Polypeptide engineering, libraries, and engineered CD98 heavy chain and transferrin receptor binding polypeptides

Abstract

The disclosure includes engineering methods and polypeptide libraries useful for introducing non-native binding sites into polypeptides. Also provided herein are polypeptides that bind to CD98hc or to the transferrin receptor (TfR) protein, methods of making such polypeptides, and methods of using the polypeptides to target compositions across the blood-brain barrier or to CD98hc- or TfR-expressing cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 291,161, filed December 17, 2021, and U.S. Provisional Patent Application No. 63 / 423,418, filed November 7, 2022, the disclosures of which are incorporated herein by reference in their entireties for all purposes. [Background technology]

[0002] background Various techniques have been developed to engineer proteins to bind to targets to which they normally do not bind. For example, libraries can be generated and screened for engineered proteins with desired binding or enzymatic activity. Summary of the Invention

[0003] overview We have developed several techniques for discovering polypeptides with novel binding sites, particularly those containing beta-sheet portions of the binding site. These techniques include the development of beta-sheet libraries and libraries that employ "limited liability" techniques to reduce the frequency of amino acids that may produce proteins with undesirable properties. As described in detail below, we have used these types of libraries to discover polypeptides that bind to targets such as CD98 heavy chain (CD98hc) and transferrin receptor (TfR). We have also developed methods for using CD98hc polypeptides to deliver to extracellular targets, particularly in the brain (e.g., across the blood-brain barrier).

[0004] In one aspect, the disclosure provides a method for engineering a non-native binding site into a polypeptide, comprising: (a) generating a library of polypeptides, at least some of the polypeptides comprising at least seven randomized positions, and 10-60% of the randomized positions having restricted diversity to exclude one or more of the following amino acids: Cys, Trp, Met, Arg, or Gly, but comprising at least eight amino acids at each position; (b) contacting the library with a target protein; (c) selecting library members that bind to the target protein; (d) isolating the selected library members, thereby engineering a non-native binding site into the polypeptide.

[0005] In some embodiments, the method includes repeating steps (b)-(d) using the library members isolated from the first step (d). In some embodiments, the library includes at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more randomized positions. In some embodiments, the primary amino acid sequence of each polypeptide includes positions with limited diversity separated by positions without limited diversity.

[0006] In some embodiments, each polypeptide comprises a beta sheet, and at least three of the randomized positions are present within a single beta sheet. In certain embodiments, at least three of the randomized positions are present within at least two beta strands forming the beta sheet. In certain embodiments, at least three of the randomized positions are present within at least one beta strand forming the beta sheet. In some embodiments, at least three of the randomized positions form a surface on one side of the beta sheet. In certain embodiments, at least three of the randomized positions are surface-exposed. In some embodiments, the beta sheet comprises at least one position with limited diversity. In certain embodiments, the beta sheet comprises at least two positions with limited diversity. In certain embodiments, the at least two positions with limited diversity are separated by a position without limited diversity. In some embodiments, the beta sheet comprises at least two positions without limited diversity. In certain embodiments, the at least two positions without limited diversity are separated by a position with limited diversity. In certain embodiments, the separation is with respect to the primary amino acid sequence of the polypeptide or with respect to the spatial three-dimensional positioning of the amino acids within the protein structure.

[0007] In some embodiments, the positions with limited diversity are encoded by degenerate codons. In certain embodiments, at least one of the degenerate codons is NHK. In some embodiments, the positions without limited diversity are encoded by the degenerate codon NNK.

[0008] In some embodiments, the polypeptide contains an immunoglobulin-like fold. In certain embodiments, the polypeptide comprises an immunoglobulin (IgG) domain. In certain embodiments, the IgG domain is derived from the IgG, IgA, IgE, IgM, or IgD family. In certain embodiments, the IgG domain is derived from an IgG1, IgG2, IgG3, or IgG4 molecule. In certain embodiments, the IgG domain comprises a VH, CH1, CH2, CH3, VL, or CL domain. In some embodiments, the randomized positions are surface-accessible. In certain embodiments, the randomized positions are selected from any of those listed in Table 1B. In certain embodiments, the polypeptide comprises fibronectin or any other protein scaffold described herein.

[0009] In another aspect, the disclosure provides a library of polypeptides, at least some of the polypeptides comprising at least seven randomized positions, wherein 10-60% of the randomized positions have restricted diversity to exclude one or more of the following amino acids: Cys, Trp, Met, Arg, or Gly, but comprise at least eight amino acids at each position.

[0010] In some embodiments, the library comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more randomized positions. In some embodiments, the primary amino acid sequence of each polypeptide includes positions with limited diversity separated by positions without limited diversity.

[0011] In some embodiments, each polypeptide comprises a beta sheet, and at least three of the randomized positions are present within a single beta sheet. In certain embodiments, at least three of the randomized positions are present within at least two beta strands forming the beta sheet. In certain embodiments, at least three of the randomized positions are present within at least one beta strand forming the beta sheet. In some embodiments, at least three of the randomized positions form a surface on one side of the beta sheet. In certain embodiments, at least three of the randomized positions are surface-exposed. In some embodiments, the beta sheet comprises at least one position with limited diversity. In certain embodiments, the beta sheet comprises at least two positions with limited diversity. In certain embodiments, the at least two positions with limited diversity are separated by a position without limited diversity. In certain embodiments, the separation is with respect to the primary sequence of the polypeptide or the spatial three-dimensional positioning of amino acids within the protein structure. In some embodiments, the beta sheet comprises at least two positions without limited diversity. In certain embodiments, the at least two positions without limited diversity are separated by a position with limited diversity. In certain embodiments, the segregation is with respect to the primary sequence of the polypeptide or with respect to the spatial three-dimensional positioning of amino acids within the protein structure.

[0012] In some embodiments, the polypeptide contains an immunoglobulin-like fold. In certain embodiments, the polypeptide comprises an immunoglobulin (IgG) domain. In certain embodiments, the IgG domain is derived from the IgG, IgA, IgE, IgM, or IgD family. In certain embodiments, the IgG domain is derived from an IgG1, IgG2, IgG3, or IgG4 molecule. In certain embodiments, the IgG domain comprises a VH, CH1, CH2, CH3, VL, or CL domain. In some embodiments, the randomized positions are surface-accessible. In certain embodiments, the randomized positions are selected from any of those listed in Table 1B. In certain embodiments, the polypeptide comprises fibronectin or any other protein scaffold described herein.

[0013] In another aspect, the present disclosure provides a polypeptide comprising a non-CDR portion of an immunoglobulin constant or variable domain having at least three modified positions within a beta sheet, (i) the modified positions are within at least two beta strands that form a beta sheet; (ii) the modified position forms at least a part of a binding site capable of binding to an antigen; (iii) providing a polypeptide whose beta sheet does not bind to an antigen that does not have the modified position;

[0014] In some embodiments, the constant domain comprises an Fc polypeptide. In some embodiments, at least two beta chains are selected from the group consisting of amino acid positions 124-128, 139-147, 155-157, 179-178, 199-203, 208-214, 239-243, 258-265, 274-278, 301-307, 319-324, 332-336, 347-351, 363-372, 378-383, 391-393, 406-412, 423-428, and 437-441, where the positions are determined according to EU numbering. In certain embodiments, the positions are surface accessible. In certain embodiments, the positions are selected from those listed in Table 1B.

[0015] In some embodiments, the modified positions form adjacent surfaces on the beta sheet. In some embodiments, the modified positions are surface-accessible residues. In certain embodiments, the surface-accessible residues are selected from the group consisting of amino acid positions 347, 349, 351, 362, 364, 366, 368, 370, 378, 380, 382, ​​405, 407, 409, 411, 424, 426, 428, 436, 438, and 440, where the positions are determined according to EU numbering. In certain embodiments, the surface-accessible residues are selected from the group consisting of amino acid positions 347, 362, 378, 380, 382, ​​411, 424, 426, 428, 436, 438, and 440, where the positions are determined according to EU numbering.

[0016] In some embodiments, the modified positions comprise 3, 4, 5, 6, or 7 amino acid substitutions at the set of amino acid positions including: 380, 382, ​​383, 424, 426, 438, and 440, where the positions are determined according to EU numbering. In some embodiments, the modified positions comprise 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions at the set of amino acid positions including: 378, 380, 382, ​​383, 422, 424, 426, 428, 438, 440, and 442, where the positions are determined according to EU numbering.

[0017] In some embodiments, the binding site comprises one or more modified positions within at least one loop region. In certain embodiments, the one or more modified positions within at least one loop region are selected from the group consisting of amino acid positions 387 and 422, the positions being determined according to EU numbering. In certain embodiments, the loop region connects two beta strands.

[0018] In another aspect, the present disclosure provides a method for introducing a non-native binding site into a non-CDR region of an immunoglobulin constant or variable domain, comprising: (a) generating a polynucleotide library encoding immunoglobulin sequences having at least three modified positions within a beta sheet, wherein the library is randomized with codons encoding amino acids at the modified positions, and the modified positions are within at least two beta strands that form the beta sheet; (b) expressing the library to generate a library of sequence variants; (c) contacting the sequence variant with a target protein; and (d) isolating sequence variants that bind to the target protein, thereby introducing a non-native binding site into a non-CDR region of an immunoglobulin constant or variable domain.

[0019] In some embodiments, the immunoglobulin sequence comprises an Fc polypeptide. In some embodiments, at least two beta chains are selected from the group consisting of amino acid positions 239-243, 258-265, 274-278, 301-307, 319-324, 332-336, 347-351, 363-372, 378-383, 391-393, 406-412, 423-428, and 437-441, wherein the positions are determined according to EU numbering.

[0020] In some embodiments, the modified positions form adjacent surfaces on the beta sheet. In some embodiments, the modified positions are surface-accessible residues. In certain embodiments, the surface-accessible residues are selected from the group consisting of amino acid positions 347, 349, 351, 362, 364, 366, 368, 370, 378, 380, 382, ​​405, 407, 409, 411, 424, 426, 428, 436, 438, and 440, where the positions are determined according to EU numbering. In certain embodiments, the surface-accessible residues are selected from the group consisting of amino acid positions 347, 362, 378, 380, 382, ​​411, 424, 426, 428, 436, 438, and 440, where the positions are determined according to EU numbering.

[0021] In some embodiments, the modified positions comprise 3, 4, 5, 6, or 7 amino acid substitutions at the set of amino acid positions including: 380, 382, ​​383, 424, 426, 438, and 440, where the positions are determined according to EU numbering. In some embodiments, the modified positions comprise 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions at the set of amino acid positions including: 378, 380, 382, ​​383, 422, 424, 426, 428, 438, 440, and 442, where the positions are determined according to EU numbering.

[0022] In some embodiments, the binding site comprises one or more modified positions within at least one loop region. In certain embodiments, the one or more modified positions within at least one loop region are selected from the group consisting of amino acid positions 387 and 422, the positions being determined according to EU numbering. In certain embodiments, the loop region connects two beta strands.

[0023] In another aspect, the disclosure provides a library of immunoglobulin variants comprising at least 10 members, wherein the variants each comprise at least three modified positions within a beta sheet forming part of the constant or non-CDR variable domain of the immunoglobulin, and the modified positions are within at least two beta strands forming the beta sheet.

[0024] In some embodiments, the library of immunoglobulin variants comprises at least 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 In some embodiments, the library contains at least eight amino acids at each position, with 10-60% of the randomized positions having diversity restricted to exclude one or more of the following amino acids: Cys, Trp, Met, Arg, or Gly. In certain embodiments, at least one of the diversity-restricting positions does not encode tryptophan or cysteine. In certain embodiments, at least two diversity-restricting positions do not encode tryptophan or cysteine. In certain embodiments, at least two diversity-restricting positions do not encode tryptophan, cysteine, or arginine.

[0025] In some embodiments, the diversity-restricting position is encoded by a degenerate codon. In certain embodiments, at least one diversity-restricting position is encoded by an NHK codon. In certain embodiments, the NHK codons are not adjacent to each other in the primary amino acid sequence or in the three-dimensional protein structure. In certain embodiments, the NHK codon alternates with one or more NNK codons.

[0026] In some embodiments, the present disclosure provides libraries of polynucleotides encoding immunoglobulin variants from the libraries described herein.

[0027] In another aspect, the present disclosure provides a method for engineering a non-native binding site for transferrin receptor (TfR) or CD98hc protein into a polypeptide, comprising: (a) generating a library of polypeptides, at least some of the polypeptides comprising at least seven randomized positions, and 10-60% of the randomized positions having restricted diversity to exclude one or more of the following amino acids: Cys, Trp, Met, Arg, or Gly, but comprising at least eight amino acids at each position; (b) contacting the library with a target protein; (c) selecting library members that bind to the target protein; (d) isolating the selected library members, thereby engineering a non-native binding site for TfR or CD98hc into the polypeptide.

[0028] In some embodiments of this aspect, the method includes repeating steps (b) through (d) using the library members isolated from the first step (d).

[0029] In some embodiments, the library comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more randomized positions.

[0030] In some embodiments, the primary amino acid sequence of each polypeptide comprises positions with limited diversity separated by positions without limited diversity. In certain embodiments, each polypeptide comprises a beta sheet, and at least three of the randomized positions are present within a single beta sheet. In certain embodiments, at least three of the randomized positions are present within at least two beta strands forming the beta sheet. In certain embodiments, at least three of the randomized positions are present within at least one beta strand forming the beta sheet. In certain embodiments, at least three of the randomized positions form a surface on one side of the beta sheet. In certain embodiments, at least three of the randomized positions are exposed to the surface.

[0031] In some embodiments of this aspect, the beta sheet comprises at least one position with limited diversity. In some embodiments, the beta sheet comprises at least two positions with limited diversity. In some embodiments, the at least two positions with limited diversity are separated by a position that does not have limited diversity.

[0032] In some embodiments, the beta sheet comprises at least two positions that do not have restricted diversity. In certain embodiments, the at least two positions that do not have restricted diversity are separated by a position that has restricted diversity.

[0033] In some embodiments, the segregation is with respect to the primary amino acid sequence of the polypeptide or with respect to the spatial three-dimensional positioning of amino acids within the protein structure.

[0034] In some embodiments, the positions with limited diversity are encoded by degenerate codons. In certain embodiments, at least one of the degenerate codons is NHK. In certain embodiments, the positions without limited diversity are encoded by the degenerate codon NNK.

[0035] In some embodiments of this aspect, the polypeptide contains an immunoglobulin-like fold. In some embodiments, the polypeptide comprises an immunoglobulin (IgG) domain. In certain embodiments, the IgG domain is derived from the IgG, IgA, IgE, IgM, or IgD family. In certain embodiments, the IgG domain is derived from an IgG1, IgG2, IgG3, or IgG4 molecule. In certain embodiments, the IgG domain comprises a VH, CH1, CH2, CH3, VL, or CL domain.

[0036] In some embodiments, the randomized positions are surface accessible. In certain embodiments, the randomized positions are selected from any of those listed in Table 1B. In certain embodiments, the polypeptide comprises fibronectin or any other protein scaffold described herein.

[0037] In another aspect, the present disclosure provides a polypeptide having at least three modified positions in a beta sheet portion, (i) the modified positions are within at least two beta strands that form a beta sheet; (ii) the modified position forms at least part of a binding site capable of binding to CD98hc; (iii) providing a polypeptide whose beta sheet does not bind to an antigen that does not have the modified position;

[0038] In some embodiments of this aspect, the polypeptide comprises at least 4 or 5 modified positions within the beta sheet. In some embodiments, the polypeptide comprises at least 7 modified positions that form at least a portion of a binding site capable of binding to CD98hc. In some embodiments, the polypeptide contains an immunoglobulin-like fold. In certain embodiments, the polypeptide comprises an immunoglobulin (IgG) domain. In certain embodiments, the IgG domain is derived from the IgG, IgA, IgE, IgM, or IgD family. In certain embodiments, the IgG domain is derived from an IgG1, IgG2, IgG3, or IgG4 molecule. In certain embodiments, the IgG domain comprises a VH, CH1, CH2, CH3, VL, or CL domain.

[0039] In some embodiments, the modified position is surface accessible. In some embodiments, the modified position is selected from any of those listed in Table 1B. In certain embodiments, the polypeptide comprises fibronectin or any other protein scaffold described herein.

[0040] In another aspect, the present disclosure provides a polypeptide comprising a non-CDR portion of an immunoglobulin constant or variable domain having at least three modified positions within a beta sheet, (i) the modified positions are within at least two beta strands that form a beta sheet; (ii) the modified position forms at least a portion of a binding site capable of binding to a TfR or CD98hc protein; (iii) providing a polypeptide whose beta sheet does not bind to an antigen that does not have the modified position;

[0041] In some embodiments of this aspect, the constant domain comprises an Fc polypeptide.

[0042] In some embodiments, at least two beta strands are selected from the group consisting of amino acid positions 124-128, 139-147, 155-157, 179-178, 199-203, 208-214, 239-243, 258-265, 274-278, 301-307, 319-324, 332-336, 347-351, 363-372, 378-383, 391-393, 406-412, 423-428, and 437-441, where the positions are determined according to EU numbering. In some embodiments, the positions are surface accessible. In certain embodiments, the positions are selected from those listed in Table 1B.

[0043] In some embodiments, the modified positions form adjacent surfaces on the beta sheet.

[0044] In some embodiments, the modified position is a surface-accessible residue. In certain embodiments, the surface-accessible residue is selected from the group consisting of amino acid positions 347, 349, 351, 362, 364, 366, 368, 370, 378, 380, 382, ​​405, 407, 409, 411, 424, 426, 428, 436, 438, and 440, where the positions are determined according to EU numbering. In certain embodiments, the surface-accessible residue is selected from the group consisting of amino acid positions 347, 362, 378, 380, 382, ​​411, 424, 426, 428, 436, 438, and 440, where the positions are determined according to EU numbering.

[0045] In some embodiments, the modified positions comprise 3, 4, 5, 6, or 7 amino acid substitutions at the set of amino acid positions including 380, 382, ​​383, 424, 426, 438, and 440, where the positions are determined according to EU numbering. In some embodiments, the modified positions comprise 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions at the set of amino acid positions including 378, 380, 382, ​​383, 422, 424, 426, 428, 438, 440, and 442, where the positions are determined according to EU numbering. In certain embodiments, the binding site comprises one or more modified positions within at least one loop region. In certain embodiments, the one or more modified positions within at least one loop region are selected from the group consisting of amino acid positions 387 and 422, where the positions are determined according to EU numbering. In certain embodiments, the loop region connects two beta strands.

[0046] In another aspect, the present disclosure provides a method for introducing a non-native binding site for a TfR or CD98hc protein into a non-CDR region of an immunoglobulin constant or variable domain, comprising: (a) generating a polynucleotide library encoding immunoglobulin sequences having at least three modified positions within a beta sheet, wherein the library is randomized with codons encoding amino acids at the modified positions, and the modified positions are within at least two beta strands that form the beta sheet; (b) expressing the library to generate a library of sequence variants; (c) contacting the sequence variant with a TfR or CD98hc protein; and (d) isolating sequence variants that bind to the TfR or CD98hc protein, thereby introducing a non-native binding site into a non-CDR region of the constant or variable domain of an immunoglobulin.

[0047] In some embodiments of this aspect, the immunoglobulin sequence comprises an Fc polypeptide.

[0048] In some embodiments, at least two beta strands are selected from the group consisting of amino acid positions 239-243, 258-265, 274-278, 301-307, 319-324, 332-336, 347-351, 363-372, 378-383, 391-393, 406-412, 423-428, and 437-441, wherein the positions are determined according to EU numbering.

[0049] In some embodiments, the modified positions form adjacent surfaces on the beta sheet.

[0050] In some embodiments, the modified position is a surface-accessible residue. In certain embodiments, the surface-accessible residue is selected from the group consisting of amino acid positions 347, 349, 351, 362, 364, 366, 368, 370, 378, 380, 382, ​​405, 407, 409, 411, 424, 426, 428, 436, 438, and 440, where the positions are determined according to EU numbering. In certain embodiments, the surface-accessible residue is selected from the group consisting of amino acid positions 347, 362, 378, 380, 382, ​​411, 424, 426, 428, 436, 438, and 440, where the positions are determined according to EU numbering.

[0051] In some embodiments, the modified positions comprise 3, 4, 5, 6, or 7 amino acid substitutions at the set of amino acid positions including: 380, 382, ​​383, 424, 426, 438, and 440, where the positions are determined according to EU numbering. In some embodiments, the modified positions comprise 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions at the set of amino acid positions including: 378, 380, 382, ​​383, 422, 424, 426, 428, 438, 440, and 442, where the positions are determined according to EU numbering.

[0052] In some embodiments, the binding site comprises one or more modified positions within at least one loop region, hi some embodiments, the one or more modified positions within at least one loop region are selected from the group consisting of amino acid positions 387 and 422, the positions being determined according to EU numbering.

[0053] In another aspect, the present disclosure provides a method for introducing a CD98hc binding site into a beta sheet containing polypeptide, comprising: (a) generating a polynucleotide library encoding polypeptide sequences having at least three modified positions within a beta sheet, wherein the library is randomized with codons encoding amino acids at the modified positions, and the modified positions are within at least two beta strands that form the beta sheet; (b) expressing the library to generate a library of sequence variants; (c) contacting the sequence variant with at least a portion of a CD98hc protein; and (d) isolating sequence variants that bind to the CD98hc protein.

[0054] In some embodiments, the polypeptide has at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modified positions in the beta sheets. In some embodiments, the polypeptide has at least 7 modified positions in the beta sheets. In some embodiments, the polypeptide has at least 10 modified positions in the beta sheets.

[0055] In certain embodiments, the binding site comprises one or more modified positions within at least one loop region.

[0056] In some embodiments, the binding site comprises one or more beta sheets and one or more loop regions, and at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modified positions within the beta sheet(s) and loop region(s).

[0057] In some embodiments, the polypeptide contains an immunoglobulin-like fold. In certain embodiments, the polypeptide comprises an immunoglobulin (IgG) domain. In certain embodiments, the IgG domain is derived from the IgG, IgA, IgE, IgM, or IgD family. In certain embodiments, the IgG domain is derived from an IgG1, IgG2, IgG3, or IgG4 molecule. In certain embodiments, the IgG domain comprises a VH, CH1, CH2, CH3, VL, or CL domain. In some embodiments, the randomized positions are surface-accessible. In certain embodiments, the randomized positions are selected from any of those listed in Table 1B. In certain embodiments, the polypeptide comprises fibronectin or any other protein scaffold described herein.

[0058] In another aspect, the present disclosure provides a polypeptide comprising a modified constant domain that specifically binds to a CD98hc protein. In some embodiments, the modified constant domain comprises a modified CH3 domain that specifically binds to a CD98hc protein. In some embodiments, the modified CH3 domain is part of an Fc polypeptide. In certain embodiments, the CD98hc protein is a human CD98hc protein. In certain embodiments, the CD98hc protein forms a complex with LAT1 (SLC7A5), LAT2 (SLC7A8), y+LAT1 (SLC7A7), y+LAT2 (SLC7A6), Asc-1 (SLC7A10), or xCT (SLC7A11). In certain embodiments, the CD98hc protein forms a complex with LAT1 (SLC7A5).

[0059] In some embodiments, the modified constant domain (e.g., the modified CH3 domain) comprises a sequence having at least 85%, 90%, or 95% sequence identity to amino acids 111-217 of any one of SEQ ID NOs: 28-45.

[0060] In another aspect, the disclosure features a polypeptide comprising a modified constant domain (e.g., a modified CH3 domain) that specifically binds to a CD98hc protein, wherein the modified constant domain comprises at least 5, 6, 7, 8, or 9 substitutions at the set of amino acid positions consisting of 382, ​​384, 385, 387, 422, 424, 426, 438, 440, where the positions are determined with reference to EU numbering. In another aspect, the substitutions are determined with reference to SEQ ID NO:1.

