic acid sequence are replaced by human exons 7-12 of the human CD98hc nucleic acid sequence.

[0129] In some embodiments, the transgenic mouse expresses a chimeric CD98hc polypeptide comprising a partially humanized CD98hc ECD, in which the partially humanized CD98hc ECD has at least 95% identity to the sequence of SEQ ID NO:9. In particular embodiments, the partially humanized CD98hc ECD comprises the sequence of SEQ ID NO:9. In some embodiments, the transgenic mouse expresses a chimeric CD98hc polypeptide comprising a partially humanized CD98hc ECD, in which the chimeric CD98hc polypeptide has at least 80% identity to the sequence of SEQ ID NO:7. In particular embodiments, the chimeric CD98hc polypeptide comprises the sequence of SEQ ID NO:7.

[0130] A transgenic mouse can comprise a nucleic acid sequence encoding the CD98hc polypeptide containing the partially humanized CD98hc ECD (z.e., a nucleic acid sequence containing exons 9-12 of a human CD98hc nucleic acid sequence), in which the nucleic acid sequence can have a sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) identity to the sequence of SEQ ID NO:8.Fully humanized CD98hc ECD

[0131] In some embodiments, the transgenic mouse expresses a chimeric CD98hc polypeptide comprising a fully humanized CD98hc ECD. A nucleic acid sequence encoding the CD98hc ECD in the transgenic mouse can be modified in several ways such that the resulting nucleic acid sequence encodes for the CD98hc ECD found in humans. One or more exons of the endogenous mouse CD98hc ECD nucleic acid sequence can be replaced by the corresponding exons of a human CD98hc ECD nucleic acid sequence. Further, point mutations can be made to exons of the endogenous mouse CD98hc ECD nucleic acid sequence, such thatthe encoded amino acid sequence match the corresponding amino acid sequence of the human CD98hc. The point mutations can be made in such a manner that resulting nucleic acid sequence in the mouse are codon-optimized for expression in the mouse.

[0132] In some embodiments, at least four exons encoding a portion of the mouse CD98hc ECD are replaced by the corresponding exons of a human CD98hc ECD. In particular embodiments, mouse exons 7-10 of an endogenous mouse CD98hc nucleic acid sequence are replaced by human exons 9-12 of the human CD98hc nucleic acid sequence. In particular embodiments, mouse exons 5-10 of an endogenous mouse CD98hc nucleic acid sequence are replaced by human exons 7-12 of the human CD98hc nucleic acid sequence. Further, the remaining exons, e.g., exons 2-4, in the nucleic acid sequence encoding the CD98hc ECD in the transgenic mouse can be modified by point mutations. The point mutations in each of the remaining exons 2-4 can alter the encoded amino acid sequence to match that of the human amino acid sequence encoded by each of exons 4-6 from a nucleic acid sequence encoding the human CD98hc ECD. The point mutations can be made such that sequences in the remaining exons 2-4 in the nucleic acid sequence encoding the CD98hc ECD in the transgenic mouse are codon-optimized for expression in the transgenic mouse. In certain embodiments, the nucleic acid sequence encoding the CD98hc polypeptide having a fully humanized CD98hc ECD can include introns that are endogenous to the transgenic mouse.

[0133] A transgenic mouse can comprise a nucleic acid sequence encoding the CD98hc polypeptide containing the fully humanized CD98hc ECD, in which the nucleic acid sequence can have a sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) identity to the sequence of SEQ ID NO: 11. In certain embodiments, the fully humanized CD98hc ECD in the transgenic mouse comprises an amino acid sequence having at least 95% (e.g., 95%, 97%, 99%, or 100%) identity to SEQ ID NO: 12. In certain embodiments, the CD98hc polypeptide containing the fully humanized CD98hc ECD in the transgenic mouse comprises an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 97%, 99%, or 100%) identity to SEQ ID NO: 10.

[0134] Further, the disclosure also includes a transgenic rat that expresses a CD98 heavy chain (CD98hc) polypeptide containing a humanized CD98hc ECD. A nucleic acid sequence encoding a rat CD98hc polypeptide has the same exon and intron structures as those found in the mouse nucleic acid sequence. A transgenic rat that comprises a nucleic acid sequenceencoding a CD98hc polypeptide containing a partially or fully humanized CD98hc ECD can be made in a similar manner as that described for the transgenic mouse herein.V. EMBRYONIC STEM CELLS AND EMBRYOS

[0135] The disclosure also provides a non-human embryonic stem (ES) cell that expresses a CD98hc polypeptide containing a humanized CD98hc ECD, in which the non-human ES cell generates the non-human transgenic animal described herein. The non-human ES cell comprises a nucleic acid sequence encoding the CD98hc polypeptide in its genome, and the nucleic acid sequence comprises at least 4 humanized exons encoding at least a portion of the humanized CD98hc ECD that replace a nucleic acid sequence encoding the corresponding portion of the CD98hc ECD endogenous to the non-human ES cell. In some embodiments, the non-human ES cell that expresses a CD98hc polypeptide containing a humanized CD98hc ECD is a mouse ES cell.

[0136] The non-human ES cell can express a CD98hc polypeptide containing a partially humanized CD98hc ECD. The partially humanized CD98hc ECD can comprise an amino acid sequence having at least 95% (e.g., 95%, 97%, 99%, or 100%) identity to SEQ ID NO:9. The CD98hc polypeptide containing the partially humanized CD98hc ECD can comprise an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 97%, 99%, or 100%) identity to SEQ ID NO:7.

[0137] The non-human ES cell can express a CD98hc polypeptide containing a fully humanized CD98hc ECD. The fully humanized CD98hc ECD can comprise an amino acid sequence having at least 95% e.g., 95%, 97%, 99%, or 100%) identity to SEQ ID NO: 12. The CD98hc polypeptide containing the fully humanized CD98hc ECD can comprise an amino acid sequence having at least 80% (e.g., 95%, 97%, 99%, or 100%) identity to SEQ ID NO: 10.

[0138] The disclosure also provides a mouse embryonic stem (ES) cell that expresses a CD98hc polypeptide containing a humanized CD98hc ECD, in which the mouse ES cell generates the transgenic mouse described herein. The mouse ES cell comprises a nucleic acid sequence encoding the CD98hc polypeptide in its genome, and the nucleic acid sequence comprises at least 4 humanized exons encoding at least a portion of the humanized CD98hc ECD that replace a nucleic acid sequence encoding the corresponding portion of the CD98hc ECD endogenous to the mouse ES cell.

[0139] The mouse ES cell can express a CD98hc polypeptide containing a partially humanized CD98hc ECD. The partially humanized CD98hc ECD can comprise an amino acid sequence having at least 95% (e.g., 95%, 97%, 99%, or 100%) identity to SEQ ID NO:9. The CD98hc polypeptide containing the partially humanized CD98hc ECD can comprise an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 97%, 99%, or 100%) identity to SEQ ID NO:7.

[0140] The mouse ES cell can express a CD98hc polypeptide containing a fully humanized CD98hc ECD. The fully humanized CD98hc ECD can comprise an amino acid sequence having at least 95% (e.g., 95%, 97%, 99%, or 100%) identity to SEQ ID NO: 12. The CD98hc polypeptide containing the fully humanized CD98hc ECD can comprise an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 97%, 99%, or 100%) identity to SEQ ID NO: 10.

[0141] The ES cell may be developed into a progeny cell or a non-human transgenic animal whose genome comprises the nucleic acid encoding the CD98hc polypeptide containing the partially or fully humanized CD98hc ECD described herein. In some embodiments, the ES cell is introduced into blastocysts and transferred into pseudo pregnant females (e.g., healthy or diseased pseudo pregnant females). In some cases, a founder male (e.g., a healthy or diseased founder male) harboring the transgene can be selected and bred to wild-type females (e.g., healthy or diseased wild-type females) to generate Fl heterozygous mice. In some cases, a founder female (e.g., a healthy or diseased founder female) harboring the transgene can be selected and bred to wild-type males (e.g., healthy or diseased wild-type males) to generate Fl heterozygous mice. In other cases, a founder female (e.g., a healthy or diseased founder female) harboring the transgene can be selected and bred to a founder male (e.g., a healthy or diseased founder male) harboring the transgene to generate Fl generation homozygous mice. Homozygous non-human animals (e.g., healthy or diseased homozygous non-human animals) can be subsequently generated from breeding of Fl generation heterozygous non-human animals. For example, an Fl generation heterozygous male (e.g., a healthy or diseased Fl generation male) harboring the transgene can be selected and bred to an Fl generation heterozygous female (e.g., a healthy or diseased Fl generation female) to generate homozygous mice harboring the transgene. In other cases, an Fl generation heterozygous male (e.g., a healthy or diseased Fl generation male) harboring the transgene can be selected and bred to wild-type females (e.g., healthy or diseased wild-type females) to generate heterozygous mice harboring the transgene. In yet other cases, an Fl generation heterozygous female (e.g., ahealthy or diseased Fl generation female) harboring the transgene can be selected and bred to wild-type males (e.g., healthy or diseased wild-type males) to generate heterozygous mice harboring the transgene.

[0142] Methods for culturing ES cells and introducing nucleotide sequences to target the genome of an ES cell to produce a transgenic animal are well known, for example, as described in Ramirez-Solis et al., “Gene targeting in mouse embryonic stem cells,” Methods Enzymol., 225:855-878 (1993); and US Pat. Pub. No. 2013 / 0318643, the disclosures of which are incorporated by reference in their entirety. In some embodiments, ES cells from a transgenic animal (e.g., a transgenic mouse) that has a CD98hc polypeptide containing a humanized CD98hc ECD as described herein can be used as a source to provide progeny of the transgenic animal.

[0143] Also provided herein is a method of generating a transgenic non-human single cell embryo, which expresses the CD98hc polypeptide containing a partially or fully humanized CD98hc ECD described herein. In some embodiments, at least a portion of the CD98hc ECD endogenous to the non-human single cell embryo can be replaced with a corresponding heterologous portion having at least 95% (e.g., 95%, 97%, 99%, or 100%) identity to SEQ ID NO: 13. In some embodiments, at least a portion of the CD98hc ECD endogenous to the non- human single cell embryo can be replaced with a corresponding heterologous portion having at least 95% (e.g., 95%, 97%, 99%, or 100%) identity to SEQ ID NO: 14. The corresponding heterologous portion can be codon-optimized for expression in the non-human single cell embryo. Moreover, replacing the portion of the CD98hc ECD can be performed by homologous recombination. In some cases, specifically, the method comprises contacting a Cas9 protein, at least one sgRNA, and a donor DNA comprising a nucleic acid sequence encoding the corresponding heterologous portion, in which the corresponding heterologous portion is flanked by a left homology arm and a right homology arm, such that the corresponding heterologous portion coding sequence replaces the portion of the CD98hc ECD domain endogenous to the non-human single cell embryo (e.g., mouse single cell embryo).

[0144] In one illustrative embodiment, sgRNAs, Cas9, and a donor polynucleotide comprising a nucleic acid sequence encoding a corresponding heterologous portion having the sequence of SEQ ID NO: 13 or 14, where the coding sequence being flanked by a left homology arm and a right homology arm, are introduced into single cell embryos via pronuclear microinjection. The recipient embryos are then transferred to pseudo pregnant females. ThesgRNAs form a complex with the Cas9 protein, which then targets the coding sequence of the portion of the CD98hc ECD to be replaced in the non-human animal embryos. As a result, the portion of the CD98hc ECD in the non-human animal is cleaved and replaced with the corresponding heterologous portion having the sequence of SEQ ID NO: 13 or 14 from the donor polynucleotide. In some cases, a founder male harboring the transgene can be selected and bred to wild-type females to generate Fl heterozygous mice. Homozygous non-human animals can be subsequently generated from breeding of Fl generation heterozygous non- human animals. The transgenic animals disclosed herein can be a rodent, for example, a mouse or a rat.

[0145] Also provided is a method that involves breeding the male or female non-human transgenic animals disclosed herein that that expresses a CD98 heavy chain (CD98hc) polypeptide containing a partially or fully humanized CD98hc ECD with another non-human transgenic animal model to generate progeny animals (e.g., mice) and the resulting progeny non-human transgenic animal. In some cases, the non-human transgenic animal is a transgenic mouse that can be crossed with various mouse models, for example, an ALS mouse model, such as described in US Pat. No. 8,476,485; an AD mouse model, such as described in US Pat. No. 5,898,094 and US Pat. No. 6,175,057; a TSPO mouse model, such as described in US Pat. Pub. No. 2016 / 0050895, or an autism spectrum disorder (ASD) mouse model, such as described in US Pat. Pub. No. 2014 / 0041062. The entire content of these aforementioned patents and patent applications are hereby incorporated by reference. These animals can be homozygous, heterozygous, or hemizygous for the nucleic acid encoding the chimeric CD98hc.VI. POLYNUCLEOTIDES AND HOST CELLS

[0146] The disclosure provides a polynucleotide comprising a nucleic acid sequence encoding a CD98 heavy chain (CD98hc) polypeptide that comprises a humanized CD98hc ECD. The humanized CD98hc ECD can be a partially humanized CD98hc ECD having an amino acid sequence of at least 95% (e.g., 95%, 97%, 99%, or 100%) identity to SEQ ID NO:9. The humanized CD98hc ECD can be a fully humanized CD98hc ECD having an amino acid sequence of at least 95% (e.g., 95%, 97%, 99%, or 100%) identity to SEQ ID NO: 12.

[0147] The nucleic acid sequence encoding a CD98hc polypeptide having a partially humanized CD98hc ECD can contain at least 4 exons (e.g., exons 9-12) from a nucleic acid sequence encoding a human CD98hc polypeptide and introns from a nucleic acid sequence encoding a non-human CD98hc polypeptide. Such a nucleic acid sequence can comprise asequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) identity to the sequence of SEQ ID NO:8.

[0148] The nucleic acid sequence encoding a CD98hc polypeptide having a fully humanized CD98hc ECD can contain at least 6 exons (e.g., exons 7-12) from a nucleic acid sequence encoding a human CD98hc polypeptide and introns from a nucleic acid sequence encoding a non-human CD98hc polypeptide. Additionally, the nucleic acid sequence also comprises exons 2-4 from a nucleic acid sequence encoding a mouse CD98hc polypeptide, in which each of exons 2-4 comprises point mutations that alter the encoding amino acid sequence to match that of the human amino acid sequence encoded by each of exons 4-6 from a nucleic acid sequence encoding a human CD98hc polypeptide. Further exons 2-4 are codon-optimized for expression in the mouse. Such a nucleic acid sequence can comprise a sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) identity to the sequence of SEQ ID NO: 11.

[0149] The polynucleotide may be single-stranded or double-stranded. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is cDNA. In some embodiments, the polynucleotide is RNA.

[0150] In some embodiments, the disclosure provides a host cell that expresses the polynucleotide described herein. In some embodiments, the host cell is a non-human mammalian cell. In some embodiments, the host cell is eukaryotic, e.g., a mouse cell. In some embodiments, the host cell is a cell from a non-primate mammal, such as a mouse, rat, rabbit, bovine, ovine, canine, feline, equine, porcine, and the like. A host cell can also be ex vivo.VII. SCREENING FOR CD98HC-BINDING POLYPEPTIDES

[0151] The CD98hc polypeptides containing a humanized CD98hc ECD disclosed herein can be used to screen for CD98hc-binding polypeptides that are capable of binding to the CD98hc polypeptides. The screening method comprises contacting a candidate CD98hc- binding polypeptide with a CD98hc polypeptide containing a humanized CD98hc ECD disclosed herein and determining the amount of candidate CD98hc-binding polypeptide that binds to the CD98hc polypeptide. In some embodiments, the step of contacting the candidate CD98hc-binding polypeptide with the CD98hc polypeptide comprises contacting the CD98hc- binding polypeptide with a host cell that expresses the CD98hc polypeptide. In some cases, the step of contacting the candidate CD98hc-binding polypeptide with the CD98hc polypeptidecomprises contacting the CD98hc-binding polypeptide with an endothelium that expresses the CD98hc polypeptide. In some embodiments, the endothelium is a BBB endothelium.

[0152] Interactions between the candidate CD98hc-binding polypeptide and the CD98hc polypeptide can be measured using methods well known in the art, for example, immunoassays or SPR. In some embodiments, the binding of the candidate CD98hc-binding polypeptide to the CD98hc polypeptide is measured by ELISA, a Biacore™ system, or coimmunoprecipitation.VIII. SCREENING FOR CD98HC-BINDING POLYPEPTIDES THAT CAN CROSS THE BBB

[0153] The non-human transgenic animals expressing the CD98hc polypeptide containing a humanized CD98hc ECD as described herein can be used to characterize the ability of CD98hc- binding polypeptide to bind the CD98hc polypeptide and ultimately the ability to cross the BBB.

[0154] Typically, to evaluate the ability of a CD98hc-binding polypeptide to cross BBB, the CD98hc-binding polypeptide is administered to the transgenic animal carrying the CD98hc polypeptide containing a humanized CD98hc ECD disclosed herein, preferably through intravenous injection. After a period of time, e.g., at least 10 min, at least 20 min, at least 30 min, at least 60 min, at least 90 min, at least 120 min, at least 180 min, or at least 240 min, the transgenic animal is sacrificed and brain tissues are analyzed to determine the presence of the CD98hc-binding polypeptide. The presence of the CD98hc-binding polypeptide can be determined by assaying for the presence of the CD98hc-binding polypeptide and / or an effector molecule joined thereto. In some embodiments, the brain tissues are perfused with saline, e.g., PBS, and fixed before detection. The presence of the effector molecule in the sections can be detected using standard imaging methods, for example, immunohistochemical or immunofluorescent methods. A positive detection of the effector molecule in the brain tissue indicates that the effector molecule can cross the BBB. In some cases, determining the presence of the CD98hc-binding polypeptide in the brain comprises performing a quantitative immunoassay. The assay for measuring transport across the BBB using the transgenic mouse is robust and can measure greater than a 10-, 20-, 30-, 40-, or 50-fold improvement in uptake of a CD98hc-binding polypeptide.

[0155] In some embodiments, in addition to using imaging methods or immunoassays to detect the presence of the CD98hc-binding polypeptide in the brain, methods of detectingchanges of a substrate of the effector molecule can also be used to evaluate the brain uptake of the effector molecule. In one illustrative embodiment, the CD98hc-binding polypeptide comprises an effector molecule that can inhibit the enzymatic activity of an enzyme in the brain. In some embodiments, the brain uptake of a CD98hc-binding polypeptide, i.e., which reflects its ability of BBB transport, can be measured by assessing enzymatic activity of an enzyme that is modulated by either the CD98hc-binding polypeptide or an effector molecule joined thereto.

[0156] In some embodiments, the brain uptake of candidate CD98hc-binding polypeptides is measured in the brain. Plasma can also be monitored and the pharmacokinetic profiles evaluated. Following the administration of a candidate effector molecule, an increase in the brain-to-plasma ratio as compared to a non-BBB penetrating molecule indicates that the candidate CD98hc-binding polypeptide can cross the BBB.