[0061] In another aspect, the disclosure features a polypeptide comprising a modified constant domain (e.g., a modified CH3 domain) that specifically binds to a CD98hc protein, wherein the modified constant domain comprises at least 11, 12, 13, 14, or 15 substitutions at the set of amino acid positions consisting of 380, 382, ​​384, 385, 386, 387, 421, 422, 424, 426, 428, 436, 438, 440, and 442, wherein the positions are determined according to EU numbering. In another aspect, the substitutions are determined with reference to SEQ ID NO:1.

[0062] In some embodiments of this aspect, the modified constant domain (e.g., the modified CH3 domain) comprises a sequence having at least 85%, 90%, or 95% sequence identity to amino acids 111-217 of any one of SEQ ID NOs: 28-43, and the modified constant domain comprises at least 11, 12, 13, 14, or 15 substitutions at the set of amino acid positions consisting of L at position 380, N at position 382, ​​R, H, or Q at position 384, F or Y at position 385, V, L, I, F, Y, or E at position 386, L at position 387, E, Q, or A at position 421, I, T, or P at position 422, A at position 424, N at position 426, Y or W at position 428, R or W at position 436, F or W at position 438, N at position 440, and A, Q, K, R, H, or M at position 442. In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:28.

[0063] In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an E at position 421, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:29.

[0064] In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises an L at position 380, an N at position 382, ​​a Q at position 384, a Y at position 385, an E at position 386, an L at position 387, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:30.

[0065] In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises an L at position 380, an N at position 382, ​​an H at position 384, a Y at position 385, an E at position 386, an L at position 387, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:31.

[0066] In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:32.

[0067] In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an E at position 421, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:33.

[0068] In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, E at position 421, I at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, and N at position 440. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:34.

[0069] In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, I at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and R at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:35.

[0070] In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, I at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and H at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:36.

[0071] In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, I at position 422, A at position 424, N at position 426, Y at position 428, R at position 436, F at position 438, N at position 440, and R at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:37.

[0072] In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises an L at position 380, an N at position 382, ​​an H at position 384, a Y at position 385, an E at position 386, an L at position 387, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:38.

[0073] In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises L at position 380, N at position 382, ​​Q at position 384, F at position 385, H at position 386, L at position 387, I at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and L at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:39.

[0074] In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, a T at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:40.

[0075] In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, I at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and K at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:41.

[0076] In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, I at position 422, A at position 424, N at position 426, Y at position 428, W at position 436, F at position 438, N at position 440, and R at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:42.

[0077] In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises an L at position 380, an N at position 382, ​​a Q at position 384, a Y at position 385, an L at position 386, an L at position 387, an E at position 421, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:43.

[0078] In another aspect, the present disclosure provides a polypeptide comprising a modified constant domain (e.g., a modified CH3 domain) that specifically binds to a CD98hc protein, wherein the modified constant domain: (i) a first amino acid sequence which is LX1NX2X3X4X5L (SEQ ID NO: 46), wherein X1 is any amino acid, X2 is R, H, or Q, X3 is F or Y, X4 is V, L, I, F, Y, or E, and X5 is any amino acid; (ii) a second amino acid sequence of X1X2X3AX4X5X6X7 (SEQ ID NO: 47), wherein X1 is E, N, Q, or A, X2 is I, V, T, or P, X3 and X4 are any amino acids, X5 is N or S, X6 is any amino acid, and X7 is Y or W; and (iii) a third amino acid sequence of X1X2X3X4NX5X6 (SEQ ID NO: 48), wherein X1 is Y, R, or W, X2 is any amino acid, X3 is F or W, X4 and X5 are any amino acids, and X6 is A, Q, K, R, H, M, or S.

[0079] In some embodiments, the polypeptide binds to human CD98hc with an affinity of 15 nM to 5 μM (e.g., 15 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM). In some embodiments, the polypeptide has cross-reactivity with cynomolgus monkeys (Macaca fascicularis). In certain embodiments, the polypeptide binds to cynomolgus CD98hc with an affinity of 80 nM to 5 μM (e.g., 80 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM).

[0080] In another aspect, the disclosure features a polypeptide comprising a modified constant domain (e.g., a modified CH3 domain) that specifically binds to a CD98hc protein, wherein the modified constant domain comprises at least 8, 9, 10, 11, 12, or 13 substitutions at the set of amino acid positions consisting of 380, 382, ​​384, 385, 386, 387, 422, 424, 426, 428, 434, 438, and 440, wherein the substitutions are determined with reference to SEQ ID NO: 1 and the positions are determined according to EU numbering. In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises D, M, N, P, F, or H at position 380, R, Y, F, S, W, Y, K, or N at position 382, ​​L, Y, A, S, or F at position 384, F, K, D, M, I, N, Y, L, or H at position 385, T, P, E, K, A, V, D, T, or F at position 386, N, L, Y, R, G, S, D, or T at position 387, I, K, R, T, F, or H at position 422, V, W, G, L, I, P, or Y at position 424, D, A, Q, W, L, or P at position 426, L at position 428, S at position 434, I, F, N, P, or S at position 438, and K, T, I, or F at position 440.

[0081] In another aspect, the disclosure features a polypeptide comprising a modified constant domain (e.g., a modified CH3 domain) that specifically binds to a CD98hc protein, wherein the modified constant domain comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitutions at the set of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 434, 436, 438, 440, and 442, wherein the substitutions are determined with reference to SEQ ID NO: 1 and the positions are determined according to EU numbering. In some embodiments, the modified constant domain (e.g., a modified CH3 domain) comprises S or V at position 378, D at position 380, R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T, Y, or F at position 389, D, E, or Q at position 421, I at position 422, V at position 424, D at position 426, L or Y at position 428, S at position 434, F at position 436, I or V at position 438, K at position 440, and Q or M at position 442.

[0082] In another aspect, the disclosure features a polypeptide comprising a modified constant domain (e.g., a modified CH3 domain) that specifically binds to a CD98hc protein, wherein the modified constant domain comprises at least 8, 9, 10, 11, 12, 13, 14, or 15 substitutions at the set of amino acid positions consisting of 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 436, 438, and 440, wherein the substitutions are determined with reference to SEQ ID NO: 1 and the positions are determined according to EU numbering. In some embodiments, the modified constant domain (e.g., the modified CH3 domain) comprises a sequence having at least 85%, 90%, or 95% sequence identity to amino acids 111-217 of any one of SEQ ID NOs: 44-45, and the modified constant domain comprises at least 8, 9, 10, 11, 12, 13, 14, or 15 substitutions at the following amino acid positions: R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T at position 389, D at position 421, I at position 422, V at position 424, D at position 426, L at position 428, F at position 436, I at position 438, and K at position 440.

[0083] In some embodiments of this aspect, the modified constant domain (e.g., the modified CH3 domain) comprises an R at position 382, ​​a T at position 383, a Y at position 384, a K at position 385, a P at position 386, a Y at position 387, a T at position 389, a D at position 421, an I at position 422, a V at position 424, a D at position 426, an F at position 436, an I at position 438, and a K at position 440. In particular embodiments, the modified constant domain (e.g., the modified CH3 domain) comprises SEQ ID NO:44.

[0084] In some embodiments of this aspect, the modified constant domain (e.g., modified CH3 domain) comprises an R at position 382, ​​a T at position 383, a Y at position 384, a K at position 385, a P at position 386, a Y at position 387, a T at position 389, a D at position 421, an I at position 422, a V at position 424, a D at position 426, an L at position 428, an F at position 436, an I at position 438, and a K at position 440. In particular embodiments, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:45.

[0085] In another aspect, the present disclosure provides a polypeptide comprising a modified constant domain (e.g., a modified CH3 domain) that specifically binds to a CD98hc protein, wherein the modified constant domain: (i) a first amino acid sequence that is X1X2YKPYX3T (SEQ ID NO: 49), wherein X1 is E or R, X2 is S or T, and X3 is any amino acid; (ii) a second amino acid sequence of X1X2X3VX4DX5X6 (SEQ ID NO: 50), wherein X1 is N or D, X2 is V or I, X3, X4, and X5 are any amino acids, and X6 is M or L; and (iii) a third amino acid sequence of X1X2IX3X4 (SEQ ID NO: 51), wherein X1 is Y or F, X2 and X3 are any amino acids, and X4 is S or K.

[0086] In another aspect, the disclosure features a polypeptide comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), wherein the modified CH3 domain comprises five, six, or seven amino acid substitutions at a set of amino acid positions comprising 422, 424, 426, 433, 434, 438, and 440 of an Fc polypeptide (e.g., SEQ ID NO: 1), wherein the modified CH3 domain does not have the combination of G at position 437, F at position 438, and D at position 440, wherein the positions are determined according to EU numbering.

[0087] In another aspect, the disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), wherein the modified CH3 domain comprises 3, 4, 5, 6, 7, or 8 amino acid substitutions and / or 1 or 2 amino acid deletions at a set of amino acid positions that includes positions 380 and 382-389 of the Fc polypeptide (e.g., SEQ ID NO: 1), and 5, 6, or 7 amino acid substitutions at a set of amino acid positions that includes positions 422, 424, 426, 433, 434, 438, and 440 of the Fc polypeptide (e.g., SEQ ID NO: 1), wherein the positions are determined according to EU numbering.

[0088] In another aspect, the disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), wherein the modified CH3 domain comprises a sequence comprising at least one (e.g., 1, 2, 3, 4, 5, 6, or 7) amino acid substitution in the sequence of VFSCSVMHEALHNHYTQKS (SEQ ID NO: 57), wherein the sequence of SEQ ID NO: 57 is between positions 422 and 440 of an Fc polypeptide (e.g., SEQ ID NO: 1), and the sequence does not have the combination of G at position 437, F at position 438, and D at position 440, the positions being determined according to EU numbering. In some embodiments of this aspect, the sequence comprises 5, 6, or 7 amino acid substitutions at the set of amino acid positions comprising 422, 424, 426, 433, 434, 438, and 440.

[0089] In another aspect, the disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), wherein the modified CH3 domain comprises a first sequence comprising at least one amino acid substitution (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions) and / or deletion in the sequence of AVEWESNGQPENN (SEQ ID NO: 56), and a second amino acid substitution comprising at least one amino acid substitution (e.g., 1, 2, 3, 4, 5, 6, or 7) in the sequence of VFSCSVMHEALHNHYTQKS (SEQ ID NO: 57), wherein the sequence of SEQ ID NO: 56 is at positions 378-390 of an Fc polypeptide (e.g., SEQ ID NO: 1) and the sequence of SEQ ID NO: 57 is at positions 422-440 of an Fc polypeptide (e.g., SEQ ID NO: 1), wherein the positions are determined according to EU numbering. In some embodiments of this aspect, the modified CH3 domain comprises 3, 4, 5, 6, 7, or 8 amino acid substitutions at the set of amino acid positions including 380 and 382-389. In some embodiments, the modified CH3 domain comprises 5, 6, or 7 amino acid substitutions at the set of amino acid positions including 422, 424, 426, 433, 434, 438, and 440. In particular embodiments, the modified CH3 domain comprises 1 or 2 amino acid deletions in the sequence of SEQ ID NO:56.

[0090] In some embodiments of the above four aspects, the modified CH3 domain is part of an Fc polypeptide.

[0091] In some embodiments, the modified CH3 domain comprises an F at position 382.

[0092] In some embodiments, the modified CH3 domain comprises an A or a polar amino acid (eg, Y or S) at position 383.

[0093] In some embodiments, the modified CH3 domain comprises a G, N, or acidic amino acid (eg, D or E) at position 384.

[0094] In some embodiments, the modified CH3 domain comprises an N, R, or polar amino acid (e.g., S or T) at position 389.

[0095] In some embodiments, the modified CH3 domain comprises at least one amino acid substitution at a beta sheet position relative to the sequence of SEQ ID NO:56.

[0096] In certain embodiments, the modified CH3 domain comprises one, two, or three amino acid substitutions at beta sheet positions relative to the sequence of SEQ ID NO: 56. In some embodiments, the beta sheet position(s) are selected from the group consisting of positions 380, 382, ​​and 383, wherein the positions are determined according to EU numbering.

[0097] In certain embodiments, the modified CH3 domain comprises an amino acid substitution at beta sheet position 380 relative to the sequence of SEQ ID NO: 56. In certain embodiments, the modified CH3 domain comprises an E, N, F, or Y (e.g., E) at position 380.

[0098] In some embodiments, the modified CH3 domain comprises an amino acid substitution at beta sheet position 382 relative to the sequence of SEQ ID NO: 56. In particular embodiments, the modified CH3 domain comprises an F at position 382.

[0099] In some embodiments, the modified CH3 domain comprises an amino acid substitution or deletion at beta sheet position 383 relative to the sequence of SEQ ID NO: 56. In certain embodiments, the modified CH3 domain comprises a Y or A (e.g., a Y) at position 383.

[0100] In some embodiments, the modified CH3 domain comprises at least one amino acid substitution at a beta-sheet position relative to the sequence of SEQ ID NO: 57. In some embodiments, the modified CH3 domain comprises one, two, three, or four amino acid substitutions at beta-sheet positions relative to the sequence of SEQ ID NO: 57. In certain embodiments, the beta-sheet position(s) is / are selected from the group consisting of positions 424, 426, 438, and 440, according to EU numbering.

[0101] In some embodiments, the modified CH3 domain comprises an amino acid substitution at beta sheet position 424 relative to the sequence of SEQ ID NO: 57. In particular embodiments, the modified CH3 domain comprises an A at position 424.

[0102] In some embodiments, the modified CH3 domain comprises an amino acid substitution at beta sheet position 426 relative to the sequence of SEQ ID NO: 57. In particular embodiments, the modified CH3 domain comprises an E at position 426.

[0103] In some embodiments, the modified CH3 domain comprises an amino acid substitution at beta sheet position 438 relative to the sequence of SEQ ID NO: 57. In particular embodiments, the modified CH3 domain comprises a Y at position 438.

[0104] In some embodiments, the modified CH3 domain comprises an amino acid substitution at beta sheet position 440 relative to the sequence of SEQ ID NO: 57. In particular embodiments, the modified CH3 domain comprises an L at position 440.

[0105] In certain embodiments, the modified CH3 domain comprises an H or E (e.g., an H) at position 433.

[0106] In some embodiments, the modified CH3 domain comprises an N or G (e.g., an N) at position 434.

[0107] In some embodiments, the modified CH3 domain comprises at least one position selected from the following: E, N, F, or Y at position 380; F at position 382; Y, S, A, or an amino acid deletion at position 383; G, D, E, or N at position 384; D, G, N, or A at position 385; Q, S, G, A, or N at position 386; K, I, R, or G at position 387; E, L, D, or Q at position 388; and N, T, S, or R at position 389. In certain embodiments, the modified CH3 domain comprises 5, 6, 7, or 8 positions selected from the following: F at position 382, ​​Y or S at position 383, G, D, or E at position 384, D, G, N, or A at position 385, Q, S, or A at position 386, K at position 387, E or L at position 388, and N, T, or S at position 389.

[0108] In some embodiments, the modified CH3 domain comprises at least one position selected from the following: L at position 422, A at position 424, E at position 426, H or E at position 433, N or G at position 434, Y at position 438, and L at position 440. In certain embodiments, the modified CH3 domain comprises five positions selected from the following: L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440.

[0109] In another aspect, the disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), the modified CH3 domain comprising: (i) the sequence AVX1WFX2X3X4X5X6X7X8N (SEQ ID NO: 65), wherein Xi is E, N, F, or Y; X2 is Y, S, A, or absent; X3 is G, D, E, or N; X4 is D, G, N, or A; X5 is Q, S, G, A, or N; X6 is K, I, R, or G; X7 is E, L, D, or Q; and X8 is N, T, S, or R; and (ii) the sequence LFACEVMHEALX1X2HYTYKL (SEQ ID NO: 67), wherein Xi is H or E; and X2 is N or G.

[0110] In some embodiments of the above five aspects, the modified CH3 domain comprises the sequence AVEWFYDDSKLTN (SEQ ID NO: 58), AVEWFYGNAKETN (SEQ ID NO: 59), AVEWFYEAQKLNN (SEQ ID NO: 60), AVEWFSEGSKETN (SEQ ID NO: 61), AVEWFSGAQKESN (SEQ ID NO: 62), or AVEWFSGAQKLTN (SEQ ID NO: 63). In some embodiments, the modified CH3 domain comprises the sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64).

[0111] In certain embodiments, the modified CH3 domain comprises the sequence AVEWFYDDSKLTN (SEQ ID NO: 58) and the sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64). In certain embodiments, the modified CH3 domain comprises the sequence AVEWFYGNAKETN (SEQ ID NO: 59) and the sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64). In certain embodiments, the modified CH3 domain comprises the sequence AVEWFYEAQKLNN (SEQ ID NO: 60) and the sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64). In certain embodiments, the modified CH3 domain comprises the sequence AVEWFSEGSKETN (SEQ ID NO: 61) and the sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64). In certain embodiments, the modified CH3 domain comprises the sequence AVEWFSGAQKESN (SEQ ID NO: 62) and the sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64). In certain embodiments, the modified CH3 domain comprises the sequence AVEWFSGAQKLTN (SEQ ID NO: 63) and the sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64).

[0112] In further embodiments of the above five aspects, the modified CH3 domain further comprises one, two, three, four, or five amino acid substitutions at positions 419-421, inclusive, 442, and 443, where the positions are determined according to EU numbering. In some embodiments, the modified CH3 domain comprises a Q or P at position 419, a G or R at position 420, an N or G at position 421, an S or G at position 442, and / or an L or E at position 443.

[0113] In some embodiments, the modified CH3 domain comprises a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 111-217 of any one of SEQ ID NOs: 72-77. In some embodiments, the modified CH3 domain comprises amino acids 111-217 of any one of SEQ ID NOs: 72-77.

[0114] In some embodiments, the polypeptide comprises a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 72-77. In some embodiments, the polypeptide comprises the sequence of any one of SEQ ID NOs: 72-77.

[0115] In another aspect, the disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), wherein the modified CH3 domain comprises F at position 382, ​​Y at position 383, D at position 384, D at position 385, S at position 386, K at position 387, L at position 388, T at position 389, P at position 419, R at position 420, G at position 421, L at position 422, A at position 424, E at position 426, Y at position 438, L at position 440, G at position 442, and E at position 443, wherein positions are determined according to EU numbering.

[0116] In another aspect, the disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), wherein the modified CH3 domain comprises F at position 382, ​​Y at position 383, G at position 384, N at position 385, A at position 386, K at position 387, T at position 389, L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440, wherein the positions are determined according to EU numbering.

[0117] In another aspect, the disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), wherein the modified CH3 domain comprises F at position 382, ​​Y at position 383, E at position 384, A at position 385, K at position 387, L at position 388, L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440, wherein the positions are determined according to EU numbering.

[0118] In another aspect, the disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), wherein the modified CH3 domain comprises F at position 382, ​​E at position 384, S at position 386, K at position 387, T at position 389, L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440, wherein the positions are determined according to EU numbering.

[0119] In another aspect, the disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), wherein the modified CH3 domain comprises F at position 382, ​​G at position 384, A at position 385, K at position 387, S at position 389, L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440, wherein the positions are determined according to EU numbering.

[0120] In another aspect, the disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), wherein the modified CH3 domain comprises F at position 382, ​​G at position 384, A at position 385, K at position 387, L at position 388, T at position 389, L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440, wherein the positions are determined according to EU numbering.

[0121] In another aspect, the disclosure provides a polypeptide comprising the sequence of any one of SEQ ID NOs: 72, 78, 84, 90, 96, 102, 108, 114, and 120.

[0122] In another aspect, the disclosure provides a polypeptide comprising the sequence of any one of SEQ ID NOs: 73, 79, 85, 91, 97, 103, 109, 115, and 121.

[0123] In another aspect, the disclosure provides a polypeptide comprising the sequence of any one of SEQ ID NOs: 74, 80, 86, 92, 98, 104, 110, 116, and 122.

[0124] In another aspect, the disclosure provides a polypeptide comprising the sequence of any one of SEQ ID NOs: 75, 81, 87, 93, 99, 105, 111, 117, and 123.

[0125] In another aspect, the disclosure provides a polypeptide comprising the sequence of any one of SEQ ID NOs: 76, 82, 88, 94, 100, 106, 112, 118, and 124.

[0126] In another aspect, the disclosure provides a polypeptide comprising the sequence of any one of SEQ ID NOs: 77, 83, 89, 95, 101, 107, 113, 119, and 125.

[0127] In another aspect, the disclosure provides an Fc polypeptide that specifically binds to TfR, comprising a modified CH3 domain, wherein the modified CH3 domain comprises a sequence at least 85% (e.g., at least 90%, 91%, 93%, 95%, 97%, 98%, or 99%) identical to amino acids 111-217 of the sequence of SEQ ID NO: 137, and wherein the modified CH3 domain is modified by replacing Ala, Asp, His, Tyr, or Phe at position 378, 380, 382, ​​383, 384, 385, 386, 387, 388, 389, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454 and Gly, Ser, Thr, or Val at position 440. In some embodiments, the modified CH3 domain has a Met or Leu at position 428.

[0128] In another aspect, the disclosure provides an Fc polypeptide that specifically binds to TfR, comprising a modified CH3 domain, wherein the modified CH3 domain comprises a sequence at least 85% (e.g., at least 90%, 91%, 93%, 95%, 97%, 98%, or 99%) identical to amino acids 111-217 of the sequence of SEQ ID NO: 137, and wherein the modified CH3 domain comprises any of the sets of substitutions provided in clones in any of Tables 32B-1, 32C, 32D, 32E, 32F, 323G, 32H, 32H-1, 32J, and 32K, or comprises a possible amino acid set forth in Table 32I.

[0129] In some embodiments of the above two aspects, the modified CH3 domain comprises Ala or His at position 378, Asp or Glu at position 380, Gly at position 382, ​​Leu at position 384, Val at position 385, Gln or Ala at position 386, Ile or Val at position 422, Ala or Pro at position 424, Thr or Ile at position 426, Ile at position 438, and Gly or Thr at position 440, according to EU numbering. The modified CH3 domain may also comprise Met or Leu at position 428.

[0130] In some embodiments, the modified CH3 domain comprises His at position 378, Glu at position 380, Gly at position 382, ​​Leu at position 384, Val at position 385, Gln at position 386, He at position 422, Pro at position 424, He at position 426, He at position 438, and Thr at position 440 according to EU numbering. The modified CH3 domain may also comprise Met or Leu at position 428.

[0131] In some embodiments, the modified CH3 domain comprises His at position 378, Glu at position 380, Gly at position 382, ​​Leu at position 384, Val at position 385, Gln at position 386, He at position 422, Pro at position 424, He at position 426, Leu at position 428, He at position 438, and Thr at position 440 according to EU numbering.

[0132] In another aspect, the disclosure provides an Fc polypeptide that specifically binds to TfR, comprising a modified CH3 domain, wherein the modified CH3 domain comprises a sequence at least 85% (e.g., at least 90%, 91%, 93%, 95%, 97%, 98%, or 99%) identical to amino acids 111-217 of SEQ ID NO: 137, and the modified CH3 domain comprises His at position 378, Glu at position 380, Gly at position 382, ​​Leu at position 384, Val at position 385, Gln at position 386, He at position 422, Pro at position 424, He at position 426, He at position 438, and Thr at position 440, according to EU numbering. The modified CH3 domain may also comprise Met or Leu at position 428.