[0157] In some cases, a non-human transgenic animal comprising a nucleic acid sequence encoding the CD98hc polypeptide containing a humanized CD98hc ECD can be crossed with a non-human transgenic animal that has been engineered to show a certain disease phenotype. In some cases, the non-human transgenic animal is a transgenic mouse that can be crossed with various mouse models, for example, an ALS mouse model, such as described in US Pat. No. 8,476,485; an AD mouse model, such as described in US Pat. No. 5,898,094 and US Pat. No. 6,175,057; a TSPO mouse model, such as described in US Pat. Pub. No. 2016 / 0050895, an autism spectrum disorder (ASD) mouse model, such as described in US Pat. Pub. No. 2014 / 0041062. The entire content of these aforementioned patents and patent applications are hereby incorporated by reference. In some cases, the hybrid mice produced by such crosses can be used to evaluate both the distribution of a CD98hc-binding polypeptide comprising an effector molecule in the brain as well as the efficacy of the CD98hc-binding polypeptide or effector molecule in treating brain diseases.IX. METHODS FOR REPLACING A PORTION OF THE NON-HUMAN CD98HC ECD WITH A DESIRED CD98HC ECD

[0158] A non-human transgenic animal comprising a knock-in of a heterologous portion of the CD98hc ECD as disclosed herein can be generated using a variety of methods, for example, a zinc finger nuclease (ZFN), a Tale-effector domain nuclease (TALEN), a transposon- mediated system, and the CRIPSR / Cas9 system. These methods typically comprise administering to the cell one or more polynucleotides encoding one or more nucleases suchthat the nuclease mediates modification of the endogenous gene by cleaving the DNA to create 5’ and 3’ cut ends in the DNA strand. In the presence of a donor sequence that is flanked by left and right homology arms that are substantially homologous to a sequence extending 5’ from the 5’ end and a sequence extending 3’ from the 3’ end, the donor is integrated into the endogenous gene targeted by the nuclease via homology-directed repair (HDR). In some embodiments, the knock-in is conducted using the CRISPR / Cas9 system. For example, a nucleic acid sequence encoding a heterologous portion of the CD98hc ECD is introduced into an endogenous CD98hc ECD gene, which results in that the naturally occurring sequence that encodes the corresponding endogenous portion of the CD98hc ECD is replaced but the overall structure of the gene is maintained.CRISPR

[0159] In some embodiments, the knock-in of a heterologous portion of the CD98hc ECD that is at least 95% (e.g., 95%, 97%, 99%, or 100%) identical to SEQ ID NO: 13 or 14 is performed using the CRIPSR / Cas9 system. The CRISPR / Cas9 system includes a Cas9 protein and at least one to two ribonucleic acids that are capable of directing the Cas9 protein to and hybridizing to a target motif in the CD98hc ECD that is to be replaced. These ribonucleic acids are commonly referred to as the “single guide RNA” or “sgRNA.” The Cas9 protein then cleaves the target motif, which results in a double-strand break or a single-strand break. In the presence of a donor DNA that comprises the heterologous portion of the CD98hc ECD coding sequence flanked by two homology arms, the donor DNA is inserted into the target CD98hc ECD DNA, replacing the corresponding endogenous portion.

[0160] The Cas9 protein used in the disclosure can be a naturally occurring Cas9 protein or a functional derivative thereof. A “functional derivative” of a native sequence polypeptide is a compound having a qualitative biological property in common with a native sequence polypeptide. “Functional derivatives” include, but are not limited to, fragments of a native sequence and derivatives of a native sequence polypeptide and its fragments, provided that they have a biological activity in common with a corresponding native sequence polypeptide. A biological activity contemplated herein is the ability of the functional derivative of Cas9 to hydrolyze a DNA substrate into fragments. Suitable functional derivatives of a Cas9 polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of Cas9 protein or a fragment thereof.

[0161] In some embodiments, the Cas9 protein is from Streptococcus pyogenes. Cas9 contains 2 endonuclease domains, including a RuvC-like domain which cleaves target DNA that is noncomplementary to the sgRNA, and an HNH nuclease domain which cleave target DNA complementary to sgRNA. The double-stranded endonuclease activity of Cas9 also requires that a short conserved sequence (2-5 nucleotides), known as a protospacer-associated motif (PAM), follows immediately 3’ of a target motif in the target sequence. In some embodiments, the PAM motif is an NGG motif. In one illustrative embodiment, a portion of the CD98hc ECD in a mouse is replaced by using the Cas9 protein, which is directed by sgRNAs to the region between exons 7-10 or between exons 5-10 of the mouse gene. A donor DNA is introduced to the reaction. In one example, the donor DNA comprises exons 9-12 from a human CD98hc sequence that is between a left homology arm that is homologous to the mouse sequence starting upstream of exon 7 and a right homology arm that is homologous to the mouse sequence starting within exon 10. As a result, the nucleotide sequence encoding exons 9-12 from a human CD98hc sequence can be inserted after the fourth mouse exon, and the inserted nucleotide sequence is flanked at the 3’ end by the appropriately following mouse exon. In another example, the donor DNA comprises exons 7-12 from a human CD98hc sequence that is between a left homology arm that is homologous to the mouse sequence starting upstream of exon 5 and a right homology arm that is homologous to the mouse sequence starting within exon 10. As a result, the nucleotide sequence encoding exons 7-12 from a human CD98hc sequence can be inserted after the fourth mouse exon, and the inserted nucleotide sequence is flanked at the 3’ end by the appropriately following mouse exon. In some embodiments, the human coding sequence that is inserted into the mouse gene is codon- optimized for mouse expression.

[0162] The sgRNAs can be selected depending on the particular CRISPR / Cas9 system employed and the sequence of the target polynucleotide. In some embodiments, the one to two ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas9 protein. In some embodiments, each of the one to two ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas9 protein, wherein the target motifs flank the genomic sequence to be replaced. Guide RNAs can be designed using software that is readily available, for example, at http: / / crispr.mit.edu.

[0163] In some embodiments, the donor DNA as disclosed herein comprises a nucleotide sequence that encodes an amino acid sequence at least 95% (e.g., 95%, 97%, 99%, or 100%)identical to SEQ ID NO: 13. In some embodiments, the donor DNA as disclosed herein comprises a nucleotide sequence that encodes an amino acid sequence at least 95% (e.g., 95%, 97%, 99%, or 100%) identical to SEQ ID NO: 14. The donor DNA as disclosed herein further comprises a left homology arm and a right homology arm that flank the nucleotide sequence and are designed to overlap the 5’ and 3’ exon sequences relative to the cleave site by the Cas9 protein. The homology arms may extend beyond the 5’ and 3’ exon sequences, and each of the homology arms may be at least 20, 30, 40, 50, 100, or 150 nucleotides in length. One of skilled in the art can readily determine the optimal length of the homology arm required for the experiment.

[0164] In some embodiments, the sgRNAs can also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, the one to two ribonucleic acids are designed to hybridize to a target motif that contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell to minimize off-target effects of the CRISPR / Cas9 system. Those skilled in the art will appreciate that a variety of techniques can be used to select suitable target motifs for minimizing off-target effects (e.g., bioinformatics analyses). Methods of using the CRISPR / Cas9 system to reduce gene expression are described in various publications, e.g., US Pat. Pub. Nos. 2014 / 0170753 and 2016 / 0257974, the disclosures of which hereby are incorporated by reference in their entirety.Zinc finger nuclease (ZFN)

[0165] In some embodiments, the CD98hc polypeptide containing a humanized CD98hc ECD is produced by knocking-in the humanized CD98hc ECD or a portion thereof using a ZFN. ZFNs are fusion proteins that comprise a non-specific cleavage domain (N) of FokI endonuclease and a zinc finger protein (ZFP). A pair of ZNFs are involved to recognize a specific locus in a target gene: one that recognizes the sequence upstream and the other that recognizes the sequence downstream of the site to be modified. The nuclease portion of the ZFN cuts at the specific locus. The donor DNA as described above can then be inserted into the specific locus. Methods of using the ZFNs to reduce gene expression are well known, for example, as disclosed in US Pat. No. 9,045,763 and also in Durai etal., “Zinc Finger Nucleases: Custom-Designed Molecular Scissors for Genome Engineering of Plant and Mammalian cells,” Nucleic Acid Research, 33 (18):5978-5990 (2005), the disclosures of which are incorporated by reference in their entirety.Transcription activator-like effector nucleases (TALENs)

[0166] In some embodiments, the CD98hc polypeptide containing a humanized CD98hc ECD is produced by knocking-in the humanized CD98hc ECD or a portion thereof with TALENs. TALENs are similar to ZFNs in that they bind as a pair around a genomic site and direct the same non-specific nuclease, FokI, to cleave the genome at a specific site, but instead of recognizing DNA triplets, each domain recognizes a single nucleotide. Methods of using the ZFNs to reduce gene expression are also well known, for example, as disclosed in US Pat. No. 9,005,973 and also Christian et al. , “Targeting DNA Double-Strand Breaks with TAL Effector Nucleases,” Genetics, 186(2): 757-761 (2010), the disclosures of which are incorporated by reference in their entirety.X. CD98HC-BINDING POLYPEPTIDES

[0167] A “CD98hc-binding polypeptide” is a polypeptide that binds to a CD98hc polypeptide having a partially or fully humanized CD98hc ECD, e.g., those as described herein. The CD98hc-binding polypeptide can be an antibody or any polypeptide that is capable of binding to the CD98hc ECD. In some embodiments, the CD98hc-binding polypeptide is an agent that is to be delivered across the blood-brain barrier. In some embodiments, the CD98hc- binding polypeptide further comprises an effector molecule coupled to it, e.g., by covalent linkage. The effector molecule may be a therapeutic agent, a labelling agent, or diagnostic agent. In certain embodiments, the effector molecule is a polypeptide, such as a therapeutic or diagnostic antibody, or a polypeptide that has an enzymatic activity or inhibitory activity on an enzyme or a signaling molecule. In certain embodiments, the effector molecule comprises a small molecule, RNA, DNA, or protein.

[0168] In some embodiments, the CD98hc-binding polypeptide is a bispecific antibody, with the binding region being an antibody that recognizes the CD98hc ECD and the effector molecule being an antibody that recognizes a different antigen, e.g., an enzyme or a signaling molecule, and the binding of the effector moiety either activates or inhibits the enzyme or the signaling molecule.XI. KITS

[0169] In some embodiments, kits comprising a CD98hc polypeptide with a humanized CD98hc ECD, or a polynucleotide encoding such polypeptides, or cells that express such polypeptides, as described herein are provided. In some embodiments, the kits are for use in screening for CD98hc-binding polypeptides as described above.

[0170] In some embodiments, the kit further comprises buffers and vessels that can be used in the assay to detect the binding between the CD98hc polypeptide with a humanized CD98hc ECD and a CD98hc-binding polypeptide. In some embodiments, the kit further comprises instructional materials containing directions (i.e., protocols) for the practice of the methods described herein (e.g., instructions for using the kit for administering a composition across the blood-brain barrier). While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD-ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.XII. EXAMPLES

[0171] The following examples are for illustrative purposes only and should not be interpreted as limitations of the claimed disclosure. There are a variety of alternative techniques and procedures available to those of skill in the art which would similarly permit one to successfully perform the intended disclosure.Example 1: Construct design and mouse model generation for full ECD knock-in CD98hc mouse

[0172] We generated a construct for replacing the ECD in mouse CD98hc with a humanized gene locus as shown in FIG. 1A. Briefly, this construct was designed to replace the entire mouse ECD with the human ECD sequence. The construct replaced a part of mouse exon 2, all of mouse exons 3-9, and the coding portion of mouse exon 10 with: the corresponding part of human exon 4, all human exons 5-11, and the corresponding coding portion of human exon 12. The construct thus retained mouse exon 1, the non-ECD coding portion of exon 2, and the non-coding portion of exon 10, as well as mouse introns 1-3.

[0173] This construct was electroporated into mouse ES cells. ES were screened for proper incorporation by PCR, and ES cells containing the properly incorporated humanized SLC3 A2 gene were injected into goGermline blastocytes (Ozgene), followed by embryo transfer to pseudo pregnant females. The resulting male mice were sterile and could not be bred, presumably due to disrupted CD98hc expression. Two possible reasons for the lack of functional CD98hc were considered: splicing or promoter disruption due to the introduction of the neomycin resistance gene, or improper splicing due to introduction of the human genomicsequence. To overcome these problems, two different mice were designed, as described in Examples 2 and 8 below.Example 2: Construct design and mouse model generation for full ECD knock-in CD98hc mouse

[0174] Following the failure of our first attempt to generate humanized CD98hc mouse model with two main priorities: first preserve the structure of the enodgenous genomic locus as much as possible to ensure both correct splicing and faithful recapitulation of CD98hc expression patterns and levels and second, if possible, humanize the entire ECD to be able to use this model to test any molecule that binds the ECD of CD9hc. The following approach aimed to achieve both of these priorities.

[0175] The construct for humanizing the extracellular domain (ECD) of CD98hc contained 5 primary elements. First, 3’ and 5’ arms homologous to the endogenous mouse SLC3A2 locus to enable homologous recombination. Next, point mutations were made in murine exons 2, 3, and 4 to humanize only those extracellular mouse residues that differ from the orthologous human residues. This second element enabled preservation of mouse introns 1, 2, and 3, which are predicted to have promoter regulatory regions, and the endogenous splice sites at the intronl-exonl / 2, intron2-exon 2 / 3, intron3-exon 3 / 4, and intron4-exon4 junctions. The third element was an FRT flanked Neo cassette (neomycin resistance gene) into murine intron 4, which served to disrupt a long region of mouse homology that could have caused incomplete incorporation of the entire construct and enabled screening for partial incorporation based on neomycin antibiotic resistance. Because intron 4 also contained predicted promoter regulatory regions we were concerned the Neo cassette could disrupt Slc3a2 expression therefore the FRT sites provided the option to remove the cassette in ES after incorporation was confirmed. The fourth element was the cDNA of human residues E335 to the STOP codon in place of the murine genomic DNA from residue S268 in exon 5 to the STOP codon in exon 10, which achieved humanization of the remainder of the CD98hc ECD while providing enough differentiation from the endogenous mouse sequence that homologous recombination of the entire construct could be achieved. The fifth element was a F3’ flanked hygro cassette (hygromycin resistance gene) downstream of the murine 3’ UTR, which enabled screening for incorporation of the entire construct by adding hygromycin in addition to neomycin to the ES cell culture medium. Because the hygromycin was after the stop codon, we did not anticipate it disrupting Slc3a2 expression and the cassette would be automatically excised in the malegermline of the resulting mice. FIG. IB illustrates this construct design for full ECD knock-in CD98hc mouse.

[0176] This construct was electroporated into ES cells from C57B16 mice. ES cells with proper homologous recombination were selected for by growing the cells in the presence of neomycin and hygromycin. Incorporation was confirmed by PCR. The Neo cassette was removed in vitro by electroporation of a Flp recombinase expressing construct. This step was critical as the Neo cassette was suspected to disrupt expression of the SLC3A2 gene, and CD98hc protein is required for sperm function. This approach resulted in surface expression of hCD98hc on the ES cells. By contrast, ES cells that retained the Neo cassette did not express huCD98hc on ES cells. ES cells containing the properly incorporated humanized SLC3 A2 gene without the Neo cassette were injected into goGermline blastocytes (Ozgene), followed by embryo transfer to pseudo pregnant females. Founder males were selected from the offspring of the female that received the embryos and bred to wild-type females to generate Fl heterozygous mice. Homozygous mice were subsequently generated from breeding of Fl generation heterozygous mice. Successful generation of viable and fertile Fl heterozygous mice supports the hypothesis that a construct that largely retains the endogenous mouse sequence upstream of exon 5 and has the Neo cassette prior to embryo transfer is important for preserving Slc3a2 expression.Example 3: Human CD98hc expression analysis of full ECD knock-in CD98hc mouseCD98hcmu / huKI or SLC3A2huECD / huECD) by IHC and Western

[0177] Wild-type (C57 / B6) control and / '3A2l'ul':c,) / l'ul':c,)(“huCD98hcKI / KI”) animals were sacrificed. Hemi-brains, kidney, testis and pancreas were drop fixed in 4% PFA overnight followed by 30% sucrose preservation. Additionally, pieces of brain, small intestine, kidney, lung, liver, and heart were snap frozen.

[0178] For fixed tissues, 10 pm sections (sagittal for brain) were cut using a cryostat and mounted on superfrost slides, blocked in 5% BSA + 0.3% Triton X-100, followed by staining with either rat anti-msCD98hc conjugated to Alex647 (Biolegend, 128202, clone RL338, 1 :500) or human anti-human CD98hc (in house antibody, 1 : 1000) followed by secondary staining with Alexa488 anti-huIgG (Jackson ImmunoReseach, 109-545-003, 1 :500). Brain sections were also co-stained with mouse anti-Glutl primary antibody (Sigma MABS132, 1 :500) (followed by Alexa 568 conjugated anti-mouse IgGl secondary, 1 :500) to identify the brain vasculature. Images of brain sections were taken using a Leica SP8 Lightning confocalmicroscope with a 20x objective. Peripheral tissue images were taken with a Zeiss Axio Scan with a 20x objective.

[0179] CD98hc protein expression was observed on endothelial cells and astrocytes in both wild-type (C57 / B6) control and huCD98hcKI / KIanimals indicating the knocked in humanized CD98hc recapitulates mouse CD98hc expression in brain (FIGS. 2A-2D). CD98hc protein expression was additionally observed on a subset of kidney tubules, Leydig cells in the testis, as well as high expression on pancreatic lobules with lower expression in both wild-type (C57 / B6) control and huCD98hcKI / KIanimals indicating the knocked in humanized CD98hc recapitulates mouse CD98hc expression in peripheral tissues (FIGS. 3A-3F).

[0180] Frozen tissues were homogenized using a Qiagen TissueLyser in lOx tissue weight of lysis buffer containing 1% NP-40 + protease inhibitors in PBS. Total protein concentration was measured with BCA and samples were diluted to Img / ml of total protein in NuPAGE LDS sample buffer (4X) with NuPAGE sample reducing agent (10X) and boiled for 10 minutes. A separate aliquot of brain lysate was diluted in NuPAGE LDS sample buffer (4X) without any reducing agent. Samples were run on NuPAGE 4-12% Bis-Tris gels. Because anti-CD98hc antibodies are either mouse or human CD98hc specific, denatured protein samples were run with dilutions (50, 10, 2, 0.4, 0.08ug / mL) of either mouse or human CD98hc recombinant protein on the same gels. Mouse CD98hc was detected with rabbit anti-mouse CD98hc (LSBio, LS-C296476, 1 : 1000) and human CD98hc was detected with rabbit anti-human CD98hc (ThermoFisher PA5-23661, 1 : 1000). Blots were cut and co-stained with rabbit anti-GAPDH (Abeam abl81603, 1 :5000) as a loading control. Primary antibodies were detected with licor secondaries and blots were imaged on a Licor Odyssey CLx. Blot images were quantified with Image Studio. For denatured protein samples, concentrations of CD98hc were calculated based on a standard curve of recombinant proteins (FIG. 4A). For non-denatured protein samples, the amount of CD98hc paired with light chain and unpaired CD98hc was quantified relative to the total amount of CD98hc (FIG. 4B).

[0181] This data further confirms that the spatial expression of humanized CD98hc is similar to that of endogenous mouse CD98hc. Additionally, this data shows that protein levels of humanized CD98hc in homozygous knock-in mice are similar to that of endogenous mouse CD98hc and that humanized CD98hc can still pair with endogenous mouse light chains.Example 4: Brain uptake of anti-CD98hc antibody by full ECD knock-in CD98hc mouse(i.e., CD98hcmu / huKI or SLC3A2huECD / huECD)

[0182] SLC3A2huECD / huECD(“huCD98hcKI / KI”) mice were intravenously injected with 50 mg / kg of either the anti-DNP02 antibody or the anti-huCD98hc / BACEl bispecific antibody. The anti-DNP02 antibody is an antibody with non-targeting Fab fragments to dinitrophenyl hapten (DNP) (See, e.g., Leahy, PNAS 3661-3665, 1988). After 48 hours, blood was collected via cardiac puncture, and the mice were perfused with PBS. Brain tissue was homogenized using a Qiagen TissueLyser in lOx tissue weight of lysis buffer containing 1% NP-40 in PBS with protease inhibitors. Blood was collected in EDTA tubes to prevent clotting and spun at 14000 rpm for 7 minutes to isolate plasma. The total therapeutic antibody concentrations in plasma and brain were quantified using a generic anti-human IgG sandwich-format ELISA. Briefly, plates were coated overnight at 4C with donkey anti-human IgG (JIR #709-006-098) at 1 ug / mL in sodium bicarbonate solution (Sigma #C3041-50CAP) with gentle agitation. Plates were then washed 3x with wash buffer (PBS + 0.05% Tween 20). Assay standards and samples were diluted in PBS + 0.05% Tween 20 + 1% BSA (10 mg / mL). Standard curve preparation ranged from 0.41 to 1,500 ng / mL or 0.003 to lOnM (BLQ < 0.03nM). Standards and diluted samples were incubated with agitation for 2 hr at room temperature. After incubation, plates were washed 3x with wash buffer. Detection antibody, goat anti-human IgG (JIR #109-036-098), was diluted in blocking buffer (PBS + 0.05% Tween 20 + 5% BSA (50 mg / mL)) to a final concentration of 0.02 ug / mL and plates were incubated with agitation for 1 hr at room temperature. After a final 3x wash, plates were developed by adding TMB substrate and incubated for 5-10 minutes. Reaction was quenched by adding 4N H2SO4 and read using 450 nM absorbance.