[0133] In another aspect, the disclosure provides an Fc polypeptide that specifically binds to TfR, comprising a modified CH3 domain, wherein the modified CH3 domain comprises a sequence at least 85% (e.g., at least 90%, 91%, 93%, 95%, 97%, 98%, or 99%) identical to amino acids 111-217 of SEQ ID NO: 138, and the modified CH3 domain comprises His at position 378, Glu at position 380, Gly at position 382, ​​Leu at position 384, Val at position 385, Gln at position 386, He at position 422, Pro at position 424, He at position 426, Leu at position 428, He at position 438, and Thr at position 440, according to EU numbering. In some embodiments of the disclosure provided herein, the modified constant domain (e.g., the modified CH3 domain) further comprises at least one modification that promotes heterodimerization. In certain embodiments, the modified constant domain (e.g., the modified CH3 domain) further comprises a T366W substitution according to EU numbering. In certain embodiments, the modified constant domain (e.g., the modified CH3 domain) further comprises T366S, L368A, and Y407V substitutions according to EU numbering.

[0134] In some embodiments of the disclosure provided herein, the modified constant domain (e.g., a modified CH3 domain) further comprises a CH2 domain (e.g., a modified CH2 domain). In some embodiments, the modified CH2 and CH3 domains form an Fc polypeptide. In certain embodiments, the modified CH2 domain comprises a modification that reduces effector function. In certain embodiments, the CH2 domain comprises Ala at position 234 and Ala at position 235 according to EU numbering. In certain embodiments, the CH2 domain comprises Ala at position 234, Ala at position 235, and Gly at position 329 according to EU numbering. In certain embodiments, the CH2 domain comprises Ala at position 234, Ala at position 235, and Ser at position 329 according to EU numbering.

[0135] In some embodiments, the CH2 domain is a human IgG1, IgG2, IgG3, or IgG4 CH2 domain.

[0136] In some embodiments of any aspect delineated herein, the polypeptide is part of a dimer. In some embodiments, the dimer is an Fc dimer. In some embodiments, the polypeptide is further linked to a Fab.

[0137] In some embodiments of any aspect described herein, the polypeptide is the first polypeptide of a dimer such that the dimer is monovalent for CD98hc binding. In other embodiments, the polypeptide is the first polypeptide of a dimer such that the dimer is bivalent for CD98hc binding.

[0138] In some embodiments of any aspect described herein, the polypeptide is the first polypeptide of a dimer such that the dimer is monovalent for TfR binding. In other embodiments, the polypeptide is the first polypeptide of a dimer such that the dimer is bivalent for TfR binding.

[0139] In some embodiments of any aspect described herein, the C-terminal lysine of the polypeptide is removed.

[0140] In another aspect, the disclosure provides a polynucleotide comprising a nucleic acid sequence encoding a polypeptide described herein.

[0141] In another aspect, the disclosure provides a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a polypeptide described herein.

[0142] In another aspect, the present disclosure provides a host cell comprising a polynucleotide comprising a nucleic acid sequence encoding a polypeptide described herein.

[0143] In another aspect, the disclosure provides a method for producing a polypeptide comprising a modified constant domain (e.g., a modified CH3 domain), comprising culturing a host cell under conditions in which a polypeptide encoded by a polynucleotide described herein is expressed.

[0144] In another aspect, the present disclosure provides a pharmaceutical composition comprising a polypeptide described herein and a pharmaceutically acceptable carrier.

[0145] In another aspect, the disclosure provides methods for transcytosis of a therapeutic agent across an endothelium. In some embodiments, the method comprises contacting the endothelium with a composition comprising a polypeptide dimer (e.g., a polypeptide dimer described herein) capable of binding to CD98hc fused to a therapeutic agent. In some embodiments, the method comprises contacting the endothelium with a composition comprising a polypeptide dimer (e.g., a polypeptide dimer described herein) capable of binding to TfR fused to a therapeutic agent. In some embodiments, the endothelium is the BBB.

[0146] In another aspect, the present disclosure provides a method for engineering a polypeptide comprising a CH3 domain modified to specifically bind to a CD98hc protein, the method comprising: (a) modifying a polynucleotide encoding a modified CH3 domain to comprise: (i) a first sequence comprising at least one substitution relative to the sequence of EWESNGQP (SEQ ID NO: 52); (ii) a second sequence comprising at least one substitution relative to the sequence of NVFSCSVM (SEQ ID NO: 53); and (iii) a third sequence comprising at least one substitution relative to the sequence of YTQKSLS (SEQ ID NO: 54); (b) expressing and recovering a polypeptide comprising the modified CH3 domain; (c) determining whether the polypeptide binds to the CD98hc protein; The sequence of SEQ ID NO: 52 is positions 380 to 387 of an Fc polypeptide (e.g., SEQ ID NO: 1), the sequence of SEQ ID NO: 53 is positions 421 to 428 of an Fc polypeptide (e.g., SEQ ID NO: 1), and the sequence of SEQ ID NO: 54 is positions 436 to 442 of an Fc polypeptide (e.g., SEQ ID NO: 1), and the positions are determined according to EU numbering.

[0147] In another aspect, the disclosure provides a method for engineering a polypeptide comprising a CH3 domain modified to specifically bind to a TfR protein, the method comprising: (a) modifying a polynucleotide encoding a modified CH3 domain to comprise (i) a first sequence comprising at least one amino acid substitution and / or deletion relative to the sequence of AVEWESNGQPENN (SEQ ID NO: 56), and (ii) a second sequence comprising at least one amino acid substitution in the sequence of VFSCSVMHEALHNHYTQKS (SEQ ID NO: 57); (b) expressing and recovering a polypeptide comprising the modified CH3 domain; (c) determining whether the polypeptide binds to the TfR protein; The method provides a method in which the sequence of SEQ ID NO: 56 is positions 378 to 390 of an Fc polypeptide (e.g., SEQ ID NO: 1), and the sequence of SEQ ID NO: 57 is positions 422 to 440 of an Fc polypeptide (e.g., SEQ ID NO: 1), the positions being determined according to EU numbering.

[0148] In some embodiments of this aspect, the steps of expressing a polypeptide comprising the modified CH3 domain and determining whether the modified CH3 domain binds to CD98hc or TfR are carried out using a display system. In certain embodiments, the display system is a cell surface display system, a viral display system, an mRNA display system, a polysome display system, or a ribosome display system.

[0149] In another aspect, the present disclosure provides a method for delivering a therapeutic agent across the BBB to the brain parenchyma, comprising contacting the BBB with a composition comprising a polypeptide dimer described herein fused to a therapeutic agent.

[0150] In another aspect, the present disclosure provides a method for delivering a therapeutic agent across the BBB to target an extracellular target, comprising contacting the BBB with a composition comprising a polypeptide dimer as described herein fused to a therapeutic agent.

[0151] In some embodiments of the above two aspects, one polypeptide in the polypeptide dimer comprises at least 11, 12, 13, 14, or 15 substitutions at the set of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 391, 421, 422, 424, 426, 428, 434, 436, 438, 440, 441, and 442 according to EU numbering.

[0152] In some embodiments of the above two aspects, one polypeptide in the polypeptide dimer comprises at least 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitutions at the set of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 434, 436, 438, 440, and 442 according to EU numbering.

[0153] In some embodiments, both polypeptides in a polypeptide dimer do not have the substitutions L234A, L235A, and P329G.

[0154] In another aspect, the disclosure provides a method for delivery across the BBB to a biological target in the brain, the method comprising: (a) a CD98hc-binding polypeptide described herein; and (b) a means for binding the biological target in the brain.

[0155] In some embodiments, the biological target is a cell surface target in the brain, such as microglial cells, astrocytes, oligodendrocytes, neurons, and cancer cells, hi some embodiments, the cell surface target is selected from the group consisting of TREM2, PILRA, CD33, CR1, ABCA1, ABCA7, MS4A4A, MS4A6A, MS4A4E, HLA-DR5, HLA-DR1, IL1RAP, TREML2, IL-34, SORL1, ADAM17, and Siglec11.

[0156] In some embodiments, the biological target is a cell surface target on a hematological cancer cell, hi certain embodiments, the cell surface target is selected from the group consisting of B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b.

[0157] In some embodiments, the target is on a tumor cell. In certain embodiments, the target is selected from the group consisting of ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR(1-4), folate receptor alpha, GAL-3BP (galectin-binding protein), GD2, GD3, GloboH (globohexacylceramide), gp100, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine-rich repeat-containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4, colon cancer kinase), RON, ROR1, TF (tissue factor), and TROP2.

[0158] In some embodiments, the target may include alpha-synuclein or a derivative or fragment thereof, a derivative of amyloid-beta peptide or a fragment thereof, Tau or a derivative or fragment thereof, pTau, huntingtin, transthyretin, or TAR DNA-binding protein 43 (TDP-43) or a derivative or fragment thereof.

[0159] In another aspect, the present disclosure provides a method of targeting an extracellular target in the brain using a CD98hc-binding polypeptide, comprising administering the CD98hc-binding polypeptide to a patient, wherein the polypeptide is transported across the BBB into the parenchyma without being transcytosed into cells in the brain. In some embodiments, the extracellular target is on or near astrocytes, microglia, oligodendrocytes, or cancer cells. In certain embodiments, the extracellular target is an antigen in the brain. In certain embodiments, the antigen is a plaque, tangle, or other non-cellular target. In some embodiments, the extracellular target is a non-neuronal target. In certain embodiments, the method comprises delivering a therapeutic agent to the extracellular target.

[0160] In another aspect, the disclosure provides a method of delivering a therapeutic agent across the BBB to astrocyte cells, the method comprising contacting the BBB with a composition comprising a polypeptide dimer described herein fused to a therapeutic agent. In some embodiments, both polypeptides of the polypeptide dimer comprise at least 11, 12, 13, 14, or 15 substitutions at the set of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 391, 421, 422, 424, 426, 428, 434, 436, 438, 440, 441, and 442 according to EU numbering.

[0161] In another aspect, the present disclosure provides a method for delivering a therapeutic agent to a peripheral CD98hc-expressing organ, comprising administering to a subject a composition comprising a polypeptide dimer described herein fused to a therapeutic agent. In certain embodiments, the peripheral CD98hc-expressing organ is the kidney, testis, bone marrow, spleen, or pancreas.

[0162] In another aspect, the disclosure provides a CD98hc-binding polypeptide, wherein, when bound to human CD98hc, the polypeptide binds to at least 7, 8, 9, 10, 11, 12, 13, or 14 of residues selected from the group consisting of positions 477, 478, 479, 480, 481, 482, 483, 486, 499, 497, 498, 500, 501, and 502 of SEQ ID NO: 134. In certain embodiments, when bound to human CD98hc, the polypeptide binds to positions 477, 478, 479, 480, 481, 482, 483, 486, 499, 497, 498, 500, 501, and 502 of SEQ ID NO: 134. In certain embodiments, when bound to human CD98hc, the polypeptide further binds to at least one additional residue selected from the group consisting of positions 229, 231, 232, 236, 235, 488, 495, and 496 of SEQ ID NO: 134. In certain embodiments, when bound to human CD98hc, the polypeptide further binds to at least one additional residue selected from the group consisting of positions 312, 315, 348, 381, 439, 444, 443, 485, 484, 476, 475, and 442 of SEQ ID NO: 134.

[0163] In another aspect, the disclosure provides a CD98hc-binding polypeptide, wherein when bound to human CD98hc, the polypeptide binds to at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 residues selected from the group consisting of positions 229, 231, 232, 236, 235, 486, 488, 495, 496, 498, 500, 499, 497, 482, 481, 483, 477, 480, 501, 502, 478, and 479 of SEQ ID NO: 134.

[0164] In another aspect, the disclosure provides a CD98hc-binding polypeptide, wherein, when bound to human CD98hc, the polypeptide binds to at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 residues selected from the group consisting of positions 312, 315, 348, 381, 439, 444, 443, 485, 484, 477, 483, 481, 480, 478, 476, 502, 499, 501, 500, 498, 497, 486, 479, 482, 475, and 442 of SEQ ID NO: 134. In certain embodiments, the polypeptide is an antibody or fragment thereof, a VHH domain, or a polypeptide comprising a modified constant domain that specifically binds to a CD98hc protein.

[0165] In another aspect, the disclosure provides a method for increasing brain exposure to a therapeutic agent in a subject relative to a reference molecule, the method comprising administering to the subject a monovalent molecule that binds to CD98hc with a binding affinity of about 20 nM to about 550 nM, wherein the molecule binds to the therapeutic agent, and the reference molecule comprises the therapeutic agent but does not comprise a CD98hc-binding moiety.

[0166] In another aspect, the disclosure provides a method for increasing brain exposure to a therapeutic agent in a subject relative to a reference molecule, the method comprising administering to the subject a bivalent molecule that binds to CD98hc with a binding affinity of about 275 nM to about 2100 nM, wherein the molecule binds to the therapeutic agent, and the reference molecule comprises the therapeutic agent but does not comprise a CD98hc-binding moiety.

[0167] In another aspect, the disclosure provides a composition for delivery across the BBB to a biological target in the brain, the composition comprising: (a) a CD98hc-binding polypeptide described herein; and (b) a means for binding the biological target in the brain.

[0168] In some embodiments, the biological target is a cell surface target in the brain, such as microglial cells, astrocytes, oligodendrocytes, neurons, and cancer cells, hi some embodiments, the cell surface target is selected from the group consisting of TREM2, PILRA, CD33, CR1, ABCA1, ABCA7, MS4A4A, MS4A6A, MS4A4E, HLA-DR5, HLA-DR1, IL1RAP, TREML2, IL-34, SORL1, ADAM17, and Siglec11.

[0169] In some embodiments, the biological target is a cell surface target on a hematological cancer cell, hi certain embodiments, the cell surface target is selected from the group consisting of B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b.

[0170] In some embodiments, the target is on a tumor cell. In certain embodiments, the target is selected from the group consisting of ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR(1-4), folate receptor alpha, GAL-3BP (galectin-binding protein), GD2, GD3, GloboH (globohexacylceramide), gp100, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine-rich repeat-containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4, colon cancer kinase), RON, ROR1, TF (tissue factor), and TROP2.

[0171] In some embodiments, the target may include alpha-synuclein or a derivative or fragment thereof, a derivative of amyloid-beta peptide or a fragment thereof, Tau or a derivative or fragment thereof, pTau, huntingtin, transthyretin, or TAR DNA-binding protein 43 (TDP-43) or a derivative or fragment thereof.

[0172] In another aspect, the present disclosure provides a method for delivery across the BBB to a biological target in the brain of a subject, comprising: (a) providing a composition comprising (i) a CD98hc-binding polypeptide described herein, and (ii) a means for binding a biological target; (b) peripherally administering the composition of step (a) to the subject.

[0173] In another aspect, the disclosure provides a method for binding a biological target in the brain of a subject, the method comprising: (a) providing a composition comprising (i) a CD98hc-binding polypeptide described herein and (ii) a means for binding a biological target; (b) peripherally administering the composition of step (a) to a subject; A method is provided wherein the composition binds to a biological target in the brain of a subject.

[0174] Unless otherwise indicated or clear from the context, all numbering of positions (e.g., "position x") throughout this specification in an Fc, CH2, or CH3 polypeptide is based on the EU numbering system. [Brief explanation of the drawings]

[0175] [Figure 1] 1 shows the plasma pharmacokinetics of LLB2 and LLB1 CD98hc binding molecules in C57 / B6 (WT) mice. [Figure 2] 1 shows the plasma pharmacokinetics of additional LLB2 and LLB1 CD98hc binding molecules in C57 / B6 (WT) mice. [Figure 3] 1 shows the plasma pharmacokinetics of affinity-matured LLB2 CD98hc binding molecules in C57 / B6 (WT) mice. [Figure 4] 1 shows the plasma pharmacokinetics of affinity-de-matured LLB2 CD98hc binding molecules in C57 / B6 (WT) mice. [Figure 5]A-C show brain uptake of LLB2 and LLB1 CD98hc binding molecules in CD98hcmu / huKI mice. (A) huIgG in plasma 48 hours after administration. (B) huIgG in whole brain lysates. (C) Ratio of huIgG in brain to plasma. [Figure 6] Figure 1 shows capillary depletion demonstrating that CD98hc-binding molecules cross the BBB and enter the brain parenchyma of CD98hcmu / huKI mice. [Figure 7] 1 shows the CNS biodistribution of LLB2 and LLB1 variants in CD98hcmu / huKI mice. [Figure 8] Immunohistochemistry shows the cell-specific biodistribution of the CD98hc binding molecule IBA1 (microglia) in CD98hcmu / huKI mice. [Figure 9] Immunohistochemistry shows the cell-specific biodistribution of CD98hc binding molecule AQPN4 (astrocytes) in CD98hcmu / huKI mice. [Figure 10] A and B show brain uptake of additional LLB2 and LLB1 variants in CD98hcmu / huKI mice. (A) huIgG in plasma 48 hours post-dose. (B) huIgG in whole brain lysates. [Figure 11A] 1 shows peripheral tissue localization of LLB2 and LLB1 variants in CD98hcmu / huKI mice. [Figure 11B] 1 shows peripheral tissue localization of LLB2 and LLB1 variants in CD98hcmu / huKI mice. [Figure 11C] 1 shows peripheral tissue localization of LLB2 and LLB1 variants in CD98hcmu / huKI mice. [Figure 11D] 1 shows peripheral tissue localization of LLB2 and LLB1 variants in CD98hcmu / huKI mice. [Figure 11E] 1 shows peripheral tissue localization of LLB2 and LLB1 variants in CD98hcmu / huKI mice. [Figure 11F]1 shows peripheral tissue localization of LLB2 and LLB1 variants in CD98hcmu / huKI mice. [Figure 12] A and B show the time course of brain uptake of monovalent and bivalent LLB2 variants in CD98hcmu / huKI mice. (A) huIgG PK in plasma up to 10 days post-dose. (B) huIgG PK in whole brain lysates. [Figure 13] Figure 1 shows capillary depletion demonstrating that monovalent and bivalent LLB2 variants cross the BBB and enter the brain parenchyma of CD98hcmu / huKI mice. [Figure 14A] huIgG pharmacokinetics (PK) in peripheral tissues of monovalent and bivalent LLB2 variants in CD98hcmu / huKI mice. [Figure 14B] huIgG pharmacokinetics (PK) in peripheral tissues of monovalent and bivalent LLB2 variants in CD98hcmu / huKI mice. [Figure 14C] huIgG pharmacokinetics (PK) in peripheral tissues of monovalent and bivalent LLB2 variants in CD98hcmu / huKI mice. [Figure 14D] huIgG pharmacokinetics (PK) in peripheral tissues of monovalent and bivalent LLB2 variants in CD98hcmu / huKI mice. [Figure 14E] huIgG pharmacokinetics (PK) in peripheral tissues of monovalent and bivalent LLB2 variants in CD98hcmu / huKI mice. [Figure 14F] huIgG pharmacokinetics (PK) in peripheral tissues of monovalent and bivalent LLB2 variants in CD98hcmu / huKI mice. [Figure 14G] huIgG pharmacokinetics (PK) in peripheral tissues of monovalent and bivalent LLB2 variants in CD98hcmu / huKI mice. [Figure 14H] huIgG pharmacokinetics (PK) in peripheral tissues of monovalent and bivalent LLB2 variants in CD98hcmu / huKI mice. [Figure 15]1 shows the biodistribution timecourse of monovalent and bivalent LLB2-10-8 by huIgG immunohistochemistry. [Figure 16] Biodistribution time courses of monovalent and bivalent LLB2-10-8 by immunohistochemistry for huIgG and Iba1 (microglia) are shown. [Figure 17] A and B show (A) plasma and (B) brain exposure after repeated administration of monovalent LLB2 variants. [Figure 18] 1 shows the biodistribution time course after repeated administration of a monovalent LLB2-10-8 variant. [Figure 19] A and B show the yeast display library surface modeled on the wild-type IgG1 Fc backbone (PDB 1hzh). [Figure 20] Figures 20A-C show the concentrations of clones and controls in the plasma (Figure 20A) and whole brain (Figure 20B) of chimeric huTfR apical knock-in mice 24 hours after administration of a 50 mg / kg dose of clone or control. Figure 20C shows the reduction of Aβ40 levels in the mouse brain. [Figure 21A] Plasma PK of clones and controls at a 10 mg / kg dose in wild-type mice is shown (Figure 21A). Brain PK (Figure 21B), brain PD (Figure 21C), and plasma PK (Figure 21D) of monovalent clone 6.5.11.5.42.2, bivalent clone 6.5.11.5.42.2, and controls at a 50 mg / kg dose in chimeric huTfR apical knock-in mice. Figures 3E and 3F show safety data showing the percent of Ter119+ erythrocytes (Figure 21E) or CD71+ bone marrow reticulocytes (Figure 21F) in the total plasma cell population for anti-BACE1 control or bivalent clone 6.5.11.5.42.2. Figure 21G shows the levels of total brain TfR compared to loading control GAPDH 24 hours after treatment. [Figure 21B]Plasma PK of clones and controls at a 10 mg / kg dose in wild-type mice is shown (Figure 21A). Brain PK (Figure 21B), brain PD (Figure 21C), and plasma PK (Figure 21D) of monovalent clone 6.5.11.5.42.2, bivalent clone 6.5.11.5.42.2, and controls at a 50 mg / kg dose in chimeric huTfR apical knock-in mice. Figures 3E and 3F show safety data showing the percent of Ter119+ erythrocytes (Figure 21E) or CD71+ bone marrow reticulocytes (Figure 21F) in the total plasma cell population for anti-BACE1 control or bivalent clone 6.5.11.5.42.2. Figure 21G shows the levels of total brain TfR compared to loading control GAPDH 24 hours after treatment. [Figure 21C] Plasma PK of clones and controls at a 10 mg / kg dose in wild-type mice is shown (Figure 21A). Brain PK (Figure 21B), brain PD (Figure 21C), and plasma PK (Figure 21D) of monovalent clone 6.5.11.5.42.2, bivalent clone 6.5.11.5.42.2, and controls at a 50 mg / kg dose in chimeric huTfR apical knock-in mice. Figures 3E and 3F show safety data showing the percent of Ter119+ erythrocytes (Figure 21E) or CD71+ bone marrow reticulocytes (Figure 21F) in the total plasma cell population for anti-BACE1 control or bivalent clone 6.5.11.5.42.2. Figure 21G shows the levels of total brain TfR compared to loading control GAPDH 24 hours after treatment. [Figure 21D] Plasma PK of clones and controls at a 10 mg / kg dose in wild-type mice is shown (Figure 21A). Brain PK (Figure 21B), brain PD (Figure 21C), and plasma PK (Figure 21D) of monovalent clone 6.5.11.5.42.2, bivalent clone 6.5.11.5.42.2, and controls at a 50 mg / kg dose in chimeric huTfR apical knock-in mice. Figures 3E and 3F show safety data showing the percent of Ter119+ erythrocytes (Figure 21E) or CD71+ bone marrow reticulocytes (Figure 21F) in the total plasma cell population for anti-BACE1 control or bivalent clone 6.5.11.5.42.2. Figure 21G shows the levels of total brain TfR compared to loading control GAPDH 24 hours after treatment. [Figure 21E]Plasma PK of clones and controls at a 10 mg / kg dose in wild-type mice is shown (Figure 21A). Brain PK (Figure 21B), brain PD (Figure 21C), and plasma PK (Figure 21D) of monovalent clone 6.5.11.5.42.2, bivalent clone 6.5.11.5.42.2, and controls at a 50 mg / kg dose in chimeric huTfR apical knock-in mice. Figures 3E and 3F show safety data showing the percent of Ter119+ erythrocytes (Figure 21E) or CD71+ bone marrow reticulocytes (Figure 21F) in the total plasma cell population for anti-BACE1 control or bivalent clone 6.5.11.5.42.2. Figure 21G shows the levels of total brain TfR compared to loading control GAPDH 24 hours after treatment. [Figure 21F] Plasma PK of clones and controls at a 10 mg / kg dose in wild-type mice is shown (Figure 21A). Brain PK (Figure 21B), brain PD (Figure 21C), and plasma PK (Figure 21D) of monovalent clone 6.5.11.5.42.2, bivalent clone 6.5.11.5.42.2, and controls at a 50 mg / kg dose in chimeric huTfR apical knock-in mice. Figures 3E and 3F show safety data showing the percent of Ter119+ erythrocytes (Figure 21E) or CD71+ bone marrow reticulocytes (Figure 21F) in the total plasma cell population for anti-BACE1 control or bivalent clone 6.5.11.5.42.2. Figure 21G shows the levels of total brain TfR compared to loading control GAPDH 24 hours after treatment. [Figure 21G] Plasma PK of clones and controls at a 10 mg / kg dose in wild-type mice is shown (Figure 21A). Brain PK (Figure 21B), brain PD (Figure 21C), and plasma PK (Figure 21D) of monovalent clone 6.5.11.5.42.2, bivalent clone 6.5.11.5.42.2, and controls at a 50 mg / kg dose in chimeric huTfR apical knock-in mice. Figures 3E and 3F show safety data showing the percent of Ter119+ erythrocytes (Figure 21E) or CD71+ bone marrow reticulocytes (Figure 21F) in the total plasma cell population for anti-BACE1 control or bivalent clone 6.5.11.5.42.2. Figure 21G shows the levels of total brain TfR compared to loading control GAPDH 24 hours after treatment. [Figure 22]1 shows size exclusion chromatography (SEC) analysis of clone 6.5.11.5.42.2 under low pH and control conditions. [Figure 23A] The structure of clone 6.5.11.5.42, which has a circularly permuted apical domain of human TfR, is shown with library residues in sticks (Figure 23A). The structure of clone 6.5.11.5.42, which has the TfR apical domain and a modeled full-length human TfR domain (Figure 23B). A zoom of Figure 23B showing clashes between human TfR domains (Figure 23C). [Figure 23B] The structure of clone 6.5.11.5.42, which has a circularly permuted apical domain of human TfR, is shown with library residues in sticks (Figure 23A). The structure of clone 6.5.11.5.42, which has the TfR apical domain and a modeled full-length human TfR domain (Figure 23B). A zoom of Figure 23B showing clashes between human TfR domains (Figure 23C). [Figure 23C] The structure of clone 6.5.11.5.42, which has a circularly permuted apical domain of human TfR, is shown with library residues in sticks (Figure 23A). The structure of clone 6.5.11.5.42, which has the TfR apical domain and a modeled full-length human TfR domain (Figure 23B). A zoom of Figure 23B showing clashes between human TfR domains (Figure 23C). [Figure 24] A and B show the concentrations of monovalent and bivalent clone 42.2.1.2, monovalent and bivalent clone 6.5.11.5.42.2, and control in the whole brain (Figure 24A) and plasma (Figure 24B) of chimeric huTfR apical knock-in mice 24 hours after administration of a 50 mg / kg dose of the clone or control to the mice. [Figure 25] 1 shows safety data showing rectal bulb levels for monovalent and bivalent clone 42.2.1.2, monovalent and bivalent clone 6.5.11.5.42.2, and controls. [Figure 26A]Plasma PK (Figures 26A and 26B) and brain PK (Figures 26C and 26D) of monovalent and bivalent clones 42.8.17, 42.8.15, 42.8.80, 42.8.196, 42.2.3-1H, and 42.2.19, and controls at a 50 mg / kg dose in chimeric huTfR apical knock-in mice are shown. [Figure 26B] Plasma PK (Figures 26A and 26B) and brain PK (Figures 26C and 26D) of monovalent and bivalent clones 42.8.17, 42.8.15, 42.8.80, 42.8.196, 42.2.3-1H, and 42.2.19, and controls at a 50 mg / kg dose in chimeric huTfR apical knock-in mice are shown. [Figure 26C] Plasma PK (Figures 26A and 26B) and brain PK (Figures 26C and 26D) of monovalent and bivalent clones 42.8.17, 42.8.15, 42.8.80, 42.8.196, 42.2.3-1H, and 42.2.19, and controls at a 50 mg / kg dose in chimeric huTfR apical knock-in mice are shown. [Figure 26D] Plasma PK (Figures 26A and 26B) and brain PK (Figures 26C and 26D) of monovalent and bivalent clones 42.8.17, 42.8.15, 42.8.80, 42.8.196, 42.2.3-1H, and 42.2.19, and controls at a 50 mg / kg dose in chimeric huTfR apical knock-in mice are shown. [Figure 27] A and B show (A) plasma and (B) brain exposure after repeated administration of bivalent LLB2 variants in CD98hcmu / huKI mice. [Figure 28] A and B show the crystal structures of bivalent CD98hc binding molecules with CD98hc: (A) LLB2-10-6 dimer (B) LLB1-3-16 dimer. [Figure 29] A and B show the combined molecular crystal structures of the co-complexes: (A) LLB2-10-6 dimer (B) LLB1-3-16 dimer. [Figure 30A]Orientation of bivalent CD98hc binding molecules crystal structures relative to the modeled CD98hc-LAT1 complex: (A) bivalent LLB2-10-6 dimer, (B) bivalent LLB1-3-16 dimer, and (C) monovalent LLB2-10-6 dimer. [Figure 30B] Orientation of bivalent CD98hc binding molecules crystal structures relative to the modeled CD98hc-LAT1 complex: (A) bivalent LLB2-10-6 dimer, (B) bivalent LLB1-3-16 dimer, and (C) monovalent LLB2-10-6 dimer. [Figure 30C] Orientation of bivalent CD98hc binding molecules crystal structures relative to the modeled CD98hc-LAT1 complex: (A) bivalent LLB2-10-6 dimer, (B) bivalent LLB1-3-16 dimer, and (C) monovalent LLB2-10-6 dimer. [Figure 31A] Figure 1 shows plasma and brain PK and capillary depletion results in CD98hcmu / huKI mice after administration with monovalent LLB2 variants: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 31B] Figure 1 shows plasma and brain PK and capillary depletion results in CD98hcmu / huKI mice after administration with monovalent LLB2 variants: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 31C] Figure 1 shows plasma and brain PK and capillary depletion results in CD98hcmu / huKI mice after administration with monovalent LLB2 variants: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 31D] Figure 1 shows plasma and brain PK and capillary depletion results in CD98hcmu / huKI mice after administration with monovalent LLB2 variants: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 31E]Figure 1 shows plasma and brain PK and capillary depletion results in CD98hcmu / huKI mice after administration with monovalent LLB2 variants: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 31F] Figure 1 shows plasma and brain PK and capillary depletion results in CD98hcmu / huKI mice after administration with monovalent LLB2 variants: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 32A] Figure 1 shows plasma and brain PK and capillary depletion results in CD98hcmu / huKI mice after administration with bivalent LLB2 variants: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 32B] Figure 1 shows plasma and brain PK and capillary depletion results in CD98hcmu / huKI mice after administration with bivalent LLB2 variants: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 32C] Figure 1 shows plasma and brain PK and capillary depletion results in CD98hcmu / huKI mice after administration with bivalent LLB2 variants: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 32D] Figure 1 shows plasma and brain PK and capillary depletion results in CD98hcmu / huKI mice after administration with bivalent LLB2 variants: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 32E] Figure 1 shows plasma and brain PK and capillary depletion results in CD98hcmu / huKI mice after administration with bivalent LLB2 variants: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 32F]Figure 1 shows plasma and brain PK and capillary depletion results in CD98hcmu / huKI mice after administration with bivalent LLB2 variants: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 33] A and B show (A) huIgG PK in plasma and (B) huIgG PK in whole brain lysates up to 21 days post-dose in affinity-matched LLB1 and LLB2 variants. [Figure 34A] Figure 1 shows the results of plasma and brain PK and capillary depletion in CD98hcmu / huKI mice after administration with the LLB2-10-8 variant: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 34B] Figure 1 shows the results of plasma and brain PK and capillary depletion in CD98hcmu / huKI mice after administration with the LLB2-10-8 variant: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 34C] Figure 1 shows the results of plasma and brain PK and capillary depletion in CD98hcmu / huKI mice after administration with the LLB2-10-8 variant: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 34D] Figure 1 shows the results of plasma and brain PK and capillary depletion in CD98hcmu / huKI mice after administration with the LLB2-10-8 variant: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 34E] Figure 1 shows the results of plasma and brain PK and capillary depletion in CD98hcmu / huKI mice after administration with the LLB2-10-8 variant: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 34F]Figure 1 shows the results of plasma and brain PK and capillary depletion in CD98hcmu / huKI mice after administration with the LLB2-10-8 variant: (A) plasma PK, (B) brain PK, (C) parenchymal fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 35] Immunohistochemistry of huIgG on brain sections from CD98hcmu / huKI mice 1, 7, 14, and 21 days after a 50mpk dose of the LLB2-10-8 variant is shown. [Figure 36] A–C show immunohistochemistry for huIgG and CNS cell type markers on brain sections from CD98hcmu / huKI 7 days after a 50 mpk dose of the LLB2-10-8 variant: (A) Iba1 for microglia, (B) AQP4 for astrocytic processes, and (C) NeuN for neurons. [Figure 37] AC show (A) plasma PK, (B) brain PK, and (C) A beta reduction (PD) using CD98hc TV with BACE1 Fab. [Figure 38A] Immunohistochemistry for huIgG, NeuN (neuronal), and LAMP2 (lysosomal) on brain sections from CD98hcmu / huKI 7 days after administration of CD98hc TV with BACE1 Fab is shown. [Figure 38B] Immunohistochemistry for huIgG, NeuN (neuronal), and LAMP2 (lysosomal) on brain sections from CD98hcmu / huKI 7 days after administration of CD98hc TV with BACE1 Fab is shown. [Figure 39] A-D show huIgG PK in plasma and whole brain lysates in mice dosed at 15 mpk: (A) Plasma PK for the monovalent variant, (B) Brain PK for the monovalent variant, (C) Plasma PK for the bivalent variant, and (D) Brain PK for the bivalent variant. [Figure 40A]Results of plasma and brain exposure in NHPs, and capillary depletion in NHP brain tissue are shown: (A) plasma exposure, (B) brain exposure, (C) brain parenchyma fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 40B] Results of plasma and brain exposure in NHPs, and capillary depletion in NHP brain tissue are shown: (A) plasma exposure, (B) brain exposure, (C) brain parenchyma fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 40C] Results of plasma and brain exposure in NHPs, and capillary depletion in NHP brain tissue are shown: (A) plasma exposure, (B) brain exposure, (C) brain parenchyma fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 40D] Results of plasma and brain exposure in NHPs, and capillary depletion in NHP brain tissue are shown: (A) plasma exposure, (B) brain exposure, (C) brain parenchyma fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 40E] Results of plasma and brain exposure in NHPs, and capillary depletion in NHP brain tissue are shown: (A) plasma exposure, (B) brain exposure, (C) brain parenchyma fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 40F] Results of plasma and brain exposure in NHPs, and capillary depletion in NHP brain tissue are shown: (A) plasma exposure, (B) brain exposure, (C) brain parenchyma fraction, (D) vasculature fraction, (E) cell-associated fraction, and (F) non-cell-associated fraction. [Figure 41] A-C show immunohistochemistry of huIgG and CNS cell type markers on NHP brain sections: (A) Iba1 for microglia, (B) AQP4 for astrocyte processes, and (C) NeuN for neurons. [Figure 42] A and B show the binding epitopes of CD98hc binding molecules: (A) LLB2 family and (B) LLB1 family. [Figure 43]A and B show quantification of cellular uptake of clone 1 with LALA, clone 3 with LALA, and a control into HEK293T human TfR-positive cells (Figure 43A) and Chinese hamster ovary (CHO) cells (Figure 43B) ectopically expressing cynomolgus monkey TfR at 37°C. [Figure 44] Plasma PK of Clone 1 with LALA and Clone 3 with LALA is shown. [Figure 45A] The concentrations of monovalent clone 1-112_L, monovalent clone 1-112_LS, monovalent clone 1-292, monovalent clone 1-321, and control are shown in the whole brain (Figure 45A) and plasma (Figure 45B) of chimeric huTfR apical knock-in mice 24 hours after administration of a 50 mg / kg dose of the clone or control to the mice. [Figure 45B] The concentrations of monovalent clone 1-112_L, monovalent clone 1-112_LS, monovalent clone 1-292, monovalent clone 1-321, and control are shown in the whole brain (Figure 45A) and plasma (Figure 45B) of chimeric huTfR apical knock-in mice 24 hours after administration of a 50 mg / kg dose of the clone or control to the mice. [Figure 46] A multiple dose study of clones and controls dosed at 50 mg / kg on days 0, 3, and 5 in chimeric huTfR apical knock-in mice 24 hours after the last dose shows brain Aβ40 levels. [Figure 47A] Shown is the structure of clone 6.5.11.5.42, which contains the TfR apical domain and a modeled full-length human TfR domain (Figure 47A), a zoom of Figure 47A showing the clash between the human TfR domains (Figure 47B), and the structure of clone 1-112, which contains the TfR apical domain and a modeled full-length human TfR domain, LALA, and M428L (Figure 47C). [Figure 47B]Shown is the structure of clone 6.5.11.5.42, which contains the TfR apical domain and a modeled full-length human TfR domain (Figure 47A), a zoom of Figure 47A showing the clash between the human TfR domains (Figure 47B), and the structure of clone 1-112, which contains the TfR apical domain and a modeled full-length human TfR domain, LALA, and M428L (Figure 47C). [Figure 47C] Shown is the structure of clone 6.5.11.5.42, which contains the TfR apical domain and a modeled full-length human TfR domain (Figure 47A), a zoom of Figure 47A showing the clash between the human TfR domains (Figure 47B), and the structure of clone 1-112, which contains the TfR apical domain and a modeled full-length human TfR domain, LALA, and M428L (Figure 47C). DETAILED DESCRIPTION OF THE INVENTION

[0176] Detailed Description I. Introduction Several techniques have been developed for generating non-natural binding sites in polypeptides by screening polypeptide libraries for novel binding agents. One challenge in introducing non-natural binding sites is that such libraries often contain a large number of sequences with undesirable properties (e.g., non-specific binding or lack of generative potential). As described below, "limited liability" techniques can reduce the frequency of amino acids associated with these undesirable properties, thus resulting in libraries that generate more useful sequences. This limited liability technique can be used with various protein scaffolds, including immunoglobulins (i.e., both CDR and non-CDR portions) and other scaffolds, such as fibronectin or any other protein scaffold described herein, to enhance and accelerate the discovery of novel polypeptide binding agents. In a specific example, as described below, a library can be used in which engineered portions of the polypeptide contain exposed sides of beta sheets within the polypeptide.

[0177] We have also developed immunoglobulin libraries modified and engineered at the beta-sheet surface. These libraries have been used to generate novel binding sites in the non-CDR portions of immunoglobulins, specifically to generate novel molecules that bind to CD98 heavy chain (CD98hc) and transferrin receptor (TfR). The present disclosure is based, in part, on the discovery that certain amino acids, particularly those at beta-sheet positions within the CH3 domain of an Fc polypeptide, can be substituted to generate modified CH3 domains containing novel binding sites specific for CD98hc (e.g., CD98hc-binding sites). Beta-sheet positions in the CH3 domain include positions 347-351, 363-372, 378-383, 391-393, 406-412, 422-428, and 437-441 according to EU numbering, as well as other beta-sheet residues in the constant domains described herein, e.g., in Table 1B. Substituting amino acids at beta-sheet positions can offer several advantages when generating immunoglobulin domains containing non-natural binding sites. First, the beta-sheet surface within the domain is stable, allowing for a variety of amino acid substitutions at the surface without disrupting the domain structural fold. In some embodiments, the amino acid substitutions are located on the solvent-exposed side of the domain beta-sheet surface. Second, making amino acid substitutions at beta-sheet positions avoids altering flexible loop regions within the domain, which can sometimes introduce undesirable conformational flexibility. Furthermore, the concave surface of the beta-sheet structure within the domain is ideal for forming protein-protein interactions, and the beta-sheet structure is also distinct from the FcRn and FcγR binding sites within the CH3 domain.

[0178] The engineered technology described herein has been used to discover specific polypeptides that bind to CD98hc or TfR. These polypeptides are transcytosed across the blood-brain barrier in mammals, as described herein. CD98 is highly expressed on brain endothelial cells and is therefore a promising target for receptor-mediated transcytosis (RMT). CD98 is a heterodimer formed between CD98hc (4F2 heavy chain) and CD98 light chain. To date, six CD98 light chains have been identified: LAT1 (SLC7A5, 4F2 light chain), LAT2 (SLC7A8), y + LAT1(SLC7A7), y + LAT2 (SLC7A6), Asc-1 (SLC7A10), or xCT (SLC7A11) have been identified. In this complex, the CD98 heavy chain transports the light chain to the cell surface, where it functions as a large neutral amino acid transporter that preferentially transports branched-chain (valine, leucine, isoleucine) and aromatic (tryptophan, tyrosine, phenylalanine) amino acids. Taking advantage of the CD98 receptor-mediated transcytosis pathway, therapeutic agents can be transported across the BBB using polypeptides containing the CD98hc binding site described herein. This technology can substantially improve the brain uptake of therapeutic agents and is therefore highly useful for treating disorders and diseases where brain delivery is advantageous. In addition, this technology can be used to provide brain uptake and delivery to specific extracellular or neurotumor targets within the brain. For example, if desired, the CD98hc-binding polypeptides provided herein may be used to target such extracellular or neurotumor targets while retaining wild-type effector function. In addition, such CD98hc-binding polypeptides provided herein can be used to target extracellular targets where neuronal uptake is undesirable (e.g., when the target is an antigen or plaque, such as Abeta, Tau, or alpha-synuclein). The CD98hc-binding polypeptides provided herein have well-defined kinetics, biodistribution, and safety profiles that can provide an optimized, fit-for-purpose BBB delivery platform for protein-based therapeutics.

[0179] Also described herein are polypeptides that bind to the transferrin receptor (TfR). TfR is highly expressed on the blood-brain barrier (BBB) ​​and naturally transports transferrin from the blood into the brain. Taking advantage of these advantages already provided by TfR, polypeptides containing the TfR binding site described herein can be used to transport therapeutic agents across the BBB. This technology can substantially improve the uptake of therapeutic agents into the brain and is therefore highly useful for treating disorders and diseases where brain delivery is advantageous.

[0180] Also provided herein are methods for producing polypeptides comprising modified CH3 domains that bind to CD98hc or TfR. Polypeptides comprising modified CH3 domains described herein can be analyzed for CD98hc binding or TfR binding and further mutated to enhance binding as described herein.

[0181] In a further aspect, also provided herein are therapeutic methods and methods in which CD98hc-binding or TfR-binding polypeptides are used to target compositions to CD98hc-expressing or TfR-expressing cells, e.g., to deliver compositions to the cells, or to deliver compositions across endothelia such as the BBB.

[0182] II. Definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "antibodies" optionally includes combinations of two or more such molecules, and the like.

[0183] As used herein, when used to modify a quantity specified by a numerical value or range, the terms "about" and "approximately" indicate numerical values ​​and reasonable deviations from the value known to one of ordinary skill in the art, e.g., ±20%, ±10%, or ±5%, within the intended meaning of the recited value.

[0184] As used herein, the term "CD98hc" or "CD98 heavy chain" refers to the 4F2 cell surface antigen heavy chain and is encoded by the SLC3A2 gene. CD98hc is also known as the 4F2 heavy chain. The human CD98hc sequence is set forth in SEQ ID NO: 55 and UNIPROT accession number P08195. CD98hc sequences from other species are also known (e.g., mouse, UNIPROT accession number P10852, and cynomolgus monkey, UNIPROT accession number G8F3Z0).

[0185] As used herein, the term "transferrin receptor" or "TfR" refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is set forth in SEQ ID NO: 127. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee, accession number XP_003310238.1; rhesus monkey, NP_001244232.1; dog, NP_001003111.1; cow, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP_990587.1). The term "transferrin receptor" also encompasses allelic variants of exemplary reference sequences, e.g., human sequences, encoded by genes at the transferrin receptor protein 1 chromosomal locus. Full-length transferrin receptor protein includes a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain.

[0186] As used herein, the terms "CH3 domain" and "CH2 domain" refer to immunoglobulin constant region domain polypeptides. For purposes of this application, a CH3 domain polypeptide refers to the segment of amino acids from approximately 341 to approximately 447 as numbered according to the EU numbering scheme, and a CH2 domain polypeptide refers to the segment of amino acids from approximately 231 to approximately 340 as numbered according to the EU numbering scheme, excluding the hinge region sequence. CH2 and CH3 domain polypeptides may also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, in which the CH2 domain is numbered from 1 to 110 and the CH3 domain is numbered from 1 to 107 according to the IMGT Scientific chart numbering (IMGT website). The CH2 and CH3 domains are part of the Fc region of an immunoglobulin. As used herein, Fc region refers to the segment of amino acids from about 231 to about 447, numbered according to the EU numbering scheme, but can include at least a portion of the hinge region of an antibody. An exemplary hinge region sequence is the human IgG1 hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO: 4).

[0187] As used herein, the terms "wild-type," "native," and "naturally occurring" when used with respect to a CH3 or CH2 domain refer to a domain having a sequence that occurs in nature.

[0188] As used herein, the term "mutant" when used with respect to a mutant polypeptide or mutant polynucleotide is used interchangeably with "variant." Variants relative to a given wild-type CH3 or CH2 domain reference sequence can include naturally occurring allelic variants. A "non-naturally" occurring CH3 or CH2 domain refers to a variant or mutant domain that does not occur in natural cells, produced by genetic modification of a native CH3 domain or CH2 domain polynucleotide or polypeptide, e.g., using genetic engineering or mutagenesis techniques. A "variant" includes any domain that contains at least one amino acid mutation relative to the wild-type. Mutations can include substitutions, insertions, and deletions.

[0189] 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 similarly to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, 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 analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. 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 amino acid. 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.

[0190] As used herein, "restricted diversity" refers to a codon or position that is restricted to allow fewer than all 20 naturally occurring amino acids in the context of a randomized codon within a polynucleotide library or any amino acid position within a polypeptide library described herein.

[0191] As used herein, a "beta sheet position" in the context of a polypeptide means an amino acid that is within a portion of the polypeptide that is predominantly beta sheet in structure.

[0192] As used herein, the term "immunoglobulin-like fold" refers to a protein domain of between about 80 and 150 amino acid residues that contains two layers of antiparallel beta sheets, with the flat hydrophobic faces of the two beta sheets packing against each other.

[0193] 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 residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as naturally occurring and non-naturally occurring amino acid polymers. An amino acid polymer can contain entirely L-amino acids, entirely D-amino acids, or a mixture of L- and D-amino acids.

[0194] As used herein, the term "constant domain" refers to a domain within the constant region of an immunoglobulin molecule (e.g., CH1, CH2, CH3, CH4, Ckappa, Clamda).

[0195] As used herein, the term "modified constant domain" refers to a constant domain that has at least one mutation, e.g., a substitution, deletion, or insertion, compared to a wild-type immunoglobulin constant domain sequence, but retains the overall Ig fold or structure of the native constant domain.

[0196] As used herein, the term "Fc polypeptide" refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide characterized by an Ig fold as a structural domain. An Fc polypeptide contains constant region sequences including at least a CH2 domain and / or a CH3 domain, and may also contain at least a portion of the hinge region, but does not contain any variable region.

[0197] As used herein, the term "protein" refers to either a polypeptide or a dimer (i.e., two) or multimer (i.e., three or more) of single polypeptide chains. The single polypeptide chains of a protein can be linked by covalent bonds, e.g., disulfide bonds, or by non-covalent interactions.