[0183] After 48 hours, the plasma levels of anti-CD98hc / BACEl were lower than the levels for anti-DNP02, likely due to clearance of this antibody via binding to peripherally-expressed huCD98hc (FIG. 5A). In whole brain, a 2-fold increase in the concentration of anti- CD98hc / BACEl compared to anti-DNP02 was observed (FIG. 5B). The brain to plasma ratio is shown in FIG. 5C. The significant accumulation of the anti-CD98hc / BACEl in the brain parenchyma is due to CD98hc mediated transcytosis at the BBB, this result validates the huCD98hcKI / KImice as a tool for measuring BBB uptake for human CD98hc ECD binding polypeptides.Example 5: Plasma and brain PK to Day 21 of full ECD knock-in CD98hc mouse (i.e., CD98hcmu / huKI or SLC3A2huECD / huECD)

[0184] huCD98hcKI / KImice were intravenously injected with 15 mg / kg of anti-DNP02 antibody (negative control) or 50 mg / kg of anti-huCD98hc / DNP02 bispecific antibody. Plasma samples were taken at 0.5hr, 6hr, 24hr, 48hr, 96hr, 168hr, 336hr, and 504hr. 0.5hr, 6hr, 24hr timepoints were collected in life via submandibular bleeds and 48hr, 96hr, 168hr, 336hr, and 504hr were terminal collections collected via cardiac puncture. Blood was collected in EDTA tubes to prevent clotting and spun at 14000 rpm for 7 minutes to isolate plasma. After terminal blood collection (48hr, 96hr, 168hr, 336hr, and 504hr), the mice were perfused with PBS. Brain tissue was collected and homogenized using a Qiagen TissueLyser in lOx tissue weight of lysis buffer containing 1% NP-40 in PBS with protease inhibitors. The concentration of huIgG was measured in plasma and brain was measured by sandwich ELISA as described above.

[0185] Results for PK in plasma and brain are shown in FIGS. 9 A and 9B. Anti- CD98hc / DNP02 cleared faster from the plasma than the anti-DNP02 control, likely due to clearance of this antibody via binding to peripherally-expressed humanized CD98hc. Anti- CD98hc / DNP02 reached highest concentration in brain 96hr post dose, and was close to control levels by 21 days post dose. The significant accumulation of the anti-CD98hc / DNP02 in the brain parenchyma is due to CD98hc mediated transcytosis at the blood brain barrier (BBB). This result further validates the huCD98hcKI / KImice as a tool for measuring BBB uptake for human CD98hc ECD binding polypeptides.Example 6: IHC of full ECD knock-in CD98hc mouse (i.e., CD98hcmu / huKI orSLC3A2hllECD / huECD)

[0186] To confirm the CD98hc specific BBB transport, SIX'3A2l'"d-f', l'" -f'>(“huCD98hcKI / KI”) mice were intravenously injected with 50 mg / kg of either anti- CD98hc / BACEl or anti-DNP02. After 48 hours, mice were perfused with PBS, and hemibrains were drop fixed in 4% PFA overnight followed by 30% sucrose preservation. Sagittal brain sections (40 pm) were cut using a microtome, blocked in 5% BSA + 0.3% Triton X-100, followed by fluorescent secondary staining with Alexa488 anti-huIgG (1 :500). Brain images were taken using a Leica SP8 Lightning confocal microscope with a 40x objective.

[0187] Broad brain vasculature and parenchymal staining was observed for anti- CD98hc / BACEl, while minimal staining was observed for anti-DNP02 (FIGS. 8A and 8B),indicating that a huCD98hc ECD-binding polypeptide is required for BBB transcytosis in these miceExample 7: Quantification of huIgG in brain vasculature and parenchymal capillary depletion fractions of full ECD knock-in CD98hc mouse

[0188] SLC3A2huECD / huECD(“huCD98hcKI / KI” or “CD98hcmu / huKI”) mice were intravenously injected with 50 mg / kg of either the anti-DNP02 antibody or the anti-huCD98hc / BACEl bispecific antibody. After 48 hours, mice were perfused with PBS and brains were dissected and the meninges and choroid plexus removed. The fresh brain was homogenized with a Dounce homogenizer in HBSS. Homogenized samples were centrifuged (1,000g for 10 min). Cell pellets were resuspended in 17% dextran and centrifuged at 4,122g for 15 min. Cell pellet contained vasculature and the supernatant contained parenchymal cells. Supernatant was added to a tube containing lOmL of HBSS and spun at 4,122g for 15 min. Cell pellet contained parenchymal cells. Both vascular and parenchymal cell pellets were resuspended in lysis buffer containing 1% NP-40 in PBS with protease inhibitors. Total protein concentrations of samples were measured using BCA. huIgG concentration was measured as described above using human IgG assay (MSD human IgG kit #K150JLD) and then normalized to total protein concentration in the sample.

[0189] In the parenchymal fraction, an 8-fold increase in the concentration of anti- CD98hc / BACEl was observed compared to the anti-DNP02 antibody (FIGS. 7A and 7B). The significant accumulation of the anti-CD98hc / BACEl in the brain parenchyma is due to CD98hc mediated transcytosis at the BBB. This result validates the huCD98hcKI / KImice as a tool for measuring BBB uptake for human CD98hc ECD binding polypeptides.Example 8: Construct design and mouse model generation for partial ECD knock-in CD98hc mouse

[0190] For the subsequently describe mouse, we aimed to preserved endogeous splicing and faithful recapitulation of Slc3a2 expression by humanizing only about half of the CD98hc ECD. The construct for partially humanizing the ECD of CD98hc contained the following elements: 3’ and 5’ arms homologous to the endogenous mouse SLC3A2 locus, the cDNA of human exons 9-12 in place of the murine exons 7-10 to the STOP codon in exon 10, and an FRT flanked Neo cassette (neomycin resistance gene) downstream of the murine 3’ UTR. By placing the Neo cassette in this position, it fell outside the putative promoter region and, further, was unlikely to disrupt gene splicing. FIG. 6A shows this construct that humanizes a portionof the ECD of CD98hc. FIG. 6B further shows the portion of the protein that is humanized with this construct.

[0191] This construct was electroporated into ES cells from C57B16 mice. ES cells with proper homologous recombination were selected for by growing the cells in the presence of neomycin and hygromycin. ES cells containing the properly incorporated humanized SLC3 A2 gene were injected into goGermline blastocytes (Ozgene), followed by embryo transfer to pseudo pregnant females. Founder males were selected from the offspring of the female that received the embryos and bred to wild-type females to generate Fl heterozygous mice. Homozygous mice were subsequently generated from breeding of Fl generation heterozygous mice. This approach supports the hypothesis that preserving the endogenous mouse sequence upstream of intron 6 results in expression of Slc3a2 and the resulting partially humanized CD98hc.Example 9: Chimeric CD98hc protein expression analysis of partially humanized ECD CD98hc knock-in mouseWestern

[0192] Wild-type (C57 / B6) control and SLC3A2hu9~12 / hu9~12(“SLC3A29'12 KI / KI”) animals were sacrificed. Hemi-brains, kidney, testis and pancreas were drop fixed in 4% PFA overnight followed by 30% sucrose preservation. Additionally, pieces of brain, small intestine, kidney, lung, liver, and heart were snap frozen.

[0193] For fixed tissues, 10 pm sections (sagittal for brain) were cut using a cryostat and mounted on superfrost slides, blocked in 5% BSA + 0.3% Triton X-100, followed by staining with either rat anti-msCD98hc conjugated to Alex647 (Biolegend, 128202, clone RL338, 1 :500) or a human anti-human CD98hc (1 : 1000) followed by secondary staining with Alexa488 anti-huIgG (Jackson ImmunoReseach, 109-545-003, 1 :500). Both anti-CD98 antibodies, i.e., anti-msCD98hc and anti-huCD98hc antibodies, bind the chimeric 9-12 protein. Images of brain sections were taken using a Leica SP8 Lightning confocal microscope with a 20x objective. Peripheral tissue images were taken with a Zeiss Axio Scan with a 20x objective.

[0194] CD98hc protein expression was observed on endothelial cells and astrocytes in both wild-type (C57 / B6) control and SLC3A2hu9~12 / hu9~12(“SLC3A29'12 KI / KI”) animals indicating the knocked in humanized CD98hc recapitulates mouse CD98hc expression in brain (FIGS. 11 A- 1 ID). CD98hc protein expression was additionally observed on a subset of kidney tubules, Leydig cells in the testis, as well as high expression on pancreatic lobules with lower expression in both wild-type (C57 / B6) control and SLC3A29'12 KI / KIanimals indicating the knocked inhumanized CD98hc recapitulates mouse CD98hc expression in peripheral tissues (FIGS. 12A- 12D, 13A-13D, 14A-14D).

[0195] Frozen tissues were homogenized, lysed and run on gels as described above and probed for mouse CD98hc with rabbit anti-mouse CD98hc (LSBio, LS-C296476, 1 :1000), which does not bind the 9-12 hu / msCD98hc chimeric protein, and human CD98hc with rabbit anti-human CD98hc (ThermoFisher PA5-23661, 1 : 1000), which is not mouse CD98hc cross- reactive and therefore can be used to detect the hu / msCD98hc chimeric protein. Separate western blots for mouse and human CD98hc were run because a mouse / human cross-reactive detection antibody is not available, and the anti-mouse CD98hc antibody does not bind the chimeric protein. Results are shown in FIG. 10. This data shows expression of chimeric hu / msCD98hc protein in 9-12 huCD98hc KI mice in tissues that should express CD98hc (brain, kidney, and testis) and not in tissues that should not (lung and liver). This shows that the spatial expression of 9-12 hu / msCD98hc chimeric protein recapitulates endogenous mouse CD98hc expression.Example 10: Brain uptake of anti-CD98hc antibody by partial ECD knock-in CD98hc mouse

[0196] SLC3A2hu9~12 / hu9~12(“SLC3A29'12 KI / KI”) mice were intravenously injected with 50 mg / kg of either the anti -B ACE 1 antibody (negative control), anti -msCD98hc / B ACE 1 bispecific antibody, or an anti-huCD98hc / BACEl bispecific antibody. Binding of the anti- msCD98hc / BACEl and anti-huCD98hc / BACEl bispecific antibody to the 9-12 hu / msCD98hc chimeric protein was measured using single cycle kinetics on a Biacore 8K instrument (FIG. 15 A). After 48 hours, blood was collected via cardiac puncture, and the mice were perfused with PBS. Brain tissue was homogenized using a Qiagen TissueLyser in lOx tissue weight of lysis buffer containing 1% NP-40 in PBS with protease inhibitors. Blood was collected in EDTA tubes to prevent clotting and spun at 14000 rpm for 7 minutes to isolate plasma. Antibody concentrations in mouse plasma and brain lysates were quantified using a sandwich huIgG ELISA as described above. Plasma and brain huIgG levels are shown in FIGS. 15B and 15C. Anti-mouse CD98hc / BACEl and anti-human CD98hc / BACEl were observed in brain at concentrations about 2-fold higher than the control anti-B ACE1 antibody 48hr post 50mg / kg dose. This data confirms chimeric 9-12 hu / ms CD98hc dependent brain uptake in the SLC3 A29' 12 KI / KImousemodel. Furthermore, this data validates the SLC3 A29'12 KI / KImouse as a tool for measuring BBB uptake for human CD98hc ECD binding polypeptides that bind the humanized half of the 9-12 hu / ms CD98hc chimeric protein.XIII. EXEMPLARY EMBODIMENTS