[0198] As used herein, the term "identical" or percent "identity" in the context of two or more polypeptide sequences refers to two or more sequences or subsequences that are identical over a particular region and have the same or a specified percentage of amino acid residues, e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more, 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.

[0199] For polypeptide sequence comparison, typically, one amino acid sequence serves as a reference sequence to which candidate sequences are compared.Alignment can be performed by various methods available to those skilled in the art, such as visual alignment, or by using publicly available software with known algorithms to achieve maximum alignment.Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR).The parameters used for alignment to achieve maximum alignment can be determined by those skilled in the art.For the purpose of polypeptide sequence comparison for the purpose of this application, the BLASTP algorithm, standard protein BLAST, is used to align two protein sequences with default parameters.

[0200] As used herein, the term "binding affinity" refers to the strength of the non-covalent interaction between two molecules, e.g., between a Fab or scFv and an antigen, or between a polypeptide described herein (or target-binding portion thereof) and a target. Thus, for example, the term can refer to a 1:1 interaction between a Fab or scFv and an antigen, or between a polypeptide described herein (or target-binding portion thereof) and a target, unless otherwise indicated or clear from the context. Binding affinity is measured by the equilibrium dissociation constant (K D ) and the equilibrium dissociation constant (K D ) is the dissociation rate constant (k d ,time -1 ) to the association rate constant (k a ,time -1 M -1 ) is divided by K. Dcan be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods, e.g., the Biacore™ system, kinetic exclusion assays such as KinExA®, and BioLayer interferometry (e.g., using the ForteBio® Octet platform). As used herein, "binding affinity" refers not only to formal binding affinity, such as that reflecting a 1:1 interaction between a Fab or scFv and an antigen, or between a polypeptide described herein (or target-binding portion thereof) and a target, but also to K values ​​that can reflect avid binding. D It also includes the apparent affinity, where is calculated.

[0201] As used herein, the term "specifically binds" refers to a molecule (e.g., a Fab, scFv, or polypeptide described herein (or target-binding portion thereof) that binds to an epitope or target in a sample with greater affinity, greater avidity, and / or longer duration than it binds to another epitope or non-target compound (e.g., a structurally different antigen). In some embodiments, a Fab, scFv, or polypeptide described herein that specifically binds to an epitope or target (or target-binding portion thereof) is a Fab, scFv, or polypeptide (or target-binding portion thereof) described herein that binds to an epitope or target with at least 5-fold greater affinity than other epitopes or non-target compounds, e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 100-fold, 1000-fold, 10,000-fold, or greater affinity. The terms "specific binding," "specifically binds," or "specific for" a particular epitope or target, as used herein, refer to, for example, the equilibrium dissociation constant K for the epitope or target to which it binds. D A molecule having, for example, 10 -4 M or less, e.g., 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10-10 M, 10 -11 M, or 10 -12 M. It will be recognized by those skilled in the art that a Fab or scFv that specifically binds to a target from one species may also specifically bind to an orthologue of that target.

[0202] As used herein, the terms "subject," "individual," and "patient" are used interchangeably to refer to mammals, including, but not limited to, humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), and other mammalian species. In one embodiment, the patient is a human.

[0203] As used herein, the terms "treatment," "treating," and the like generally refer to obtaining a desired pharmacological and / or physiological effect. "Treating" or "treatment" can refer to any indication of success in treating or ameliorating a neurodegenerative disease (e.g., Alzheimer's disease or another neurodegenerative disease described herein), including any objective or subjective parameter, such as relief, alleviation, improved patient survival, increased survival time or rate, alleviation of symptoms or making the disease more tolerable for the patient, slowing the rate of deterioration or decline, or improving the patient's physical and mental health. The treatment or amelioration of symptoms can be based on objective or subjective parameters. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient at different time points before or during treatment.

[0204] As used herein, the term "pharmaceutically acceptable excipient" refers to inactive pharmaceutical ingredients, such as, but not limited to, buffers, carriers, or preservatives, that are biologically or pharmacologically compatible for use in humans or animals.

[0205] As used herein, the term "therapeutic agent" refers to any molecule, drug, or agent used in the treatment and / or prevention of disease. A therapeutic agent can be a small organic molecule or compound, a polypeptide, a protein, a nucleic acid, and / or a combination of any of the above. In some embodiments, a therapeutic agent can be a known molecule, drug, or agent. In some embodiments, a therapeutic agent is a polypeptide containing an antigen-binding domain, e.g., an antibody variable domain polypeptide having one or more complementarity-determining regions (CDRs), or an antigen-binding fragment thereof. In certain embodiments, a therapeutic agent can be a Fab (e.g., a Fab that binds to a target other than TfR or CD98hc). In some embodiments, a therapeutic agent can bind to a target (e.g., a biological target, a therapeutic target, a target other than TfR or CD98hc) to treat and / or prevent the disease, depending on the disease being treated. Such targets can include cell surface targets in the brain, e.g., cell surface targets on microglial cells, astrocytes, oligodendrocytes, neurons, and cancer cells. For example, such targets include TREM2, PILRA, CD33, CR1, ABCA1, ABCA7, MS4A4A, MS4A6A, MS4A4E, HLA-DR5, HLA-DR1, IL1RAP, TREML2, IL-34, SORL1, ADAM17, and Siglec11. In some embodiments, the target may comprise alpha-synuclein or a derivative or fragment thereof, a derivative of amyloid-beta peptide or a fragment thereof, Tau or a derivative or fragment thereof, pTau, huntingtin, transthyretin, or TAR DNA-binding protein 43 (TDP-43) or a derivative or fragment thereof.In some embodiments, the target is on a tumor cell and is selected from the group consisting of ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR(1-4), folate receptor alpha, GAL-3BP (galectin-binding protein), GD2, GD3, GloboH (globohexacylceramide), gp100, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine-rich repeat-containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4, colon cancer kinase), RON, ROR1, TF (tissue factor), and TROP2. In some embodiments, the cell is a hematological cancer cell and the cell surface receptor is selected from the group consisting of B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b. Known therapeutic agents for the treatment of cancer include, for example, lorlatinib, crizotinib, cabozantinib, basiliximab, daclizumab, bivatuzumab, promiximab, lorvotuzumab, polatuzumab, tusamitamab, sunitinib, cetuximab, panitumumab, nimotuzumab, necitumumab, rindopepimto (CDX-110), amivantamab, pemigatinib, erdafitinib, STRO-002, bevacizumab, naxitamab, and ipilimumab. , tebentafusp, glenbatumumab, margetuximab-cmkb, enhertu, trastuzumab, pertuzumab, patritumab, seribantumab, lumletuzumab, elgemtumab, U3-1402, AV-203, KTN3379, AVE1642, MK-0646, cixutumumab, radilatuzumab, gemtuzumab, pembrolizumab, sacituzumab, samlotamab, amivantamab-vmjw, TEPMETKO, rifastuzumab,. 177These include lutetium-PSMA-617, cofetuzumab, Zt / g4-MMAE, VLS-101, brexcabutadine, CS5001, tisotumab, sacituzumab, teclistamab, atezolizumab, avelumab, cosibelimab, durvalumab, belantamab, benralizumab, tafasitamab, roncastoximab, obinutuzumab, ofatumumab, rituximab, MEN1309 / OBT076, inotuzumab, and brentuximab.

[0206] Additional known targets in the brain, as well as agents that bind to such targets, are described in the following references, which are incorporated herein by reference: WO2016 / 023019, WO2017 / 062672, WO2018 / 195506, WO2019 / 118513, WO2019 / 023292, WO2019 / 079529, WO2019 / 180224, US2019 / 0040130 ... 019 / 0174730, WO2020 / 069050, US2017 / 0137518, US2012 / 0258110, WO2019 / 126472, US8,691,227, WO2019 / 152715, US2007 / 026425, WO2019 / 028283, US2018 / 016066, US9,079,958, WO2020 / 069050, WO2022 / 258841, J Immunol 2000 165:1197-1209;Translational Neurodegeneration,11,18(2022).

[0207] As used herein, a "therapeutic amount" or "therapeutically effective amount" of an agent is an amount of an agent (e.g., any of the proteins described herein) that treats a disease in a subject.

[0208] As used herein, the term "administering" refers to a method of delivering an agent, compound, or composition to a desired site of biological action. These methods include, but are not limited to, topical, oral, parenteral, intravenous, intradermal, intramuscular, intrathecal, intracolonic, intrarectal, or intraperitoneal delivery. In one embodiment, the proteins described herein are administered intravenously.

[0209] III. Polypeptide Engineering We have developed "limited liability" design techniques for polypeptide libraries and libraries of polypeptides containing substantial beta-sheet content, particularly in immunoglobulin molecules, which are described in detail in the following sections. We also describe engineering methods that can be used with these libraries and library design techniques to generate polypeptides with non-natural binding sites, including, for example, sites that bind to CD98hc or TfR.

[0210] Limited Liability Library When used to screen potential targets, we observed that large (9 or more positions) combinatorial libraries of polypeptides produce a significant number of polypeptides that bind nonspecifically (e.g., via hydrophobic interactions) or have burdens that make them difficult to manipulate (e.g., low expression, excessive hydrophobicity, low stability). To reduce, but not eliminate, the occurrence of amino acid residues associated with these properties in the library, we employed what we call the "limited liability" technique. This technique involves reducing the frequency with which certain amino acids (e.g., Cys, Trp, Met, Arg, and Gly) appear in the library without completely eliminating their presence, while maintaining variability at these restricted positions, allowing, for example, at least 8, 10, 12, 14, 15, or 16 amino acids at these positions. Specifically, this involves reducing the occurrence of at least one of these amino acids in 10-60% (e.g., 20-60%, 30-60%, or 40-60%) of the randomized positions in the library, particularly while allowing some or even all of the other randomized positions to allow all 20 naturally occurring amino acids. In certain cases, positions with limited diversity alternate with positions allowing all 20 amino acids. This can avoid having excess amino acids in close proximity that may contribute to undesirable properties. In some cases, the alternation is positioned relative to the primary sequence of the polypeptide. If the structure of the protein is known (e.g., if the crystal structure has been solved), the placement of limited liability positions can be spaced relative to positions with greater or complete diversity in three-dimensional space. As described in the Examples below, this technique led to the discovery of the specific CD98hc-binding polypeptides described herein.

[0211] Limited liability libraries can be generated using any known technique for peptide library development. The libraries described in the Examples herein were generated from a polynucleotide library encoding a polypeptide of interest using degenerate codons, specifically the NNK codon (allowing all 20 amino acids) interspersed with limited liability codons such as NHK, which do not allow Arg, Cys, Trp, or Gly. The present invention also contemplates the use of other codons that offer the advantage of limited liability. Potential codons can be selected from any known codons that offer "limited liability," as described in Mena et al., Protein Eng Des Sel 18:559-61, 2005, as set forth below. Table II from Mena et al. is set forth below in Table 1A.

[0212] (Table 1A) Degenerate codons calculated by LibDesign at each position from most inclusive to least inclusive * TIFF2025503437000001.tif190165TIFF2025503437000002.tif158165

[0213] In addition to codon-based techniques for generating libraries based on degenerate codons, limited-response libraries can also be generated using trinucleotide mutagenesis techniques. These techniques involve high-throughput techniques that allow specific proportions of each trinucleotide base pair encoding a single amino acid to be added to the library to precisely control the ratio of amino acids at a given position. Technologies using such techniques are commercially available from companies such as Sloning BioTechnology GmbH (Germany) and Azenta Life Sciences (Chelmsford, Mass.). These polynucleotide libraries can be expressed to generate polypeptide libraries useful for screening against targets including TfR and CD98hc.

[0214] Beta Sheet Library Also described herein is a library that contains randomized amino acids in the beta-sheet secondary structure of polypeptide.Generally, these libraries use the exposed part of beta-sheet, and the randomized amino acids form a surface that can be used to create an antigen binding site.In addition to the beta-sheet surface, antigen binding site can also include residues from adjacent regions on polypeptide, such as the loop region connecting beta-strands, or other structural features of proteins that are adjacent in three-dimensional space.

[0215] The use of beta-sheet regions has certain advantages, including those described herein, including greater structural stability of the antigen-binding site (compared to loop regions or other less structured portions of the polypeptide) and, in certain contexts, the formation of distinct surface topologies (e.g., flat, elongated, concave) suitable for forming several protein-protein interactions.

[0216] Specific examples of beta-sheet libraries include those generated from the beta-sheet portions of immunoglobulin proteins. In some examples, beta-sheet libraries are generated within constant domains of immunoglobulins, such as the CH1, CH2, CH3, CH4, or CL domains. Other examples include beta-sheet portions of variable domains, which may include non-CDR portions of the variable region.

[0217] For the constant domain of the human IgG1 molecule, the positions shown in Table 1B are useful for generating beta-sheet libraries.

[0218] (Table 1B) Surface-accessible beta-sheet positions in the IgG1 heavy chain constant domain TIFF2025503437000003.tif85165

[0219] Based on these positions in the IgG1 heavy chain constant region, corresponding positions can be identified in different domains (e.g., variable regions and light chains), different subtypes (e.g., IgG2, IgG3, IgG4), different species (e.g., mouse, rat, cynomolgus monkey), and other Ig species (e.g., IgA, IgM, IgE). As an example, alignment of primary amino acid sequences from different domains to corresponding domains in the IgG1 heavy chain constant region can be used to determine analogous positions within additional domains that are useful for generating beta-sheet libraries. Alternatively, structural alignment of domains to one or more of the Ig domain structures within the IgG1 heavy chain constant region can be used to determine potential beta-sheet library positions within domains for which structural information exists or is predictable. Whether an identified residue is surface-exposed and can be included in the library, or buried in a protein-protein interface (e.g., CH3-CH3 interface, VH-VL interface), can similarly be determined using structural information about the particular domain, which can be found in databases such as the Protein Data Bank (Berman et al., Nucleic Acids Res, 28:235-242, 2000) or based on predictions such as the AlphaFold protein structure database (Jumper et al., Nature, 596:583-589, 2021).

[0220] Protein scaffolds for use in libraries The library design and diversification techniques described herein, as well as limited liability techniques for libraries containing beta-sheet secondary structure, can be used to generate libraries on any suitable polypeptide scaffold, which may include any polypeptide with beta-sheet secondary structure, including immunoglobulins, fibronectin type III domains, anticalins, kunitz domains, nanophytins, centrins, affimers, and lipocalins, as well as many other proteins with this canonical beta-sheet structure.

[0221] Generation of binding proteins from polypeptide libraries As described below, in some cases, a beta-sheet polypeptide library was used in which a limited library concept could be used to discover polypeptides engineered to bind to target proteins, specifically CD98hc or TfR.

[0222] Typically, a polypeptide library is expressed (e.g., on a cell surface) and tested for binding to a target protein. This can be done by any suitable method, and various aspects of screening techniques are described in Kariolis et al., Sci Transl Med 12(545):eaay1359, 2020. In one technique, the polypeptide library is expressed as a surface-display library (e.g., phage display or yeast display), incubated with the target protein, which may be conjugated to magnetic beads (MACS) or fluorescently labeled to facilitate library selection using fluorescence-activated cell sorting (FACS). After incubation with the target antigen, binders are separated from non-binders, and the process is repeated to enrich the library for desired polypeptide clones that interact with the target antigen.

[0223] Following identification of initial binders from a polypeptide library, they can be further engineered to improve various biochemical and biophysical properties. Some of these improvements can include, but are not limited to, stronger antigen binding, specificity (e.g., binding to cynomolgus monkey and human forms of the antigen), or increased structural (e.g., thermal) stability. To achieve this, maturation libraries (e.g., as described herein) can be designed and screened to isolate variants with the desired improved properties. Approaches for designing these libraries can include extending the epitope by mutating amino acid positions adjacent to the original library position, randomizing the sequence of the initial binder using techniques biased toward retaining portions of the original sequence, or using error-prone PCR to randomly incorporate mutations throughout the domain to explore additional sequence space both within and proximal to the binding epitope. These libraries are then screened using the methods described above to isolate clones with the desired properties.

[0224] IV. CD98 Heavy Chain Binding Polypeptides This section describes the production of polypeptides according to the present disclosure that bind to the CD98hc protein (i.e., polypeptides having a CD98hc binding site), and these polypeptides are capable of being transported across the blood-brain barrier (BBB).

[0225] The polypeptides provided herein can comprise a modified CH3 domain that specifically binds to a CD98hc protein. As described herein, when describing a polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain comprising amino acids 111-217 of a particular SEQ ID NO(s), or a modified CH3 domain comprising amino acid substitutions or deletions relative to amino acids 111-217 of a particular SEQ ID NO(s), or a sequence having percent identity to amino acids 111-217 of a particular SEQ ID NO(s), such description is directed to the sequence of the modified CH3 domain and should not be construed as limiting the polypeptide to containing amino acids 1-110 of the listed SEQ ID NO(s).

[0226] Those skilled in the art will understand that the CH3 domains of other immunoglobulin isotypes, such as IgM, IgA, IgE, IgD, etc., can be similarly modified by identifying amino acids within those domains that correspond to the amino acid substitutions at the positions described herein. Modifications can also be made to corresponding domains from immunoglobulins from other species, such as non-human primates, monkeys, mice, rats, or other non-human mammals.

[0227] CD98hc binding site modification In one embodiment, provided herein is a polypeptide comprising a modified constant domain (e.g., a modified CH3 domain) that specifically binds to a CD98hc protein, wherein the modified constant domain comprises at least 5, 6, 7, 8, or 9 substitutions at the set of amino acid positions consisting of 382, ​​384, 385, 387, 422, 424, 426, 438, 440, wherein the substitutions are determined with reference to SEQ ID NO: 1 and the positions are determined with reference to EU numbering.

[0228] In some embodiments, the polypeptide that binds to CD98hc is from the LLB2 family. In some embodiments, the polypeptide comprises at least 11, 12, 13, 14, or 15 substitutions at the set of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 391, 421, 422, 424, 426, 428, 434, 436, 438, 440, 441, and 442. In some embodiments, the substitutions are S, V, D, E, or Y at position 378; L, I, M, A, Q, V, or K at position 380; N, S, L, M, P, Y, K, A, or T at position 382; T, F, N, P, D, L, H, or Q at position 383; K, R, H, I, L, F, Y, V, or Q at position 384; F or Y at position 385; V, L, A, I, F, Y, S, T, H, R, or E at position 386; L or I at position 387; D, Q, A, T, H at position 389; is selected from V, T, V, or A at position 391, E, Q, or A at position 421, L, M, I, T, or P at position 422, A at position 424, N at position 426, L, T, P, Y, F, I, A, K, H, or W at position 428, S at position 434, L, V, H, F, P, R, or W at position 436, F or W at position 438, L, P, E, N, V, A, I, or D at position 440, P at position 441, and A, V, M, Q, F, P, L, Y, K, R, H, or M at position 442.

[0229] In some embodiments, the polypeptide that binds to CD98hc is from the LLB1 family. In some embodiments, the polypeptide comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitutions at the set of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 434, 436, 438, 440, and 442. In some embodiments, the substitutions are S or V at position 378, D, M, N, P, F, or H at position 380, R, Y, F, S, W, Y, K, or N at position 382, ​​T at position 383, L, Y, A, S, or F at position 384, F, K, D, M, I, N, Y, L, or H at position 385, T, P, E, K, A, V, D, T, or F at position 386, N, L, Y, R, F, G, S, D, or T at position 387, selected from T, Y, or F at position 9, D, E, or Q at position 421, I, K, L, R, T, F, or H at position 422, V, W, G, L, I, P, or Y at position 424, D, A, Q, W, L, or P at position 426, L or Y at position 428, S at position 434, F at position (436), I, V, F, N, P, or S at position 438, and K, T, P, I, or F at position 440, and Q or M at position 442.

[0230] In one embodiment, the modified polypeptide comprising a modified constant domain (e.g., a modified CH3 domain) comprises a sequence having at least 80%, 85%, 90%, or 95% sequence identity to amino acids 111-217 of any one of SEQ ID NOs: 28-45.

[0231] V. Transferrin Receptor Binding Polypeptides This section describes the production of polypeptides according to the present disclosure that bind to the transferrin receptor (TfR) (i.e., polypeptides having a TfR binding site), and these polypeptides are capable of being transported across the blood-brain barrier (BBB).

[0232] The polypeptides provided herein can comprise a modified CH3 domain that specifically binds to TfR. As described herein, when describing a polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain comprising amino acids 111-217 of a particular SEQ ID NO(s), or a modified CH3 domain comprising amino acid substitutions or deletions relative to amino acids 111-217 of a particular SEQ ID NO(s), and / or a sequence having percent identity to amino acids 111-217 of a particular SEQ ID NO(s), such description is directed to the sequence of the CH3 domain and should not be construed as limiting the polypeptide to containing amino acids 1-113 of the listed SEQ ID NO(s).

[0233] Those skilled in the art will understand that the CH3 domains of other immunoglobulin isotypes, such as IgM, IgA, IgE, IgD, etc., can be similarly modified by identifying amino acids within those domains that correspond to the amino acid substitutions at the positions described herein. Modifications can also be made to corresponding domains from immunoglobulins from other species, such as non-human primates, monkeys, mice, rats, or other non-human mammals.

[0234] TfR binding site modification In one embodiment, provided herein is a polypeptide comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), wherein the modified CH3 domain comprises five, six, or seven amino acid substitutions at a set of amino acid positions including 422, 424, 426, 433, 434, 438, and 440. The modified CH3 domain does not have the combination of G at position 437, F at position 438, and D at position 440, wherein the positions are determined according to EU numbering.

[0235] Also provided herein are polypeptides comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), wherein the modified CH3 domain comprises 3, 4, 5, 6, 7, or 8 amino acid substitutions and / or 1 or 2 amino acid deletions at the set of amino acid positions including 380 and 382-389, and 5, 6, or 7 amino acid substitutions at the set of amino acid positions including 422, 424, 426, 433, 434, 438, and 440, wherein the positions are determined according to EU numbering.

[0236] Also provided herein are polypeptides comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), wherein the modified CH3 domain comprises a sequence comprising at least one amino acid substitution in the sequence of VFSCSVMHEALHNHYTQKS (SEQ ID NO:57), where the sequence of SEQ ID NO:57 is between positions 422 and 440 of an Fc polypeptide (e.g., SEQ ID NO:1), and the sequence does not have the combination of G at position 437, F at position 438, and D at position 440, wherein the positions are determined according to EU numbering. In some embodiments, the modified CH3 domain comprises a sequence comprising 5, 6, or 7 amino acid substitutions at the set of amino acid positions including 422, 424, 426, 433, 434, 438, and 440.

[0237] Also provided herein are polypeptides comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), wherein the modified CH3 domain comprises a first sequence comprising at least one amino acid substitution and / or deletion in the sequence AVEWESNGQPENN (SEQ ID NO:56) and a second amino acid substitution comprising at least one amino acid substitution in the sequence VFSCSVMHEALHNHYTQKS (SEQ ID NO:57), wherein the sequence of SEQ ID NO:56 is at positions 378-390 of an Fc polypeptide (e.g., SEQ ID NO:1) and the sequence of SEQ ID NO:57 is at positions 422-440 of an Fc polypeptide (e.g., SEQ ID NO:1), the positions being determined according to EU numbering. In some embodiments, the modified CH3 domain comprises 3, 4, 5, 6, 7, or 8 amino acid substitutions at the set of amino acid positions including 380 and 382-389. In certain embodiments, the modified CH3 domain comprises 5, 6, or 7 amino acid substitutions at a set of amino acid positions including 422, 424, 426, 433, 434, 438, and 440.

[0238] Modifications at positions 380 and 382-389 Provided herein are polypeptides comprising a modified CH3 domain having at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 (e.g., 3, 4, 5, 6, 7, or 8)) amino acid substitution and / or at least one (e.g., 1 or 2) amino acid deletion at a set of amino acid positions including 380 and 382 to 389 according to EU numbering. The modified CH3 domain can comprise a sequence comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 (e.g., 3, 4, 5, 6, 7, or 8)) amino acid substitution and / or at least one (e.g., 1 or 2) amino acid deletion in the sequence AVEWESNGQPENN (SEQ ID NO: 56) at positions 378 to 390 of an Fc polypeptide (e.g., SEQ ID NO: 1). In some embodiments, the modified CH3 domain can include a sequence that includes at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 (e.g., 3, 4, 5, 6, 7, or 8) amino acid substitution and / or at least one (e.g., 1 or 2) amino acid deletion at a set of amino acid positions including 380 and 382-389 relative to the sequence of SEQ ID NO: 56, where the positions are numbered according to EU numbering.

[0239] In some embodiments, the modified CH3 domain in the polypeptide comprises an F at position 382. In certain embodiments, the modified CH3 domain comprises an A or a polar amino acid at position 383. In certain embodiments, the modified CH3 domain comprises an A at position 383. In certain embodiments, the modified CH3 domain comprises a polar amino acid (e.g., Y, S, N, Q, T, H, K, D, E, or W (e.g., Y or S)) at position 383. In certain embodiments, the modified CH3 domain comprises a Y or S at position 383. In some embodiments, the modified CH3 domain comprises a G, N, or an acidic amino acid at position 384. In some embodiments, the modified CH3 domain comprises a G or N at position 384. In some embodiments, the modified CH3 domain comprises an acidic amino acid (e.g., D or E) at position 384. In some embodiments, the modified CH3 domain comprises an N, R, or a polar amino acid at position 389. In some embodiments, the modified CH3 domain comprises an N or R at position 389. In some embodiments, the modified CH3 domain comprises a polar amino acid (e.g., Y, S, N, Q, T, H, K, D, E, or W (e.g., S or T)) at position 389. In some embodiments, the modified CH3 domain comprises an S or T at position 389.

[0240] In certain embodiments, at least one of the amino acid substitutions in the set of amino acid positions including 380 and 382-389 is at a beta-sheet position relative to the sequence of SEQ ID NO: 56. In some embodiments, the modified CH3 domain comprises one, two, or three amino acid substitutions at beta-sheet positions relative to the sequence of SEQ ID NO: 56. In certain embodiments, the beta-sheet position(s) is / are selected from the group consisting of positions 380, 382, ​​and 383, according to EU numbering. In certain embodiments, the modified CH3 domain comprises an amino acid substitution at position 380 relative to the sequence of SEQ ID NO: 56, e.g., E, N, F, or Y. In certain embodiments, the amino acid substitution at position 380 is E. In certain embodiments, the modified CH3 domain comprises an amino acid substitution (e.g., F) at position 382 relative to the sequence of SEQ ID NO: 56. In certain embodiments, the modified CH3 domain comprises an amino acid substitution at position 383 relative to the sequence of SEQ ID NO: 56, e.g., Y or A. In certain embodiments, the amino acid substitution at position 383 is Y.

[0241] In some embodiments of the polypeptides described herein, the polypeptide can comprise a modified CH3 domain comprising at least one position selected from the following: E, N, F, or Y at position 380; F at position 382; Y, S, A, or an amino acid deletion at position 383; G, D, E, or N at position 384; D, G, N, or A at position 385; Q, S, G, A, or N at position 386; K, I, R, or G at position 387; E, L, D, or Q at position 388; and N, T, S, or R at position 389, wherein the positions are numbered according to EU numbering. In certain embodiments, the modified CH3 domain can comprise 5, 6, 7, or 8 positions selected from the following: F at position 382, ​​Y or S at position 383, G, D, or E at position 384, D, G, N, or A at position 385, Q, S, or A at position 386, K at position 387, E or L at position 388, and N, T, or S at position 389. In certain embodiments, the modified CH3 domain can comprise the following five positions: F at position 382, ​​E at position 384, S at position 386, K at position 387, and T at position 389. In certain embodiments, the modified CH3 domain can comprise the following five positions: F at position 382, ​​G at position 384, A at position 385, K at position 387, and S at position 389. In certain embodiments, the modified CH3 domain can comprise the following six positions: F at position 382, ​​G at position 384, A at position 385, K at position 387, L at position 388, and T at position 389. In certain embodiments, the modified CH3 domain can comprise the following six positions: F at position 382, ​​Y at position 383, E at position 384, A at position 385, K at position 387, and L at position 388. In certain embodiments, the modified CH3 domain can comprise the following seven positions: F at position 382, ​​Y at position 383, G at position 384, N at position 385, A at position 386, K at position 387, and T at position 389. In certain embodiments, the modified CH3 domain can include the following eight positions: F at position 382, ​​Y at position 383, D at position 384, D at position 385, S at position 386, K at position 387, L at position 388, and T at position 389.

[0242] Modifications at positions 422, 424, 426, 433, 434, 438, and / or 440 Provided herein are polypeptides comprising a modified CH3 domain having at least one (e.g., 1, 2, 3, 4, 5, 6, or 7 (e.g., 5, 6, or 7)) amino acid substitution(s) at a set of amino acid positions including, according to EU numbering, 422, 424, 426, 433, 434, 438, and 440. The modified CH3 domain can comprise a sequence comprising at least one (e.g., 1, 2, 3, 4, 5, 6, or 7 (e.g., 5, 6, or 7)) amino acid substitution(s) in the sequence of VFSCSVMHEALHNHYTQKS (SEQ ID NO: 57) at positions 422 to 440 of an Fc polypeptide (e.g., SEQ ID NO: 1). In some embodiments, the modified CH3 domain can comprise a sequence that includes at least one (e.g., 1, 2, 3, 4, 5, 6, or 7 (e.g., 5, 6, or 7) amino acid substitution at a set of amino acid positions including 422, 424, 426, 433, 434, 438, and 440 relative to the sequence of SEQ ID NO: 57, where the positions are numbered according to EU numbering. The modified CH3 domain does not have the combination of G at position 437, F at position 438, and D at position 440, where the positions are determined according to EU numbering.

[0243] In some embodiments of a modified CH3 domain in a polypeptide, at least one of the amino acid substitutions at a set of amino acid positions including 422, 424, 426, 433, 434, 438, and 440 is at a beta-sheet position relative to the sequence of SEQ ID NO: 57. In some embodiments, the modified CH3 domain comprises one, two, three, or four amino acid substitutions at beta-sheet positions relative to the sequence of SEQ ID NO: 57. In certain embodiments, the beta-sheet position(s) is selected from the group consisting of positions 424, 426, 438, and 440, according to EU numbering. In certain embodiments, the modified CH3 domain comprises an amino acid substitution at beta-sheet position 424 relative to the sequence of SEQ ID NO: 57. The amino acid substitution at beta-sheet position 424 in the modified CH3 domain can be A. In certain embodiments, the modified CH3 domain comprises an amino acid substitution at beta-sheet position 426 relative to the sequence of SEQ ID NO: 57. The amino acid substitution at beta-sheet position 426 can be E. In certain embodiments, the modified CH3 domain comprises an amino acid substitution at beta sheet position 438 relative to the sequence of SEQ ID NO: 57. The amino acid substitution at beta sheet position 438 may be Y. In some embodiments, the modified CH3 domain comprises an amino acid substitution at beta sheet position 440 relative to the sequence of SEQ ID NO: 57. The amino acid substitution at beta sheet position 440 may be L.

[0244] In some embodiments of a modified CH3 domain in a polypeptide, the modified CH3 domain comprises an H or E (e.g., H) at position 433. In some embodiments, the modified CH3 domain comprises an N or G (e.g., N) at position 434.

[0245] In some embodiments of the polypeptides described herein, the polypeptide can comprise a modified CH3 domain comprising at least one position selected from the following: L at position 422, A at position 424, E at position 426, H or E at position 433, N or G at position 434, Y at position 438, and L at position 440. In particular, the modified CH3 domain can comprise five positions selected from the following: L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440.

[0246] TfR-binding polypeptides The present disclosure provides polypeptides comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), the modified CH3 domain comprising: (i) the sequence AVX1WFX2X3X4X5X6X7X8N (SEQ ID NO: 65), wherein X1 is E, N, F, or Y; X2 is Y, S, A, or absent; X3 is G, D, E, or N; X4 is D, G, N, or A; X5 is Q, S, G, A, or N; X6 is K, I, R, or G; X7 is E, L, D, or Q; and X8 is N, T, S, or R; and (ii) the sequence LFACEVMHEALX1X2HYTYKL (SEQ ID NO: 67), wherein X1 is H or E; and X2 is N or G. The present disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to the transferrin receptor (TfR), wherein the modified CH3 domain comprises: (i) the sequence AVEWFX1X2X3X4KX5X6N (SEQ ID NO: 66), wherein Xi is Y or S, X2 is G, D, or E, X3 is D, G, N, or A, X4 is Q, S, or A, X5 is E or L, and X6 is N, T, or S; and (ii) the sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64).

[0247] In some embodiments, the modified CH3 domain comprises the sequence AVEWFYDDSKLTN (SEQ ID NO: 58), AVEWFYGNAKETN (SEQ ID NO: 59), AVEWFYEAQKLNN (SEQ ID NO: 60), AVEWFSEGSKETN (SEQ ID NO: 61), AVEWFSGAQKESN (SEQ ID NO: 62), or AVEWFSGAQKLTN (SEQ ID NO: 63). In some embodiments, the modified CH3 domain comprises the sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64).

[0248] A modified CH3 domain in a polypeptide described herein can comprise the sequence AVEWFYDDSKLTN (SEQ ID NO: 58) and the sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64). A modified CH3 domain in a polypeptide described herein can comprise the sequence AVEWFYGNAKETN (SEQ ID NO: 59) and the sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64). A modified CH3 domain in a polypeptide described herein can comprise the sequence AVEWFYEAQKLNN (SEQ ID NO: 60) and the sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64). A modified CH3 domain in a polypeptide described herein can comprise the sequence AVEWFSEGSKETN (SEQ ID NO: 61) and the sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64). A modified CH3 domain in a polypeptide described herein can comprise the sequence AVEWFSGAQKESN (SEQ ID NO: 62) and the sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64). The modified CH3 domain in the polypeptides described herein can include the sequence AVEWFSGAQKLTN (SEQ ID NO: 63) and the sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64).

[0249] In some embodiments of the polypeptide, the modified CH3 domain further comprises one, two, three, four, or five amino acid substitutions at positions 419-421, inclusive, 442, and 443, wherein the positions are determined according to EU numbering. In particular embodiments, the modified CH3 domain comprises a Q or P at position 419, a G or R at position 420, an N or G at position 421, an S or G at position 442, and / or an L or E at position 443. In certain embodiments, the modified CH3 domain comprises a P at position 419, an R at position 420, a G at position 421, a G at position 442, and an E at position 443.

[0250] This disclosure relates to APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVX1WFX2X3X4X5X6X7X8NYKTTPPVLDSDGSFFLYSKLTVDKSRWQX9X 10 X 11 LFACEVMHEALX 12 X 13 HYTYKLLX 14 X 15 1. A polypeptide comprising the sequence of SPGK (SEQ ID NO: 68), wherein X1 is E, N, F, or Y, X2 is Y, S, A, or absent, X3 is G, D, E, or N, X4 is D, G, N, or A, X5 is Q, S, G, A, or N, X6 is K, I, R, or G, X7 is E, L, D, or Q, X8 is N, T, S, or R, X9 is Q or P, and X 10 is G or R, and X 11 is N or G and X 12 is H or E, and X 13 is N or G and X 14 is S or G, and X 15 is L or E.

[0251] In some embodiments, the present disclosure provides: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVX1WFX2X3X4X5X6X7X8NYKTTPPVLDSDGSFFLYSKLTVDKSRWQX9X 10 X 11 LFACEVMHEALHNHYTYKLLX 12 X 13 1. A polypeptide comprising the sequence of SPGK (SEQ ID NO: 69), wherein X1 is E, N, F, or Y, X2 is Y, S, A, or absent, X3 is G, D, E, or N, X4 is D, G, N, or A, X5 is Q, S, G, A, or N, X6 is K, I, R, or G, X7 is E, L, D, or Q, X8 is N, T, S, or R, X9 is Q or P, and X 10 is G or R, and X 11 is N or G and X 12 is S or G, and X 13 is L or E.

[0252] In some embodiments, the present disclosure provides: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVX1WFX2X3X4X5X6X7X8NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNLFACE X is E, N, F, or Y; X is Y, S, A, or absent; X is G, D, E, or N; X is D, G, N, or A; X is Q, S, G, A, or N; X is K, I, R, or G; X is E, L, D, or Q; and X is N, T, S, or R.

[0253] In some embodiments, the present disclosure provides: X1 is Y or S, X2 is G, D, or E, X3 is D, G, N, or A, X4 is Q, S, or A, X5 is E or L, and X6 is N, T, or S.

[0254] In some embodiments, the modified CH3 domain comprises a sequence having at least 85% identity, at least 90% identity, or at least 95% identity (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identity) to amino acids 111-217 of any one of SEQ ID NOs: 72-77. In some embodiments, the modified CH3 domain comprises a sequence having at least 85% identity, at least 90% identity, or at least 95% identity (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identity) to amino acids 111-217 of any one of SEQ ID NOs: 72-77, wherein amino acids at positions 380, 382-389, 422, 424, 426, 433, 434, 438, and / or 440 according to EU numbering are unchanged in each of SEQ ID NOs: 72-77. In certain embodiments, the modified CH3 domain comprises amino acids 111-217 of any one of SEQ ID NOs: 72-77.

[0255] In some embodiments, a polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein comprises a sequence having at least 85% identity, at least 90% identity, or at least 95% identity (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identity) to any one of SEQ ID NOs: 72-77. In some embodiments, a polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein comprises a sequence having at least 85% identity, at least 90% identity, or at least 95% identity (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identity) to any one of SEQ ID NOs: 72-77, wherein the amino acids at positions 380, 382-389, 422, 424, 426, 433, 434, 438, and / or 440 according to EU numbering are unchanged in each of SEQ ID NOs: 72-77. In certain embodiments, a polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein comprises the sequence of any one of SEQ ID NOs: 72-77.

[0256] A polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein can include F at position 382, ​​Y at position 383, D at position 384, D at position 385, S at position 386, K at position 387, L at position 388, T at position 389, P at position 419, R at position 420, G at position 421, L at position 422, A at position 424, E at position 426, Y at position 438, L at position 440, G at position 442, and E at position 443, wherein the positions are determined according to EU numbering.

[0257] A polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein can include F at position 382, ​​Y at position 383, G at position 384, N at position 385, A at position 386, K at position 387, T at position 389, L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440, where the positions are determined according to EU numbering.

[0258] A polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein can comprise an F at position 382, ​​a Y at position 383, an E at position 384, an A at position 385, a K at position 387, an L at position 388, an L at position 422, an A at position 424, an E at position 426, a Y at position 438, and an L at position 440, wherein the positions are determined according to EU numbering.

[0259] A polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein can include F at position 382, ​​E at position 384, S at position 386, K at position 387, T at position 389, L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440, where the positions are determined according to EU numbering.

[0260] A polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein can include F at position 382, ​​G at position 384, A at position 385, K at position 387, S at position 389, L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440, where the positions are determined according to EU numbering.

[0261] A polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein can include F at position 382, ​​G at position 384, A at position 385, K at position 387, L at position 388, T at position 389, L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440, where the positions are determined according to EU numbering.

[0262] In some embodiments, the above polypeptides can further comprise a W at position 366. In some embodiments, the above polypeptides can further comprise an S at position 366, an A at position 368, and a V at position 407. In certain embodiments, the above polypeptides can further comprise an A at position 234 and an A at position 235. In certain embodiments, the above polypeptides can further comprise a Gly or Ser at position 329. In some embodiments, the above polypeptides can further comprise an L at position 428 and an S at position 434. Positions are determined according to EU numbering.

[0263] VI. Additional Polypeptide Modifications Polypeptides (e.g., Fc polypeptides) comprising a modified CH3 domain provided herein can also include additional mutations, e.g., to effect knob and hole heterodimerization of the polypeptide, to modulate effector function, to extend serum half-life, to affect glycosylation, and / or to reduce immunogenicity in humans.

[0264] Polypeptide Modification for Heterodimerization In some embodiments, polypeptides (e.g., Fc polypeptides) comprising a modified CH3 domain described herein contain mutations that promote heterodimer formation and prevent homodimer formation. These modifications are useful, for example, when it is desired that only one of the polypeptides of a dimer has a CD98hc or TfR binding site (i.e., a monovalent CD98hc or TfR binder).

[0265] The knobs-into-holes approach generally involves introducing a protrusion ("knob") and a corresponding cavity ("hole") into the interface of a polypeptide (e.g., an Fc polypeptide) so that the protrusion can be positioned within the cavity to promote heterodimer formation and thus prevent homodimer formation. The protrusion is constructed by replacing small amino acid side chains from the interface of the first polypeptide (e.g., an Fc polypeptide) with larger side chains (e.g., Tyr or Trp). A compensatory cavity of identical or similar size to the protrusion is created in the interface of the second polypeptide (e.g., an Fc polypeptide) by replacing the large amino acid side chains with smaller ones (e.g., Ala or Thr). In some embodiments, such additional mutations are at locations within the polypeptide (e.g., an Fc polypeptide) that do not adversely affect binding of the polypeptide to CD98hc or TfR.

[0266] In one exemplary embodiment of the knob-and-hole technique for dimerization, position 366 of one of the polypeptides (e.g., an Fc polypeptide) contains a Trp instead of the native Thr. The other polypeptide in the dimer has a Val at position 407 instead of the native Tyr. The other polypeptide (e.g., an Fc polypeptide) may further contain a substitution in which the native Thr at position 366 is substituted with a Ser and the native Leu at position 368 is substituted with an Ala. Thus, one of the polypeptides (e.g., an Fc polypeptide) has a T366W knob mutation and the other polypeptide (e.g., an Fc polypeptide) has a Y407V hole mutation, which is typically accompanied by T366S and L368A hole mutations. As above, all positions are numbered per EU numbering.

[0267] In some embodiments, one or both polypeptides (e.g., Fc polypeptides) present in a polypeptide dimer (e.g., an Fc polypeptide dimer) can also be engineered to contain other modifications for heterodimerization, such as electrostatic manipulation of contact residues within the CH3-CH3 interface that are naturally charged or hydrophobic patch modifications.

[0268] The knob-into-hole approach (e.g., a T366W knob substitution on one polypeptide (e.g., an Fc polypeptide) and T366S, L368A, and Y407V hole substitutions on another polypeptide (e.g., an Fc polypeptide)) can be used with any of the polypeptides described herein (e.g., a CD98hc-binding polypeptide having the sequence of any one of SEQ ID NOs: 28-45, or a TfR-binding polypeptide having the sequence of any one of SEQ ID NOs: 72-77, or a TfR-binding polypeptide having the sequence of any one of the clones listed in Table 29).

[0269] In some embodiments, only one of the two polypeptides (e.g., Fc polypeptides) contains a CD98hc-binding site (e.g., a CD98hc-binding polypeptide having the sequence of any one of SEQ ID NOs: 28-45), while the other polypeptide (e.g., Fc polypeptide) does not contain a CD98hc-binding site. In specific embodiments, one of the polypeptides (e.g., Fc polypeptides) is a CD98hc-binding polypeptide and contains a knob mutation (e.g., T366W), while the other polypeptide (e.g., Fc polypeptide) does not bind to CD98hc and contains a hole mutation (e.g., T366S, L368A, and Y407V). In other embodiments, one of the polypeptides (e.g., Fc polypeptide) is a CD98hc-binding polypeptide and contains a hole mutation (e.g., T366S, L368A, and Y407V), while the other polypeptide (e.g., Fc polypeptide) does not bind to CD98hc and contains a knob mutation (e.g., T366W).

[0270] In some embodiments, only one of the two polypeptides (e.g., Fc polypeptides) contains a TfR-binding site (e.g., a TfR-binding polypeptide having the sequence of any one of SEQ ID NOs: 72-77 or the sequence of any one of the clones listed in Table 29), while the other polypeptide (e.g., Fc polypeptide) does not contain a TfR-binding site. In certain embodiments, one of the polypeptides (e.g., Fc polypeptides) is a TfR-binding polypeptide and contains a knob mutation (e.g., T366W), while the other polypeptide (e.g., Fc polypeptide) does not bind to TfR and contains a hole mutation (e.g., T366S, L368A, and Y407V). In other embodiments, one of the polypeptides (e.g., Fc polypeptide) is a TfR-binding polypeptide and contains hole mutations (e.g., T366S, L368A, and Y407V), while the other polypeptide (e.g., Fc polypeptide) does not bind to TfR and contains a knob mutation (e.g., T366W).

[0271] In certain embodiments, a polypeptide dimer (e.g., an Fc polypeptide dimer) that specifically binds to CD98hc can have a first polypeptide (e.g., an Fc polypeptide) having a T366W knob mutation and being at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to any one of SEQ ID NOs: 28-45, and a second polypeptide (e.g., an Fc polypeptide) having T366S, L368A, and Y407V hole mutations and being at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to SEQ ID NO: 1. In other embodiments, a polypeptide dimer (e.g., an Fc polypeptide dimer) that specifically binds to CD98hc can have a first polypeptide (e.g., an Fc polypeptide) having a T366W knob mutation and being at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to SEQ ID NO: 1, and a second polypeptide (e.g., an Fc polypeptide) having T366S, L368A, and Y407V hole mutations and being at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to any one of SEQ ID NOs: 28-45.In other embodiments, a polypeptide dimer (e.g., an Fc polypeptide dimer) that specifically binds to CD98hc has a T366W knob mutation and comprises a first polypeptide (e.g., an Fc polypeptide dimer) that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to SEQ ID NOs: 28-45. and a second polypeptide (e.g., an Fc polypeptide) having T366S, L368A, and Y407V hole mutations and being at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to any one of SEQ ID NOs: 28-45.

[0272] In certain embodiments, a polypeptide dimer (e.g., an Fc polypeptide dimer) that specifically binds to TfR has a T366W knob mutation and is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to any one of SEQ ID NOs: 72-77 or any one of the clones listed in Table 29. and a second polypeptide (e.g., an Fc polypeptide) that has T366S, L368A, and Y407V hole mutations and is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to SEQ ID NO: 1. In other embodiments, a polypeptide dimer (e.g., an Fc polypeptide dimer) that specifically binds to TfR comprises a first polypeptide (e.g., an Fc polypeptide) that has a T366W knob mutation and is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to SEQ ID NO:1, and a second polypeptide (e.g., an Fc polypeptide) that has a T366S, L368A knob mutation and is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to SEQ ID NO:1. , and a Y407V hole mutation, and a second polypeptide (e.g., an Fc polypeptide) that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to any one of SEQ ID NOs: 72-77 or any one of the clones listed in Table 29.In other embodiments, an Fc polypeptide dimer (e.g., an Fc polypeptide dimer) that specifically binds to TfR has a T366W knob mutation and comprises a first polypeptide (e.g., a polypeptide dimer) that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to any one of SEQ ID NOs: 72-77 or any one of the clones listed in Table 29. and a second polypeptide (e.g., an Fc polypeptide) having T366S, L368A, and Y407V hole mutations and that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to any one of SEQ ID NOs: 72-77 or the sequence of any one of the clones listed in Table 29.