[0197] Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments:1. A non-human transgenic animal that expresses a chimeric CD98 heavy chain (CD98hc) polypeptide comprising a partially or fully humanized CD98hc extracellular domain (ECD), a transmembrane domain (TD), and an intracellular domain (ICD), wherein the animal comprises a nucleic acid sequence encoding the chimeric CD98hc polypeptide, and wherein:(1) a portion of the partially or fully humanized CD98hc ECD comprises an amino acid sequence having at least 90% identity to the amino acid sequence encoded by human exons 9-12 of a human CD98hc nucleic acid sequence;(2) the TD comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the TD endogenous to the animal; and(3) the ICD comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the ICD endogenous to the animal.2. The non-human transgenic animal of embodiment 1, wherein the chimeric CD98hc polypeptide is expressed in the brain of the animal.3. The non-human transgenic animal of embodiment 1 or 2, wherein the animal comprises a partially humanized CD98hc ECD.4. The non-human transgenic animal of embodiment 3, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises four humanized exons, wherein the four humanized exons encode a portion of the partially humanized CD98hc ECD and replace a nucleic acid sequence encoding the corresponding portion of the CD98hc ECD endogenous to the animal.5. The non-human transgenic animal of embodiment 4, wherein the four humanized exons comprise human exons 9-12 of the human CD98hc nucleic acid sequence.6. The non-human transgenic animal of any one of embodiments 3 to 5, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide, or a portion thereof, is codon-optimized for expression in the animal.7. The non-human transgenic animal of any one of embodiments 3 to 6, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises introns that are endogenous to the animal.8. The non-human transgenic animal of any one of embodiments 3 to 5, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises a sequence having at least 70% identity to the sequence of SEQ ID NO:8.9. The non-human transgenic animal of any one of embodiments 3 to 8, wherein the partially humanized CD98hc ECD comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 9.10. The non-human transgenic animal of any one of embodiments 3 to 9, wherein the chimeric CD98hc polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 7.11. The non-human transgenic animal of embodiment 1 or 2, wherein the animal comprises a fully humanized CD98hc ECD.12. The non-human transgenic animal of embodiment 11, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises six humanized exons, wherein the six humanized exons encode a portion of the fully humanized CD98hc ECD and replace a nucleic acid sequence encoding the corresponding portion of the CD98hc ECD endogenous to the animal.13. The non-human transgenic animal of embodiment 12, wherein the six humanized exons comprise human exons 7-12 of the human CD98hc nucleic acid sequence.14. The non-human transgenic animal of any one of embodiments 11 to 13, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide further comprises humanizing point mutations.15. The non-human transgenic animal of embodiment 14, wherein the humanizing point mutations alter the encoded amino acid sequence to match that of the amino acid sequence encoded by human exons 4-6 of the human CD98hc nucleic acid sequence.16. The non-human transgenic animal of any one of embodiments 11 to 15, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide, or a portion thereof, is codon-optimized for expression in the animal.17. The non-human transgenic animal of any one of embodiments 11 to 16, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises introns that are endogenous to the animal.18. The non-human transgenic animal of any one of embodiments 11 to 17, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises a sequence having at least 70% identity to the sequence of SEQ ID NO: 11.19. The non-human transgenic animal of any one of embodiments 11 to 18, wherein the fully humanized CD98hc ECD comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 12.20. The non-human transgenic animal of any one of embodiments 11 to 19, wherein the chimeric CD98hc polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 10.21. The non-human transgenic animal of any one of embodiments 1 to 20, wherein the animal is a rodent.22. The non-human transgenic animal of embodiment 21, wherein the animal is a mouse.23. The non-human transgenic animal of embodiment 21, wherein the animal is a rat.24. The non-human transgenic animal of embodiment 3, wherein the animal is a mouse, and wherein the mouse exons 7-10 of an endogenous mouse CD98hc nucleic acid sequence are replaced by human exons 9-12 of the human CD98hc nucleic acid sequence.25. The non-human transgenic animal of embodiment 11, wherein the animal is a mouse, andwherein the mouse exons 5-10 of an endogenous mouse CD98hc nucleic acid sequence are replaced by human exons 7-12 of the human CD98hc nucleic acid sequence.26. The non-human transgenic animal of any one of embodiments 1 to 25, wherein the animal is homozygous for the chimeric CD98hc polypeptide.27. The non-human transgenic animal of any one of embodiments 1 to 25, wherein the animal is heterozygous for the chimeric CD98hc polypeptide.28. A genetically modified mouse comprising an amino acid sequence encoded by at least human exons 9-12 of a human CD98hc nucleic acid sequence, wherein the human exons 9-12 replace mouse exons 7-10 of an endogenous mouse CD98hc nucleic acid sequence, and wherein the mouse expresses a chimeric CD98hc polypeptide.29. The genetically modified mouse of embodiment 28, wherein the mouse comprises human exons 7-12 of the human CD98hc nucleic acid sequence, wherein the human exons 7-12 replace mouse exons 5-10 of an endogenous mouse CD98hc nucleic acid sequence.30. The genetically modified mouse of embodiment 29, wherein the mouse further comprises one or more humanizing point mutations in mouse exons 2-4.31. The genetically modified mouse of embodiment 30, wherein the humanizing point mutations alter the amino acid sequence encoded by mouse exons 2-4 of the endogenous mouse CD98hc nucleic acid sequence to match that of the amino acid sequence encoded by human exons 4-6 of the human CD98hc nucleic acid sequence.32. The genetically modified mouse of any one of embodiments 28 to 31, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide, or a portion thereof, is codon-optimized for expression in the mouse.33. The genetically modified mouse of any one of embodiments 28 to 32, wherein the nucleic acid encoding the chimeric CD98hc polypeptide is under the control of a promoter endogenous to the mouse.34. A genetically modified mouse comprising one or more humanizing point mutations introduced into an endogenous mouse CD98hc nucleic acid sequence, wherein the humanizing point mutations alter the encoded amino acids such that at least 70% of the amino acids selected from positions: 327, 295, 376, 383, 514, 478, 253, 214, 338, 384, 481, 491, 512, 507, 471, 220, 516, 135, 234, 235, 280, 182, 320, 139, 148, 342, 346,146, 527, 347, 186, 212, 136, 251, 173, 306, 224, 397, 524, 254, 400, 202, 500, 250, 493, 145,509, 172, 134, 396, 315, 528, 505, 256, 269, 177, 231, 308, 191, 501, 190, 289, 114, 412, 309,288, 170, 391, 328, 116, 313, 219, 257, 152, 420, 268, 506, 171, 518, 120, 401, 404, 469, 473, and 476 of SEQ ID NO: 15 are humanized.35. A genetically modified mouse comprising one or more humanizing point mutations introduced into an endogenous mouse CD98hc nucleic acid sequence, wherein the humanizing point mutations alter the encoded amino acids such that at least 70% of the amino acids selected from positions: 327, 295, 376, 383, 514, 478, 253, 214, 338, 384, 481, 491, 512, 507, 471, 220, 516, 135, 234, 235, 280, 182, 320, 139, 148, 342, 346,146, 527, 347, 186, 212, 136, 251, 173, 306, 224, 397, 524, 254, 400, 202, 500, 250, 493, 145,509, 172, 134, 396, 315, 528, 505, 256, 269, 177, 231, 308, 191, 501, 190, 289, 114, 412, 309,288, 170, 391, 328, 116, 313, 219, 257, 152, 268, 506, 171, 518, 120, 401, 404, 469, 473, 476 of SEQ ID NO: 15 are humanized.36. A genetically modified mouse comprising one or more humanizing point mutations introduced into an endogenous mouse CD98hc nucleic acid sequence, wherein the humanizing point mutations alter the encoded amino acids such that at least 70% of the amino acids selected from positions: 327, 295, 376, 383, 514, 478, 253, 214, 338, 384, 481, 491, 512, 507, 471, 220, 516, 135, 234, 235, 280, 182, 320, 139, 148, 342, 346,146, 527, 347, 186, 212, 136, 251, 173, 306, 224, 397, 524, 254, 400, 202, 500, 250, 493, 145,509, 172, 134, 396, 315, 528, 505, 256, 269, 485, 177, 231, 308, 191, 501, 190, 289, 114, 412,180, 216, 309, 288, 170, 391, 328, 116, 430, 313, 219, 257, 152, 420, 268, 506, 258, 171, 518,120, 387, 401, 404, 469, 473, and 476 of SEQ ID NO: 15 are humanized.37. The genetically modified mouse of embodiment 34, wherein the humanizing point mutations alter the amino acid sequence encoded by mouse exons 2-4 of the endogenous mouse CD98hc nucleic acid sequence to match that of the amino acid sequence encoded by human exons 4-6 of a human CD98hc nucleic acid sequence.38. The genetically modified mouse of any one of embodiments 34 to 37, wherein the mouse comprises an amino acid sequence encoded by at least human exons 9-12 of a human CD98hc nucleic acid sequence, wherein the human exons 9-12 replace mouse exons 7-10 of the endogenous mouse CD98hc nucleic acid sequence.39. The genetically modified mouse of any one of embodiments 34 to 38, wherein the mouse comprises an amino acid sequence encoded by human exons 7-12 of a human CD98hc nucleic acid sequence, wherein the human exons 7-12 replace mouse exons 5-10 of the endogenous mouse CD98hc nucleic acid sequence.40. A non-human embryonic stem (ES) cell that expresses a chimeric CD98hc polypeptide comprising a partially or fully humanized CD98hc extracellular domain (ECD), a transmembrane domain (TD), and an intracellular domain (ICD) in a non-human transgenic animal generated from the non-human ES cell, wherein:(1) a portion of the partially or fully humanized CD98hc ECD comprises an amino acid sequence having at least 90% identity to the amino acid sequence encoded by human exons 9-12 of a