[0273] Polypeptide modifications to modulate effector function In some embodiments, the polypeptide dimer described herein is an Fc polypeptide dimer comprising two Fc polypeptides. In some embodiments, both Fc polypeptides of the Fc polypeptide dimer can comprise a modification that reduces or eliminates effector function, i.e., a modification that reduces the ability to induce a specific biological function upon binding to an Fc receptor expressed on an effector cell that mediates effector function. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and cytotoxic T cells. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0274] Exemplary Fc polypeptide mutations that reduce effector function include, but are not limited to, substitutions in the CH2 domain, e.g., at positions 234 and 235 and / or at position 329, according to the EU numbering scheme. For example, in some embodiments, both Fc polypeptides comprise Ala residues at positions 234 and 235 (also referred to herein as "LALA"). In some embodiments, both Fc polypeptides comprise a Gly residue at position 329 (also referred to herein as "P329G" or "PG") or a Ser residue at position 329 (also referred to herein as "P329S" or "PS"). In some embodiments, both Fc polypeptides comprise an Ala residue at positions 234 and 235 and a Gly residue at position 329 (also referred to herein as "LALA PG"). In some embodiments, both Fc polypeptides comprise an Ala residue at positions 234 and 235 and a Ser residue at position 329 (also referred to herein as "LALA PS").

[0275] Additional Fc polypeptide mutations that modulate effector function include, but are not limited to, the following: position 329 can be mutated to substitute Pro with Gly, Ala, Ser, or Arg, or an amino acid residue large enough to disrupt the Fc / Fcγ receptor interface formed between proline 329 of Fc and Trp residues Trp87 and Trp110 of FcγRIII. Further exemplary substitutions include S228P, E233P, L235E, N297A, N297D, and P331S according to the EU numbering scheme. According to the EU numbering scheme, multiple substitutions may also be present, for example, L234A, L235A, and P329G in human IgG1, S228P and L235E in human IgG4, L234A and G237A in human IgG1, L234A, L235A and G237A in human IgG1, V234A and G237A in human IgG2, L235A, G237A and E318A in human IgG4, and S228P and L236E in human IgG4.

[0276] In some embodiments, a polypeptide (e.g., an Fc polypeptide) that specifically binds to CD98hc comprises a LALA substitution and a sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to any one of SEQ ID NOs: 28-45.

[0277] In some embodiments, a polypeptide (e.g., an Fc polypeptide) that specifically binds to CD98hc comprises a LALA and a P329G or P329S substitution and a sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to any one of SEQ ID NOs: 28-45.

[0278] In some embodiments, a polypeptide (e.g., an Fc polypeptide) that specifically binds to TfR comprises a LALA substitution and a sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to any one of SEQ ID NOs:72-77 or any one of the clones listed in Table 29.

[0279] In some embodiments, a polypeptide (e.g., an Fc polypeptide) that specifically binds to TfR comprises a LALA and a P329G or P329S substitution and a sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to any one of SEQ ID NOs: 72-77 or any one of the clones listed in Table 29.

[0280] Polypeptide modifications to extend serum half-life In some embodiments, modifications to enhance serum half-life can be introduced into any polypeptide described herein. For example, in some embodiments, a polypeptide dimer described herein is an Fc polypeptide dimer comprising two Fc polypeptides. In some embodiments, both Fc polypeptides in an Fc polypeptide dimer can contain M428L and N434S substitutions (also referred to as LS substitutions) numbered according to the EU numbering scheme. Alternatively, both Fc polypeptides in an Fc polypeptide dimer can have an N434S or N434A substitution. Alternatively, both Fc polypeptides in an Fc polypeptide dimer can have an M428L substitution. In other embodiments, both Fc polypeptides in an Fc polypeptide dimer can contain M252Y, S254T, and T256E substitutions.

[0281] In any of the embodiments described herein, a polypeptide (e.g., an Fc polypeptide) that specifically binds to CD98hc can further comprise an LS substitution. For example, in some embodiments, a polypeptide (e.g., an Fc polypeptide) that specifically binds to CD98hc comprises an LS substitution and a sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to any one of SEQ ID NOs: 28-45.

[0282] In any of the embodiments described herein, a polypeptide (e.g., an Fc polypeptide) that specifically binds to TfR can further comprise an LS substitution. For example, in some embodiments, a polypeptide (e.g., an Fc polypeptide) that specifically binds to TfR comprises an LS substitution and a sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to any one of SEQ ID NOs: 72-77 or any one of the clones listed in Table 29.

[0283] Polypeptides with C-terminal lysine residues removed In some embodiments, one or both of the polypeptides (e.g., Fc polypeptides) can have their C-terminal lysine removed (e.g., Lys residue at position 447 of the Fc polypeptide according to EU numbering). C-terminal lysine residues are highly conserved in immunoglobulins across many species and may be completely or partially removed by cellular machinery during protein production. In some embodiments, removal of the C-terminal lysine in the Fc polypeptide can improve protein stability.

[0284] VII. Exemplary Polypeptides that Bind CD98HC The modified CH3 domain of the present disclosure may be a naturally occurring CH2 domain or a variant CH2 domain, typically C-terminal to the CH2 domain, and can be attached to the CH2 domain to form a polypeptide (e.g., an Fc polypeptide) that binds to CD98hc. In some embodiments, the polypeptide (e.g., an Fc polypeptide) further comprises a partial or complete hinge region of an antibody attached to the N-terminus of the CH2 domain. The hinge region can be from any immunoglobulin subclass or isotype. An exemplary immunoglobulin hinge is an IgG1 hinge region, e.g., an IgG hinge region such as the human IgG1 hinge amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 4).

[0285] In certain embodiments, provided herein are CD98hc-binding polypeptides that, when bound to human CD98hc, bind to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 residues selected from the group consisting of positions 477, 478, 479, 480, 481, 482, 483, 486, 499, 497, 498, 500, 501, and 502 of SEQ ID NO: 134. In certain embodiments, when bound to human CD98hc, the antibody binds to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 residues selected from the group consisting of positions 477, 478, 479, 480, 481, 482, 483, 486, 499, 497, 498, 500, 501, and 502 of SEQ ID NO: 134, and optionally positions 229, 231, 232, 236, 235, 488, 495, 506, 508, 509, 510, 511, 512, 513, or 514 of SEQ ID NO: 134. and 496, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 additional residues selected from the group consisting of positions 312, 315, 348, 381, 439, 444, 443, 485, 484, 476, 475, and 442 of SEQ ID NO: 134. In some embodiments, provided herein are CD98hc binding polypeptides, wherein the polypeptide binds to positions 477, 478, 479, 480, 481, 482, 483, 486, 499, 497, 498, 500, 501, and 502 of SEQ ID NO: 134 when bound to human CD98hc. In certain embodiments, provided herein are CD98hc-binding polypeptides that, when bound to human CD98hc, bind to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 residues selected from the group consisting of positions 229, 231, 232, 236, 235, 486, 488, 495, 496, 498, 500, 499, 497, 482, 481, 483, 477, 480, 501, 502, 478, and 479 of SEQ ID NO: 134.In some embodiments, provided herein are CD98hc-binding polypeptides that, when bound to human CD98hc, bind to residues 229, 231, 232, 236, 235, 486, 488, 495, 496, 498, 500, 499, 497, 482, 481, 483, 477, 480, 501, 502, 478, and 479 of SEQ ID NO: 134. In certain embodiments, provided herein are CD98hc-binding polypeptides that, when bound to human CD98hc, bind to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 residues selected from the group consisting of positions 312, 315, 348, 381, 439, 444, 443, 485, 484, 477, 483, 481, 480, 478, 476, 502, 499, 501, 500, 498, 497, 486, 479, 482, 475, and 442 of SEQ ID NO: 134. In some embodiments, provided herein are CD98hc-binding polypeptides that, when bound to human CD98hc, bind to residues 312, 315, 348, 381, 439, 444, 443, 485, 484, 477, 483, 481, 480, 478, 476, 502, 499, 501, 500, 498, 497, 486, 479, 482, 475, and 442 of SEQ ID NO: 134.

[0286] In some embodiments, a polypeptide (e.g., an Fc polypeptide) may comprise a sequence from Table 2A, and the polypeptide (e.g., an Fc polypeptide) may be further modified to contain a CD98hc binding site within a modified CH3 domain described herein.

[0287] Table 2A: Fc sequences for further CD98hc or TfR binding site modifications TIFF2025503437000004.tif79165

[0288] In further embodiments, a polypeptide (e.g., an Fc polypeptide) described herein can be further conjugated to another moiety, e.g., a Fab fragment, thereby generating a CD98hc-binding Fc-Fab fusion. In some embodiments, the CD98hc-binding Fc-Fab fusion comprises a modified CH3 domain, a CH2 domain, a hinge region, and a Fab fragment. The Fab fragment can be directed to any target of interest, e.g., a therapeutic neurological target, where the Fab can be delivered to the target by transcytosis across the BBB mediated by binding of the modified CH3 domain polypeptide to CD98hc.

[0289] A CD98hc-binding polypeptide (e.g., a CD98hc-binding Fc polypeptide) may also be fused to a polypeptide of interest other than a Fab. For example, in some embodiments, a CD98hc-binding polypeptide (e.g., a CD98hc-binding Fc polypeptide) may be fused to a polypeptide that is desired to target CD98hc-expressing cells or deliver across the endothelium, e.g., the BBB, by transcytosis. In some embodiments, a CD98hc-binding polypeptide (e.g., a CD98hc-binding Fc polypeptide) is fused to a soluble protein. In yet other embodiments, a CD98hc-binding polypeptide (e.g., a CD98hc-binding Fc polypeptide) may be fused to a peptide or protein useful for protein purification, e.g., polyhistidine, an epitope tag, such as FLAG, c-Myc, a hemagglutinin tag, glutathione S-transferase (GST), thioredoxin, protein A, protein G, or maltose-binding protein (MBP). In some cases, the peptide or protein to which the CD98hc binding polypeptide (eg, CD98hc binding Fc polypeptide) is fused may contain a protease cleavage site, such as a cleavage site for factor Xa or thrombin.

[0290] CD98hc-binding polypeptide LLB2 In some embodiments, the polypeptide that binds to CD98hc is from the LLB2 family. In some embodiments, the polypeptide comprises at least 11, 12, 13, 14, or 15 substitutions at the set of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 391, 421, 422, 424, 426, 428, 434, 436, 438, 440, 441, and 442. In some embodiments, the substitutions are S, V, D, E, or Y at position 378; L, I, M, A, Q, V, or K at position 380; N, S, L, M, P, Y, K, A, or T at position 382; T, F, N, P, D, L, H, or Q at position 383; K, R, H, I, L, F, Y, V, or Q at position 384; F or Y at position 385; V, L, A, I, F, Y, S, T, H, R, or E at position 386; L or I at position 387; D, Q, A, T, H at position 389; is selected from V, T, V, or A at position 391, E, Q, or A at position 421, L, M, I, T, or P at position 422, A at position 424, N at position 426, L, T, P, Y, F, I, A, K, H, or W at position 428, S at position 434, L, V, H, F, P, R, or W at position 436, F or W at position 438, L, P, E, N, V, A, I, or D at position 440, P at position 441, and A, V, M, Q, F, P, L, Y, K, R, H, or M at position 442.In some embodiments, the polypeptide comprises any one of the sequences of SEQ ID NOs: 5-27 and at position 378, S, V, D, E, or Y; at position 380, L, I, M, A, Q, V, or K; at position 382, ​​N, S, L, M, P, Y, K, A, or T; at position 383, T, F, N, P, D, L, H, or Q; at position 384, K, R, H, I, L, F, Y, V, or Q; at position 385, F or Y; at position 386, V, L, A, I, F, Y, S, T, H, R, or E; at position 387, L or I; at position 389, D, Q, A, T, H, or V; at position 391, and at least 11, 12, 13, 14, or 15 substitutions at the set of amino acid positions consisting of: T, V, or A at position 421; E, Q, or A at position 421; L, M, I, T, or P at position 422; A at position 424; N at position 426; L, T, P, Y, F, I, A, K, H, or W at position 428; S at position 434; L, V, H, F, P, R, or W at position 436; F or W at position 438; L, P, E, N, V, A, I, or D at position 440; P at position 441; and A, V, M, Q, F, P, L, Y, K, R, H, or M at position 442.

[0291] In some embodiments, the polypeptide comprises a modified constant domain (e.g., a modified CH3 domain) comprising a sequence having at least 85%, 90%, or 95% sequence identity to amino acids 111-217 of SEQ ID NOs: 28-43. In some embodiments, the polypeptide comprises a modified constant domain (e.g., a modified CH3 domain) comprising a sequence having at least 85%, 90%, or 95% sequence identity to amino acids 111-217 of SEQ ID NOs: 28-43, wherein the modified constant domain is selected from the group consisting of S, V, D, E, or Y at position 378; L, I, M, A, Q, V, or K at position 380; N, S, L, M, P, Y, K, A, or T at position 382; T, F, N, P, D, L, H, or Q at position 383; K, R, H, I, L, F, Y, V, or Q at position 384; F or Y at position 385; and at least 11, 12, 13, 14, or 15 substitutions at the set of amino acid positions consisting of: H, R, or E; L or I at position 387; D, Q, A, T, H, or V at position 389; T, V, or A at position 391; E, Q, or A at position 421; L, M, I, T, or P at position 422; A at position 424; N at position 426; L, T, P, Y, F, I, A, K, H, or W at position 428; S at position 434; L, V, H, F, P, R, or W at position 436; F or W at position 438; L, P, E, N, V, A, I, or D at position 440; P at position 441; and A, V, M, Q, F, P, L, Y, K, R, H, or M at position 442.

[0292] In some embodiments, the polypeptide comprises at least 11, 12, 13, 14, or 15 substitutions at the set of amino acid positions consisting of 380, 382, ​​384, 385, 386, 387, 421, 422, 424, 426, 428, 436, 438, 440, and 442. In some embodiments, the substitution is selected from L at position 380, N at position 382, ​​R, H, or Q at position 384, F or Y at position 385, V, L, I, F, Y, or E at position 386, L at position 387, E, Q, or A at position 421, I, T, or P at position 422, A at position 424, N at position 426, Y or W at position 428, R or W at position 436, F or W at position 438, N at position 440, and A, Q, K, R, H, or M at position 442. In some embodiments, the polypeptide comprises any one of SEQ ID NOs: 5-27 and at least 11, 12, 13, 14, or 15 substitutions at the following amino acid positions: L at position 380, N at position 382, ​​R, H, or Q at position 384, F or Y at position 385, V, L, I, F, Y, or E at position 386, L at position 387, E, Q, or A at position 421, I, T, or P at position 422, A at position 424, N at position 426, Y or W at position 428, R or W at position 436, F or W at position 438, N at position 440, and A, Q, K, R, H, or M at position 442.

[0293] In some embodiments, the polypeptide comprises a sequence having at least 85%, 90%, or 95% sequence identity to amino acids 111-217 of SEQ ID NOs: 28-43, and the modified constant domain comprises a modified constant domain (e.g., a modified CH3 domain) that includes at least 11, 12, 13, 14, or 15 substitutions at the following amino acid positions: L at position 380, N at position 382, ​​R, H, or Q at position 384, F or Y at position 385, V, L, I, F, Y, or E at position 386, L at position 387, E, Q, or A at position 421, I, T, or P at position 422, A at position 424, N at position 426, Y or W at position 428, R or W at position 436, F or W at position 438, N at position 440, and A, Q, K, R, H, or M at position 442.

[0294] In some embodiments, a polypeptide (e.g., an Fc polypeptide) that specifically binds to CD98hc comprises a modified CH3 domain, the modified CH3 domain having (i) a first amino acid sequence that is LX1NX2X3X4X5L (SEQ ID NO: 46), wherein X1 is any amino acid, X2 is R, H, or Q, X3 is F or Y, X4 is V, L, I, F, Y, or E, and X5 is any amino acid; and (ii) a second amino acid sequence that is X1X2X3AX4X5X6X7 (SEQ ID NO: 47). and (iii) a third amino acid sequence of X1X2X3X4NX5X6 (SEQ ID NO: 48), wherein X1 is Y, R, or W, X2 is any amino acid, X3 is F or W, X4 and X5 are any amino acid, and X6 is A, Q, K, R, H, M, or S.

[0295] LLB2-10-6 In certain embodiments, the polypeptide comprises SEQ ID NO: 28. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In one embodiment, the polypeptide (e.g., an Fc polypeptide) further comprises a T366W knob mutation.

[0296] LLB2-10-8 In certain embodiments, the polypeptide comprises SEQ ID NO: 29. In one embodiment, a monovalent dimer (e.g., a monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an E at position 421, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In another embodiment, a bivalent dimer (e.g., a bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each having an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an E at position 421, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In one embodiment, the polypeptide (e.g., Fc polypeptide) further comprises a T366W knob mutation.

[0297] LLB2-10-8-d18 In certain embodiments, the polypeptide comprises SEQ ID NO: 30. In one embodiment, a bivalent dimer (e.g., a bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each having an L at position 380, an N at position 382, ​​a Q at position 384, a Y at position 385, an E at position 386, an L at position 387, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442.

[0298] LLB2-10-8-d12 In certain embodiments, the polypeptide comprises SEQ ID NO: 31. In one embodiment, a bivalent dimer (e.g., a bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides), each having an L at position 380, an N at position 382, ​​an H at position 384, a Y at position 385, an E at position 386, an L at position 387, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442.

[0299] LLB2-10-8-d6 In certain embodiments, the polypeptide comprises SEQ ID NO: 32. In one embodiment, a monovalent dimer (e.g., a monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In another embodiment, a bivalent dimer (e.g., a bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each having an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In one embodiment, the polypeptide (e.g., Fc polypeptide) further comprises a T366W knob mutation.

[0300] LLB2-10-8-d3 In certain embodiments, the polypeptide comprises SEQ ID NO: 33. In one embodiment, a monovalent dimer (e.g., a monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an E at position 421, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In another embodiment, a bivalent dimer (e.g., a bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each having an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an E at position 421, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In one embodiment, the polypeptide (e.g., Fc polypeptide) further comprises a T366W knob mutation.

[0301] LLB2-10-8-d1 In certain embodiments, the polypeptide comprises SEQ ID NO: 34. In one embodiment, a bivalent dimer (e.g., a bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each having an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an E at position 421, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, and an N at position 440.

[0302] LLB2.10.8.10.3 In certain embodiments, the polypeptide comprises SEQ ID NO: 35. In one embodiment, the monovalent dimer (e.g., a monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an R at position 442. In one embodiment, the polypeptide (e.g., an Fc polypeptide) further comprises a T366W knob mutation.

[0303] LLB2.10.8.10.8 In certain embodiments, the polypeptide comprises SEQ ID NO: 36. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an H at position 442. In one embodiment, the polypeptide (e.g., an Fc polypeptide) further comprises a T366W knob mutation.

[0304] LLB2.10.8.14.3 In certain embodiments, the polypeptide comprises SEQ ID NO: 37. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an R at position 436, an F at position 438, an N at position 440, and an R at position 442. In one embodiment, the polypeptide (e.g., an Fc polypeptide) further comprises a T366W knob mutation.

[0305] LLB2.10.8.12.5 In certain embodiments, the polypeptide comprises SEQ ID NO: 38. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an L at position 380, an N at position 382, ​​an H at position 384, a Y at position 385, an E at position 386, an L at position 387, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442. In one embodiment, the polypeptide (e.g., an Fc polypeptide) further comprises a T366W knob mutation.

[0306] LLB2-37 In certain embodiments, the polypeptide comprises SEQ ID NO: 39. In one embodiment, a monovalent dimer (e.g., a monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an L at position 380, an N at position 382, ​​a Q at position 384, an F at position 385, an H at position 386, an L at position 387, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an L at position 442. In another embodiment, a bivalent dimer (e.g., a bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each having an L at position 380, an N at position 382, ​​a Q at position 384, an F at position 385, an H at position 386, an L at position 387, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an L at position 442. In one embodiment, the polypeptide (e.g., Fc polypeptide) further comprises a T366W knob mutation.

[0307] LLB2.10.8.9.11.N In certain embodiments, the polypeptide comprises SEQ ID NO: 40. In one embodiment, a monovalent dimer (e.g., a monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, a T at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442.

[0308] LLB2.10.8.10.1 In certain embodiments, the polypeptide comprises SEQ ID NO: 41. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and a K at position 442. In one embodiment, the polypeptide (e.g., an Fc polypeptide) further comprises a T366W knob mutation.

[0309] LLB2.10.8.4.12 In certain embodiments, the polypeptide comprises SEQ ID NO: 42. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, a W at position 436, an F at position 438, an N at position 440, and an R at position 442. In one embodiment, the polypeptide (e.g., an Fc polypeptide) further comprises a T366W knob mutation.

[0310] LLB2.10.8.2.1 In certain embodiments, the polypeptide comprises SEQ ID NO: 43. In one embodiment, a monovalent dimer (e.g., a monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an L at position 380, an N at position 382, ​​a Q at position 384, a Y at position 385, an L at position 386, an L at position 387, an E at position 421, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442.

[0311] Additional polypeptides from the LLB2 family (eg, Fc polypeptides) that specifically bind to CD98hc are shown in Tables A2-A8 and A12.

[0312] Table 2B. Exemplary CD98hc binding site modifications TIFF2025503437000005.tif204165TIFF2025503437000006.tif105165

[0313] LLB1 In some embodiments, the polypeptide (e.g., Fc polypeptide) that binds to CD98hc is from the LLB1 family. In some embodiments, the polypeptide (e.g., Fc polypeptide) comprises at least 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitutions at the set of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 434, 436, 438, 440, and 442. In some embodiments, the substitutions are S or V at position 378, D, M, N, P, F, or H at position 380, R, Y, F, S, W, Y, K, or N at position 382, ​​T at position 383, L, Y, A, S, or F at position 384, F, K, D, M, I, N, Y, L, or H at position 385, T, P, E, K, A, V, D, T, or F at position 386, N, L, Y, R, F, G, S, D, or T at position 387, selected from T, Y, or F at position 9, D, E, or Q at position 421, I, K, L, R, T, F, or H at position 422, V, W, G, L, I, P, or Y at position 424, D, A, Q, W, L, or P at position 426, L or Y at position 428, S at position 434, F at position (436), I, V, F, N, P, or S at position 438, and K, T, P, I, or F at position 440, and Q or M at position 442.In some embodiments, a polypeptide (e.g., an Fc polypeptide) comprises any one of the sequences of SEQ ID NOs: 5-27 and one of the following: S or V at position 378; D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; T at position 383; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; T, P, E, K, A, V, D, T, or F at position 386; N, L, Y, R, F, G, S, D, or T at position 387; T, Y, or comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitutions at the set of amino acid positions consisting of F, D, E, or Q at position 421, I, K, L, R, T, F, or H at position 422, V, W, G, L, I, P, or Y at position 424, D, A, Q, W, L, or P at position 426, L or Y at position 428, S at position 434, F at position (436), I, V, F, N, P, or S at position 438, and K, T, P, I, or F at position 440, and Q or M at position 442.

[0314] In some embodiments, a polypeptide (e.g., an Fc polypeptide) comprises a sequence having at least 85%, 90%, or 95% sequence identity to amino acids 111-217 of a sequence set forth in SEQ ID NOs: 44-45. In some embodiments, a polypeptide (e.g., an Fc polypeptide) comprises a modified constant domain (e.g., a modified CH3 domain) comprising a sequence having at least 85%, 90%, or 95% sequence identity to amino acids 111-217 of a sequence set forth in SEQ ID NOs: 44-45, wherein the modified constant domain is selected from the group consisting of S or V at position 378, D, M, N, P, F, or H at position 380, R, Y, F, S, W, Y, K, or N at position 382, ​​T at position 383, L, Y, A, S, or F at position 384, F, K, D, M, I, N, Y, L, or H at position 385, and T, P, E, K, A, V, D, T at position 386. or F at position 387, N, L, Y, R, F, G, S, D, or T, T, Y, or F at position 389, D, E, or Q at position 421, I, K, L, R, T, F, or H at position 422, V, W, G, L, I, P, or Y at position 424, D, A, Q, W, L, or P at position 426, L or Y at position 428, S at position 434, F at position (436), I, V, F, N, P, or S at position 438, and K, T, P, I, or F at position 440, and Q or M at position 442.