human CD98hc nucleic acid sequence;(2) the TD comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the TD endogenous to the animal; and(3) the ICD comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the ICD endogenous to the animal.41. The non-human ES cell of embodiment 40, wherein the chimeric CD98hc polypeptide is expressed in the brain of the animal generated from the non-human ES cell.42. The non-human ES cell of embodiment 40 or 41, wherein the partially humanized CD98hc ECD comprises an amino acid sequence having at least 95% identity to SEQ ID NO:9.43. The non-human ES cell of embodiment 42, wherein the chimeric CD98hc polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:7.44. The non-human ES cell of embodiment 40 or 41, wherein the fully humanized CD98hc ECD comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 12.45. The non-human ES cell of embodiment 44, wherein the CD98hc polypeptide comprises an amino acid sequence having at least 80%% identity to SEQ ID NO:10.46. A polynucleotide encoding a chimeric CD98hc polypeptide, comprising a nucleic acid sequence encoding a partially or fully humanized CD98hc ECD and a nucleic acid sequence encoding a non-human CD98hc TD and / or non-human CD98hc ICD.47. The polynucleotide of embodiment 46, wherein the polynucleotide comprises a nucleic acid sequence encoding a partially humanized CD98hc ECD.48. The polynucleotide of embodiment 47, wherein the nucleic acid sequence comprises human exons 9-12 of a human CD98hc nucleic acid sequence.49. The polynucleotide of embodiment 47 or 48, wherein the polynucleotide comprises a nucleic acid sequence having at least 70% identity to the sequence of SEQ ID NO:8.50. The polynucleotide of embodiment 46, wherein the polynucleotide comprises a nucleic acid sequence encoding a fully humanized CD98hc ECD.51. The polynucleotide of embodiment 47, wherein the nucleic acid sequence comprises human exons 7-12 of a human CD98hc nucleic acid sequence.52. The polynucleotide of embodiment 47 or 48, wherein the polynucleotide further comprises humanizing point mutations.53. The polynucleotide of embodiment 52, wherein the humanizing point mutations alter the encoded amino acid sequence to match that of the amino acid sequence encoded by human exons 4-6 of the human CD98hc nucleic acid sequence.54. The polynucleotide of any one of embodiments 50 to 53, wherein the polynucleotide comprises a nucleic acid sequence having at least 70% identity to the sequence of SEQ ID NO: 11.55. The polynucleotide of any one of embodiments 46 to 54, wherein the polynucleotide, or a portion thereof, is codon optimized for expression in a non-human animal.56. The polynucleotide of any one of embodiments 46 to 55, wherein the polynucleotide comprises introns that are endogenous to the non-human animal.57. The polynucleotide of any one of embodiments 46 to 56, wherein the non-human animal is a mouse.58. A chimeric CD98 heavy chain (CD98hc) polypeptide or a portion thereof encoded by the polynucleotide of any one of embodiments 46 to 57.59. An expression cassette for expressing a polynucleotide of any one of embodiments 46 to 57 in a non-human transgenic animal, comprising an endogenous promoter operably linked to the polynucleotide, wherein the introduction of the expression cassette into the animal results in the animal expressing a chimeric CD98hc polypeptide.60. An expression cassette for expressing a polynucleotide encoding a chimeric CD98hc polypeptide in a transgenic mouse, wherein the polynucleotide comprises a nucleic acid sequence having at least 70% identity to the sequence of SEQ ID NO:8 or 11.61. A host cell comprising the expression cassette of embodiment 59 or 60.62. The host cell of embodiment 61, wherein the host cell is a mouse cell.63. The host cell of embodiment 61 or 62, wherein the host cell is ex vivo.64. The host cell of any one of embodiments 61 to 63, wherein the host cell is an embryonic stem cell.65. A non-human transgenic mouse comprising the host cell of any one of embodiments 61 to 64.66. A method of screening for a chimeric CD98hc-binding polypeptide that crosses the blood-brain barrier, the method comprising:(a) administering a chimeric CD98hc-binding polypeptide to the animal of any one of embodiments 1 to 27; and(b) measuring the presence or an activity of the chimeric CD98hc-binding polypeptide in the brain of the animal.67. The method of embodiment 66, wherein the chimeric CD98hc-binding polypeptide is coupled to an effector molecule.68. The method of embodiment 67, wherein the effector molecule is a small molecule, RNA, DNA, or polypeptide.69. The method of embodiment 68, wherein the polypeptide is an antibody or an antigen-binding fragment thereof.70. The method of embodiment 67, wherein the measuring step comprises contacting the brain or brain tissue of the animal with an agent that binds to the effector molecule and determining the level of the effector molecule present in the brain.71. The method of embodiment 67, wherein the measuring step comprises measuring a pharmacodynamic (PD) effect of the effector molecule.72. The method of embodiment 67, wherein the effector molecule is an antibody or an antigen-binding fragment thereof that binds to a target in the brain.73. A method of screening for a CD98hc-binding polypeptide that binds to a chimeric CD98hc polypeptide comprising a partially or fully humanized CD98hc extracellular domain (ECD), the method comprising contacting a candidate CD98hc-binding polypeptide with a chimeric CD98hc polypeptide of embodiment 58; and determining theamount of the candidate CD98hc-binding polypeptide that binds to the chimeric CD98hc polypeptide.74. The method of embodiment 73, wherein the step of contacting the candidate CD98hc-binding polypeptide with the chimeric CD98hc polypeptide comprises contacting the candidate CD98hc-binding polypeptide with a host cell that expresses the chimeric CD98hc polypeptide.75. The method of embodiment 73, wherein the step of contacting the candidate CD98hc-binding polypeptide with the chimeric CD98hc polypeptide comprises contacting the candidate CD98hc-binding polypeptide with an endothelium that expresses the chimeric CD98hc polypeptide.76. The method of embodiment 75, wherein the endothelium is a bloodbrain barrier endothelium.77. The method of any one of embodiments 73 to 76, wherein the amount of the candidate CD98hc-binding polypeptide that binds the chimeric CD98hc polypeptide is determined by immunoassay.78. The method of any one of embodiments 73 to 77, wherein the amount of the candidate CD98hc-binding polypeptide that binds the chimeric CD98hc polypeptide is determined by surface plasmon resonance.79. The method of any one of embodiments 73 to 78, wherein the contacting step is performed in vivo.80. The method of any one of embodiments 73 to 79, wherein the candidate CD98hc-binding polypeptide is coupled to an effector molecule.81. The method of embodiment 80, wherein the effector molecule is a small molecule, RNA, DNA, or polypeptide.82. The method of embodiment 81, wherein the effector molecule is a polypeptide.83. The method of embodiment 82, wherein the polypeptide is an antibody or an antigen-binding fragment thereof.84. A method of generating a transgenic non-human single cell embryo that expresses a chimeric CD98hc polypeptide comprising a partially or fully humanized CD98hc extracellular domain (ECD), the method comprises replacing at least a portion of the CD98hc ECD endogenous to the embryo with a corresponding heterologous portion having at least 95% identity to SEQ ID NO: 13 or 14.85. The method of embodiment 84, wherein replacing the portion of the CD98hc ECD is performed by homologous recombination.86. The method of embodiment 84 or 85, wherein the method comprises contacting a Cas9 protein, at least one sgRNA, and a donor DNA comprising a nucleic acid sequence encoding the corresponding heterologous portion, wherein the corresponding heterologous portion is flanked by a left homology arm and a right homology arm, such that the corresponding heterologous portion coding sequence replaces the portion of the CD98hc ECD endogenous to the embryo.87. The method of any one of embodiments 84 to 86, wherein the corresponding heterologous portion is codon-optimized for expression in the embryo.88. A method of generating a transgenic non-human single cell embryo that expresses a chimeric CD98hc polypeptide comprising a partially or fully humanized CD98hc extracellular domain (ECD), the method comprises introducing one or more humanizing point mutations into the exons of the CD98hc ECD nucleic acid sequence endogenous to the embryo, wherein the humanizing point mutations alter the amino acid sequence encoded by the exons to match that of the amino acid sequence encoded by human exons of the human CD98hc ECD nucleic acid sequence.89. The method of any one of embodiments 84 to 88, wherein the embryo is a mouse embryo.90. A method of generating a non-human transgenic animal comprising:(a) transferring the embryo of any one of embodiments 84 to 89 to a pseudo pregnant female of the same animal species as the embryo, and(b) selecting a transgenic animal from the progeny produced by the female, wherein the animal comprises a CD98hc polypeptide in which the CD98hc ECD endogenousto the embryo has been replaced with a heterologous ECD having an amino acid sequence of at least 95% identity to SEQ ID NO:9 or 12.91. A method of generating a transgenic animal comprising modifying the genome of a non-human animal such that the modified genome comprises a nucleic acid sequence encoding a chimeric CD98hc polypeptide, wherein the CD98hc ECD endogenous to the non-human animal has been replaced with a heterologous ECD having at least 95% identity to the amino acid sequence of SEQ ID NO:9 or 12.92. The method of embodiment 91, wherein the transgenic animal expresses the chimeric CD98hc polypeptide comprising the heterologous ECD.93. The method of embodiment 91 or 92, wherein the transgenic animal has functional sperm cells, functional ES cells, functional osteoclasts, functional keratinocytes, functional B cells, and functional T cells.94. The method of any one of embodiments 91 to 93, wherein the transgenic animal is a rodent.95. The method of any one of embodiments 91 to 93, wherein the transgenic animal is a mouse.96. The method of any one of embodiments 91 to 93, wherein the transgenic animal is a rat.

[0198] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.XIV. INFORMAL SEQUENCE LISTING

Claims

WHAT IS CLAIMED IS:

1. A non-human transgenic animal that expresses a chimeric CD98 heavy chain (CD98hc) polypeptide comprising a partially or fully humanized CD98hc extracellular domain (ECD), a transmembrane domain (TD), and an intracellular domain (ICD), wherein the animal comprises a nucleic acid sequence encoding the chimeric CD98hc polypeptide, and wherein:(1) a portion of the partially or fully humanized CD98hc ECD comprises an amino acid sequence having at least 90% identity to the amino acid sequence encoded by human exons 9-12 of a human CD98hc nucleic acid sequence;(2) the TD comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the TD endogenous to the animal; and(3) the ICD comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the ICD endogenous to the animal.

2. The non-human transgenic animal of claim 1, wherein the chimeric CD98hc polypeptide is expressed in the brain of the animal.

3. The non-human transgenic animal of claim 1, wherein the animal comprises a partially humanized CD98hc ECD.

4. The non-human transgenic animal of claim 3, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises four humanized exons, wherein the four humanized exons encode a portion of the partially humanized CD98hc ECD and replace a nucleic acid sequence encoding the corresponding portion of the CD98hc ECD endogenous to the animal.

5. The non-human transgenic animal of claim 4, wherein the four humanized exons comprise human exons 9-12 of the human CD98hc nucleic acid sequence.

6. The non-human transgenic animal of claim 3, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide, or a portion thereof, is codon-optimized for expression in the animal.

7. The non-human transgenic animal of claim 3, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises introns that are endogenous to the animal.

8. The non-human transgenic animal of claim 3, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises a sequence having at least 70% identity to the sequence of SEQ ID NO:8.

9. The non-human transgenic animal of claim 3, wherein the partially humanized CD98hc ECD comprises an amino acid sequence having at least 95% identity to SEQ ID NO:9.

10. The non-human transgenic animal of claim 3, wherein the chimeric CD98hc polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID N0:7.

11. The non-human transgenic animal of claim 1, wherein the animal comprises a fully humanized CD98hc ECD.

12. The non-human transgenic animal of claim 11, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises six humanized exons, wherein the six humanized exons encode a portion of the fully humanized CD98hc ECD and replace a nucleic acid sequence encoding the corresponding portion of the CD98hc ECD endogenous to the animal.

13. The non-human transgenic animal of claim 12, wherein the six humanized exons comprise human exons 7-12 of the human CD98hc nucleic acid sequence.

14. The non-human transgenic animal of claim 11, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide further comprises humanizing point mutations.

15. The non-human transgenic animal of claim 14, wherein the humanizing point mutations alter the encoded amino acid sequence to match that of the amino acid sequence encoded by human exons 4-6 of the human CD98hc nucleic acid sequence.

16. The non-human transgenic animal of claim 11, wherein the nucleic acid sequence encoding the chimeric CD98hc polypeptide comprises a sequence having at least 70% identity to the sequence of SEQ ID NO: 11.

17. The non-human transgenic animal of claim 11, wherein the fully humanized CD98hc ECD comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 12.

18. The non-human transgenic animal of claim 11, wherein the chimeric CD98hc polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 10.

19. The non-human transgenic animal of claim 1, wherein the animal is a rodent, a mouse, or a rat.

20. The non-human transgenic animal of claim 3, wherein the animal is a mouse, and wherein the mouse exons 7-10 of an endogenous mouse CD98hc nucleic acid sequence are replaced by human exons 9-12 of the human CD98hc nucleic acid sequence.

21. The non-human transgenic animal of claim 11, wherein the animal is a mouse, and wherein the mouse exons 5-10 of an endogenous mouse CD98hc nucleic acid sequence are replaced by human exons 7-12 of the human CD98hc nucleic acid sequence.

22. The non-human transgenic animal of claim 1, wherein the animal is homozygous or heterozygous for the chimeric CD98hc polypeptide.

23. A genetically modified mouse comprising an amino acid sequence encoded by at least human exons 9-12 of a human CD98hc nucleic acid sequence, wherein the human exons 9-12 replace mouse exons 7-10 of an endogenous mouse CD98hc nucleic acid sequence, and wherein the mouse expresses a chimeric CD98hc polypeptide.

24. The genetically modified mouse of claim 23, wherein the mouse comprises human exons 7-12 of the human CD98hc nucleic acid sequence,wherein the human exons 7-12 replace mouse exons 5-10 of an endogenous mouse CD98hc nucleic acid sequence.

25. The genetically modified mouse of claim 24, wherein the mouse further comprises one or more humanizing point mutations in mouse exons 2-4.

26. The genetically modified mouse of claim 25, wherein the humanizing point mutations alter the amino acid sequence encoded by mouse exons 2-4 of the endogenous mouse CD98hc nucleic acid sequence to match that of the amino acid sequence encoded by human exons 4-6 of the human CD98hc nucleic acid sequence.

27. A genetically modified mouse comprising one or more humanizing point mutations introduced into an endogenous mouse CD98hc nucleic acid sequence,(a) wherein the humanizing point mutations alter the encoded amino acids such that at least 70% of the amino acids selected from positions: 327, 295, 376, 383, 514, 478, 253, 214, 338, 384, 481, 491, 512, 507, 471, 220, 516, 135, 234, 235, 280, 182, 320, 139, 148, 342,346, 146, 527, 347, 186, 212, 136, 251, 173, 306, 224, 397, 524, 254, 400, 202, 500, 250, 493,145, 509, 172, 134, 396, 315, 528, 505, 256, 269, 177, 231, 308, 191, 501, 190, 289, 114, 412,309, 288, 170, 391, 328, 116, 313, 219, 257, 152, 420, 268, 506, 171, 518, 120, 401, 404, 469,473, and 476 of SEQ ID NO: 15 are humanized; or(b) wherein the humanizing point mutations alter the encoded amino acids such that at least 70% of the amino acids selected from positions: 327, 295, 376, 383, 514, 478, 253, 214, 338, 384, 481, 491, 512, 507, 471, 220, 516, 135, 234, 235, 280, 182, 320, 139, 148, 342,346, 146, 527, 347, 186, 212, 136, 251, 173, 306, 224, 397, 524, 254, 400, 202, 500, 250, 493,145, 509, 172, 134, 396, 315, 528, 505, 256, 269, 177, 231, 308, 191, 501, 190, 289, 114, 412,309, 288, 170, 391, 328, 116, 313, 219, 257, 152, 268, 506, 171, 518, 120, 401, 404, 469, 473,476 of SEQ ID NO: 15 are humanized; or(c) wherein the humanizing point mutations alter the encoded amino acids such that at least 70% of the amino acids selected from positions: 327, 295, 376, 383, 514, 478, 253, 214, 338, 384, 481, 491, 512, 507, 471, 220, 516, 135, 234, 235, 280, 182, 320, 139, 148, 342,346, 146, 527, 347, 186, 212, 136, 251, 173, 306, 224, 397, 524, 254, 400, 202, 500, 250, 493,145, 509, 172, 134, 396, 315, 528, 505, 256, 269, 485, 177, 231, 308, 191, 501, 190, 289, 114,412, 180, 216, 309, 288, 170, 391, 328, 116, 430, 313, 219, 257, 152, 420, 268, 506, 258, 171,518, 120, 387, 401, 404, 469, 473, and 476 of SEQ ID NO: 15 are humanized.

28. The genetically modified mouse of claim 27, wherein the humanizing point mutations alter the amino acid sequence encoded by mouse exons 2-4 of the endogenous mouse CD98hc nucleic acid sequence to match that of the amino acid sequence encoded by human exons 4-6 of a human CD98hc nucleic acid sequence.

29. The genetically modified mouse of claim 27, wherein:(a) the mouse comprises an amino acid sequence encoded by at least human exons 9-12 of a human CD98hc nucleic acid sequence, wherein the human exons 9-12 replace mouse exons 7-10 of the endogenous mouse CD98hc nucleic acid sequence; and / or(b) the mouse comprises an amino acid sequence encoded by human exons 7- 12 of a human CD98hc nucleic acid sequence, wherein the human exons 7-12 replace mouse exons 5-10 of the endogenous mouse CD98hc nucleic acid sequence.

30. A polynucleotide encoding a chimeric CD98hc polypeptide, comprising a nucleic acid sequence encoding a partially or fully humanized CD98hc ECD and a nucleic acid sequence encoding a non-human CD98hc TD and / or non-human CD98hc ICD.

31. The polynucleotide of claim 30, wherein the polynucleotide comprises a nucleic acid sequence encoding a partially or fully humanized CD98hc ECD.

32. A chimeric CD98 heavy chain (CD98hc) polypeptide or a portion thereof encoded by the polynucleotide of claim 30.

33. An expression cassette for expressing a polynucleotide of any one of claim 30 in a non-human transgenic animal, comprising an endogenous promoter operably linked to the polynucleotide, wherein the introduction of the expression cassette into the animal results in the animal expressing a chimeric CD98hc polypeptide.

34. An expression cassette for expressing a polynucleotide encoding a chimeric CD98hc polypeptide in a transgenic mouse, wherein the polynucleotide comprises a nucleic acid sequence having at least 70% identity to the sequence of SEQ ID NO:8 or 11.

35. A host cell comprising the expression cassette of claim 33.

36. A non-human transgenic mouse comprising the host cell of claim 35.

37. A method of screening for a chimeric CD98hc-binding polypeptide that crosses the blood-brain barrier, the method comprising:(a) administering a chimeric CD98hc-binding polypeptide to the animal of claim 1; and(b) measuring the presence or an activity of the chimeric CD98hc-binding polypeptide in the brain of the animal.

38. A method of generating a transgenic non-human single cell embryo that expresses a chimeric CD98hc polypeptide comprising a partially or fully humanized CD98hc extracellular domain (ECD), the method comprises:(a) replacing at least a portion of the CD98hc ECD endogenous to the embryo with a corresponding heterologous portion having at least 95% identity to SEQ ID NO: 13 or 14; or(b) introducing one or more humanizing point mutations into the exons of the CD98hc ECD nucleic acid sequence endogenous to the embryo, wherein the humanizing point mutations alter the amino acid sequence encoded by the exons to match that of the amino acid sequence encoded by human exons of the human CD98hc ECD nucleic acid sequence.

39. A method of generating a non-human transgenic animal comprising:(a) transferring the embryo of claim 38 to a pseudo pregnant female of the same animal species as the embryo, and(b) selecting a transgenic animal from the progeny produced by the female, wherein the animal comprises a CD98hc polypeptide in which the CD98hc ECD endogenous to the embryo has been replaced with a heterologous ECD having an amino acid sequence of at least 95% identity to SEQ ID NO:9 or 12.

40. A method of generating a transgenic animal comprising modifying the genome of a non-human animal such that the modified genome comprises a nucleic acid sequence encoding a chimeric CD98hc polypeptide, wherein the CD98hc ECD endogenous to the non-human animal has been replaced with a heterologous ECD having at least 95% identity to the amino acid sequence of SEQ ID NO:9 or 12.

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