[0315] In some embodiments, the polypeptide (e.g., Fc polypeptide) that binds to CD98hc is from the LLB1 family. In some embodiments, the polypeptide (e.g., Fc polypeptide) comprises at least 8, 9, 10, 11, 12, or 13 substitutions at the set of amino acid positions consisting of 380, 382, ​​384, 385, 386, 387, 422, 424, 426, 428, 434, 438, and 440. In some embodiments, the substitutions are selected from D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; T, P, E, K, A, V, D, T, or F at position 386; N, L, Y, R, G, S, D, or T at position 387; I, K, R, T, F, or H at position 422; V, W, G, L, I, P, or Y at position 424; D, A, Q, W, L, or P at position 426; L at position 428; S at position 434; I, F, N, P, or S at position 438; and K, T, I, or F at position 440. In some embodiments, a polypeptide (e.g., an Fc polypeptide) comprises any one of the sequences of SEQ ID NOs: 5-27 and at position 380, D, M, N, P, F, or H; at position 382, ​​R, Y, F, S, W, Y, K, or N; at position 384, L, Y, A, S, or F; at position 385, F, K, D, M, I, N, Y, L, or H; at position 386, T, P, E, K, A, V, D, T, or F; at position 387, N, , L, Y, R, G, S, D, or T, I, K, R, T, F, or H at position 422, V, W, G, L, I, P, or Y at position 424, D, A, Q, W, L, or P at position 426, L at position 428, S at position 434, I, F, N, P, or S at position 438, and K, T, I, or F at position 440, and at least 8, 9, 10, 11, 12, or 13 substitutions in the set of amino acid positions consisting of: V, W, G, L, I, P, or Y at position 426, L at position 428, S at position 434, I, F, N, P, or S at position 438, and K, T, I, or F at position 440.

[0316] In some embodiments, the polypeptide (e.g., an Fc polypeptide) comprises a modified constant domain (e.g., a modified CH3 domain) comprising a sequence having at least 85%, 90%, or 95% sequence identity to amino acids 111-217 of SEQ ID NOs: 44-45, wherein the modified constant domain comprises at least one of the following: D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; L, Y, A, S, or F at position 384; F, K, D, M, I, or I at position 385; and K, T, I, or F at position 440.

[0317] In some embodiments, the polypeptide (e.g., an Fc polypeptide) comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitutions at the set of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 434, 436, 438, 440, and 442. In some embodiments, the substitution is selected from S or V at position 378, D at position 380, R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T, Y, or F at position 389, D, E, or Q at position 421, I at position 422, V at position 424, D at position 426, L or Y at position 428, S at position 434, F at position 436, I or V at position 438, K at position 440, and Q or M at position 442. In some embodiments, a polypeptide (e.g., an Fc polypeptide) comprises the sequence of any one of SEQ ID NOs: 5-27 and at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 sequences at the set of amino acid positions consisting of S or V at position 378, D at position 380, R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T, Y, or F at position 389, D, E, or Q at position 421, I at position 422, V at position 424, D at position 426, L or Y at position 428, S at position 434, F at position 436, I or V at position 438, K at position 440, and Q or M at position 442.

[0318] In some embodiments, a polypeptide (e.g., an Fc polypeptide) comprises a modified constant domain (e.g., a modified CH3 domain) comprising a sequence having at least 85%, 90%, or 95% sequence identity to amino acids 111-217 of SEQ ID NOs: 44-45, wherein the modified constant domain comprises an S or V at position 378, a D at position 380, an R at position 382, ​​a T at position 383, a Y at position 384, a Y at position 385, a S or V at position 386, a D at position 387, an R at position 388, a T at position 389, a Y at position 390, an R at position 391, a T at position 392, a Y at position 393, a Y at position 394, a Y at position 395, a Y at position 396, a Y at position 397, a Y at position 398, a Y at position 399, a Y at position 400, a Y at position 401, a Y at position 402, a Y at position 403, a Y at position 404, a Y at position 405, a Y at position 406, a Y at position 407, a Y at position 408, a Y at position 409, a Y at position 410, a Y at position 411, a Y at position 412, a Y at position 413, a Y at position 414, a Y at position 415, a Y at position 416, a Y at position 417, a Y at position 418, a Y at position 419, a Y at position 420, a Y at position 421, a Y at position 422, a Y at position 423, a Y at position 424, a Y at position 425, a Y at position 42 and at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids in the set of amino acid positions consisting of K at position 386, P at position 387, Y at position 389, T, Y, or F at position 389, D, E, or Q at position 421, I at position 422, V at position 424, D at position 426, L or Y at position 428, S at position 434, F at position 436, I or V at position 438, K at position 440, and Q or M at position 442.

[0319] In some embodiments, the polypeptide (e.g., an Fc polypeptide) comprises at least 8, 9, 10, 11, 12, 13, 14, or 15 substitutions at the set of amino acid positions consisting of 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 436, 438, and 440. In some embodiments, the substitutions are selected from: R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T at position 389, D at position 421, I at position 422, V at position 424, D at position 426, L at position 428, F at position 436, I at position 438, and K at position 440. In some embodiments, a polypeptide (e.g., an Fc polypeptide) comprises any one of sequences 5-27 and at least 8, 9, 10, 11, 12, 13, 14, or 15 of the set of amino acid positions consisting of R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T at position 389, D at position 421, I at position 422, V at position 424, D at position 426, L at position 428, F at position 436, I at position 438, and K at position 440.

[0320] In some embodiments, a polypeptide (e.g., an Fc polypeptide) comprises a modified constant domain (e.g., a modified CH3 domain) comprising a sequence having at least 85%, 90%, or 95% sequence identity to amino acids 111-217 of SEQ ID NOs: 28-43, wherein the modified constant domain comprises at least 8, 9, 10, 11, 12, 13, 14, or 15 of the following amino acid positions: R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T at position 389, D at position 421, I at position 422, V at position 424, D at position 426, L at position 428, F at position 436, I at position 438, and K at position 440.

[0321] In some embodiments, a polypeptide (e.g., an Fc polypeptide) that specifically binds to CD98hc comprises a modified CH3 domain, the modified CH3 domain comprising: (i) a first amino acid sequence that is X1X2YKPYX3T (SEQ ID NO: 49), where X1 is E or R, X2 is S or T, and X3 is any amino acid; (ii) a second amino acid sequence that is X1X2X3VX4DX5X6 (SEQ ID NO: 50), where X1 is N or D, X2 is V or I, X3, X4, and X5 are any amino acid, and X6 is M or L; and (iii) a third amino acid sequence that is X1X2IX3X4 (SEQ ID NO: 51), where X1 is Y or F, X2 and X3 are any amino acid, and X4 is S or K.

[0322] LLB1-3-16-2 In certain embodiments, the polypeptide comprises SEQ ID NO: 44. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an R at position 382, ​​a T at position 383, a Y at position 384, a K at position 385, a P at position 386, a Y at position 387, a T at position 389, a D at position 421, an I at position 422, a V at position 424, a D at position 426, an F at position 436, an I at position 438, and a K at position 440. In one embodiment, the polypeptide (e.g., an Fc polypeptide) further comprises a T366W knob mutation.

[0323] LLB1-3-16 In certain embodiments, the polypeptide comprises SEQ ID NO: 45. In one embodiment, the monovalent dimer (e.g., a monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an R at position 382, ​​a T at position 383, a Y at position 384, a K at position 385, a P at position 386, a Y at position 387, a T at position 389, a D at position 421, an I at position 422, a V at position 424, a D at position 426, an L at position 428, an F at position 436, an I at position 438, and a K at position 440. In one embodiment, the polypeptide (e.g., an Fc polypeptide) further comprises a T366W knob mutation.

[0324] Additional polypeptides (eg, Fc polypeptides) from the LLB1 family that specifically bind to CD98hc are shown in Tables A9-A11 and A13.

[0325] VIII. Exemplary Polypeptides that Bind TFR The disclosed CH3 domain is typically C-terminal to the CH2 domain and may be a naturally occurring CH2 domain or a variant CH2 domain, and can be linked to the CH2 domain to form a polypeptide (e.g., an Fc polypeptide) that binds to TfR. In some embodiments, the polypeptide (e.g., an Fc polypeptide) further comprises a partial or complete hinge region of an antibody linked to the N-terminus of the CH2 domain. The hinge region can be from any immunoglobulin subclass or isotype. An exemplary immunoglobulin hinge is an IgG1 hinge region, e.g., an IgG hinge region such as the human IgG1 hinge amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 4).

[0326] In some embodiments, a polypeptide (e.g., an Fc polypeptide) may comprise a sequence from Table 2A, and the polypeptide (e.g., an Fc polypeptide) may be further modified to contain a TfR binding site within a modified CH3 domain as described herein.

[0327] In further embodiments, the polypeptide (e.g., an Fc polypeptide) can be further conjugated to another moiety, e.g., a Fab fragment, thereby generating a TfR-binding Fc-Fab fusion. In some embodiments, the TfR-binding Fc-Fab fusion comprises a modified CH3 domain, a CH2 domain, a hinge region, and a Fab fragment. The Fab fragment can be directed to any target of interest, e.g., a therapeutic neurological target, where the Fab can be delivered to the target by transcytosis across the BBB mediated by binding of the modified CH3 domain polypeptide to TfR.

[0328] TfR-binding polypeptides (e.g., TfR-binding Fc polypeptides) may also be fused to polypeptides of interest other than Fabs. For example, in some embodiments, TfR-binding polypeptides (e.g., TfR-binding Fc polypeptides) may be fused to polypeptides that are desired to target TfR-expressing cells or deliver across the endothelium, e.g., the BBB, by transcytosis. In some embodiments, TfR-binding polypeptides (e.g., TfR-binding Fc polypeptides) are fused to soluble proteins. In yet other embodiments, TfR-binding polypeptides (e.g., TfR-binding Fc polypeptides) may be fused to peptides or proteins useful for protein purification, such as polyhistidine, epitope tags, e.g., FLAG, c-Myc, hemagglutinin tags, glutathione S-transferase (GST), thioredoxin, protein A, protein G, or maltose-binding protein (MBP). In some cases, the peptide or protein to which the TfR-binding polypeptide (eg, a TfR-binding Fc polypeptide) is fused may contain a protease cleavage site, such as a cleavage site for factor Xa or thrombin.

[0329] TfR-binding polypeptides 42.2.19 In certain embodiments, the polypeptide comprises the sequence of SEQ ID NO: 72. Further, the polypeptide can comprise the sequence of any one of SEQ ID NOs: 78, 84, 90, 96, 102, 108, 114, and 120. In one embodiment, a monovalent dimer (e.g., a monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having an F at position 382, ​​a Y at position 383, a D at position 384, a D at position 385, an S at position 386, a K at position 387, an L at position 388, a T at position 389, a P at position 419, an R at position 420, a G at position 421, an L at position 422, an A at position 424, an E at position 426, a Y at position 438, an L at position 440, a G at position 442, and an E at position 443, wherein the positions are determined according to EU numbering. In one embodiment, a polypeptide (e.g., an Fc polypeptide) in the monovalent dimer (e.g., a monovalent Fc dimer) further comprises a T366W knob mutation. In another embodiment, a polypeptide (e.g., an Fc polypeptide) in the monovalent dimer (e.g., a monovalent Fc dimer) further comprises T366S, L368A, and Y407V hole mutations. In another embodiment, a bivalent dimer (e.g., a bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each having an F at position 382, ​​a Y at position 383, a D at position 384, a D at position 385, an S at position 386, a K at position 387, an L at position 388, a T at position 389, a P at position 419, an R at position 420, a G at position 421, an L at position 422, an A at position 424, an E at position 426, a Y at position 438, an L at position 440, a G at position 442, and an E at position 443, wherein the positions are determined according to EU numbering.

[0330] 42.2.3-1H In certain embodiments, the polypeptide comprises the sequence of SEQ ID NO: 73. Further, the polypeptide can comprise the sequence of any one of SEQ ID NOs: 79, 85, 91, 97, 103, 109, 115, and 121. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., Fc polypeptide) having F at position 382, ​​Y at position 383, G at position 384, N at position 385, A at position 386, K at position 387, T at position 389, L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440, wherein the positions are determined according to EU numbering. In one embodiment, the polypeptide (e.g., Fc polypeptide) in the monovalent dimer (e.g., monovalent Fc dimer) further comprises a T366W knob mutation. In another embodiment, a polypeptide (e.g., Fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises T366S, L368A, and Y407V hole mutations. In another embodiment, a bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each having an F at position 382, ​​a Y at position 383, a G at position 384, an N at position 385, an A at position 386, a K at position 387, a T at position 389, an L at position 422, an A at position 424, an E at position 426, a Y at position 438, and an L at position 440, wherein the positions are determined according to EU numbering.

[0331] 42.8.196 In certain embodiments, the polypeptide comprises the sequence of SEQ ID NO: 74. Further, the polypeptide can comprise the sequence of any one of SEQ ID NOs: 80, 86, 92, 98, 104, 110, 116, and 122. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., Fc polypeptide) having F at position 382, ​​Y at position 383, E at position 384, A at position 385, K at position 387, L at position 388, L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440, wherein the positions are determined according to EU numbering. In one embodiment, the polypeptide (e.g., Fc polypeptide) in the monovalent dimer (e.g., monovalent Fc dimer) further comprises a T366W knob mutation. In another embodiment, a polypeptide (e.g., Fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises T366S, L368A, and Y407V hole mutations. In another embodiment, a bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each having an F at position 382, ​​a Y at position 383, an E at position 384, an A at position 385, a K at position 387, an L at position 388, an L at position 422, an A at position 424, an E at position 426, a Y at position 438, and an L at position 440, wherein the positions are determined according to EU numbering.

[0332] 42.8.80 In certain embodiments, the polypeptide comprises the sequence of SEQ ID NO: 75. Further, the polypeptide can comprise the sequence of any one of SEQ ID NOs: 81, 87, 93, 99, 105, 111, 117, and 123. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., Fc polypeptide) having F at position 382, ​​E at position 384, S at position 386, K at position 387, T at position 389, L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440, wherein the positions are determined according to EU numbering. In one embodiment, the polypeptide (e.g., Fc polypeptide) in the monovalent dimer (e.g., monovalent Fc dimer) further comprises a T366W knob mutation. In another embodiment, a polypeptide (e.g., Fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises T366S, L368A, and Y407V hole mutations. In another embodiment, a bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each having an F at position 382, ​​an E at position 384, an S at position 386, a K at position 387, a T at position 389, an L at position 422, an A at position 424, an E at position 426, a Y at position 438, and an L at position 440, wherein the positions are determined according to EU numbering.

[0333] 42.8.15 In certain embodiments, the polypeptide comprises the sequence of SEQ ID NO: 76. Further, the polypeptide can comprise the sequence of any one of SEQ ID NOs: 82, 88, 94, 100, 106, 112, 118, and 124. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., Fc polypeptide) having F at position 382, ​​G at position 384, A at position 385, K at position 387, S at position 389, L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440, wherein the positions are determined according to EU numbering. In one embodiment, the polypeptide (e.g., Fc polypeptide) in the monovalent dimer (e.g., monovalent Fc dimer) further comprises a T366W knob mutation. In another embodiment, a polypeptide (e.g., Fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises T366S, L368A, and Y407V hole mutations. In another embodiment, a bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each having an F at position 382, ​​a G at position 384, an A at position 385, a K at position 387, an S at position 389, an L at position 422, an A at position 424, an E at position 426, a Y at position 438, and an L at position 440, wherein the positions are determined according to EU numbering.

[0334] 42.8.17 In certain embodiments, the polypeptide comprises the sequence of SEQ ID NO: 77. Further, the polypeptide can comprise the sequence of any one of SEQ ID NOs: 83, 89, 95, 101, 107, 113, 119, and 125. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., Fc polypeptide) having F at position 382, ​​G at position 384, A at position 385, K at position 387, L at position 388, T at position 389, L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440, wherein the positions are determined according to EU numbering. In one embodiment, the polypeptide (e.g., Fc polypeptide) in the monovalent dimer (e.g., monovalent Fc dimer) further comprises a T366W knob mutation. In another embodiment, a polypeptide (e.g., Fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises T366S, L368A, and Y407V hole mutations. In another embodiment, a bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each having an F at position 382, ​​a G at position 384, an A at position 385, a K at position 387, an L at position 388, a T at position 389, an L at position 422, an A at position 424, an E at position 426, a Y at position 438, and an L at position 440, wherein the positions are determined according to EU numbering.

[0335] IX. Dimers for CD98HC Binding Site Modification In some embodiments, a polypeptide (e.g., an Fc polypeptide) that binds to CD98hc can form a dimer (e.g., an Fc dimer) comprising two polypeptides (e.g., Fc polypeptides). The dimer can be a heterodimer or a homodimer.

[0336] Dimers that bind bivalently to CD98hc In some embodiments, the dimer is an Fc dimer comprising two Fc polypeptides, each containing a CD98hc-binding site, i.e., bivalently binding to CD98hc. In an Fc dimer that bivalently binds to CD98hc, the first and second Fc polypeptides may contain the same modified CH3 domain. In other embodiments, the second Fc polypeptide may contain a modified CH3 domain that is different from that in the first Fc polypeptide to provide a second CD98hc-binding site.

[0337] In some embodiments, a bivalent Fc dimer that specifically binds to CD98hc described herein comprises a first and second Fc polypeptide pair from Table 2C, wherein (i) each of the first and second Fc polypeptides is further modified to contain a CD98hc-binding site in a modified CH3 domain described herein, or (ii) the first and second Fc polypeptides contain a CD98hc-binding site in a modified CH3 domain described herein. In other embodiments, a bivalent Fc dimer that specifically binds to CD98hc described herein comprises a first and second Fc polypeptide pair from Table 2C, wherein the first polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a sequence from the first Fc polypeptide sequence from Table 2C. and the second polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a second Fc polypeptide sequence from Table 2C, and each of the first and second Fc polypeptides is further modified to contain a CD98hc binding site within a modified CH3 domain as described herein. In one embodiment, a bivalent Fc dimer that specifically binds to CD98hc described herein comprises a first and second Fc polypeptide pair from Table 2C, wherein the first polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a sequence from the first Fc polypeptide sequence from Table 2C, and and wherein the first and second polypeptides have at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a second Fc polypeptide sequence from Table 2C, and each of the first and second Fc polypeptides is selected from the group consisting of: (i) 380, 382, ​​384, 385, 386, 387, 421, 422, 424, 426, 428, 436, 438,and (ii) a set of amino acid substitutions consisting of L at position 380, N at position 382, ​​R, H, or Q at position 384, F or Y at position 385, V, L, I, F, Y, or E at position 386, L at position 387, E, Q, or A at position 421, I, T, or P at position 422, A at position 424, N at position 426, Y or W at position 428, R or W at position 436, F or W at position 438, N at position 440, and A, Q, K, R, H, or M at position 442; , M, A, Q, V, or K, N, S, L, M, P, Y, K, A, or T at position 382, ​​T, F, N, P, D, L, H, or Q at position 383, K, R, H, I, L, F, Y, V, or Q at position 384, F or Y at position 385, V, L, A, I, F, Y, S, T, H, R, or E at position 386, L or I at position 387, D, Q, A, T, H, or V at position 389, T, V, or A at position 391, E, Q, or A at position 421, L, M, I, T, or P at position 422, A at position 424, a set of amino acid substitutions consisting of N at position 426, L, T, P, Y, F, I, A, K, H, or W at position 428, S at position 434, L, V, H, F, P, R, or W at position 436, F or W at position 438, L, P, E, N, V, A, I, or D at position 440, P at position 441, and A, V, M, Q, F, P, L, Y, K, R, H, or M at position 442, or at least 11, 12, 13, 14, or 15 substitutions; or S or V at position 378, D, M, N, P, F, or H at position 380, D, M, N, P, F, or H at position 382, R, Y, F, S, W, Y, K, or N at position 383; T at position 384; L, Y, A, S, or F at position 385; F, K, D, M, I, N, Y, L, or H at position 386; T, P, E, K, A, V, D, T, or F at position 387; N, L, Y, R, F, G, S, D, or T at position 389; T, Y, or F at position 421; D, E, or Q at position 422; I, K, L, R, T, F, or H at position 424; V, W, G, L, I, P, or Y at position 426; D, A, Q, W, L, or P at position 426.At least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitutions are selected from the set of amino acid substitutions consisting of L or Y at position 428, S at position 434, F at position 436, I, V, F, N, P, or S at position 438, and K, T, P, I, or F at position 440, and Q or M at position 442.

[0338] In one embodiment, a bivalent Fc dimer that specifically binds to CD98hc described herein comprises a first and second Fc polypeptide pair from Table 2C, wherein the first polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a sequence from the first Fc polypeptide sequence from Table 2C, and the second The polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a second Fc polypeptide sequence from Table 2C, wherein each of the first and second Fc polypeptides is further modified to contain a CD98hc binding site within a modified CH3 domain comprising a set of modifications selected from Table 2B.

[0339] Dimer that binds monovalently to CD98hc In some embodiments, the dimer is a monovalent Fc dimer comprising two Fc polypeptides, wherein only one of the two Fc polypeptides in the monovalent Fc dimer comprises a CD98hc-binding site, and the other Fc polypeptide does not bind to CD98hc. In addition, the Fc polypeptides may contain modifications (e.g., T366W, and T366S, L368A, and Y407V) to promote heterodimerization of the Fc dimer. In some embodiments, the monovalent Fc dimers that specifically bind to CD98hc described herein comprise a first and second Fc polypeptide pair from Table 2D, wherein the first Fc polypeptide is further modified to contain a CD98hc-binding site within a modified CH3 domain described herein. In other embodiments, a monovalent Fc dimer that specifically binds to CD98hc described herein comprises a first and second Fc polypeptide pair from Table 2D, wherein the first polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a sequence from the first Fc polypeptide sequence from Table 2D. and the second polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a second Fc polypeptide sequence from Table 2D, and the first Fc polypeptide is further modified to contain a CD98hc binding site within a modified CH3 domain as described herein. In one embodiment, a monovalent Fc dimer that specifically binds to CD98hc described herein comprises a first and second Fc polypeptide pair from Table 2D, wherein the first polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a sequence from the first Fc polypeptide sequence from Table 2D, and the second polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99%) or 100% identity to the first Fc polypeptide, wherein the first Fc polypeptide is further modified to contain a CD98hc binding site within a modified CH3 domain comprising at least 11, 12, 13, 14, or 15 substitutions in the set of amino acid substitutions consisting of 380, 382, ​​384, 385, 386, 387, 421, 422, 424, 426, 428, 436, 438, 440, and 442. In some embodiments, the substitutions are: L at position 380, N at position 382, ​​R, H, or Q at position 384, F or Y at position 385, V, L, I, F, Y, or E at position 386, L at position 387, E, Q, or A at position 421, I, T, or P at position 422, A at position 424, N at position 426, Y or W at position 428, R or W at position 436, F or W at position 438, N at position 440, and A, Q, K, R, H, or M at position 442; (ii) 3 S, V, D, E, or Y at rank 78; L, I, M, A, Q, V, or K at rank 380; N, S, L, M, P, Y, K, A, or T at rank 382; T, F, N, P, D, L, H, or Q at rank 383; K, R, H, I, L, F, Y, V, or Q at rank 384; F or Y at rank 385; V, L, A, I, F, Y, S, T, H, R, or E at rank 387; L or I at rank 389; D, Q, A, T, H, or V at rank 391 an amino acid sequence consisting of T, V, or A at position 421, E, Q, or A at position 421, L, M, I, T, or P at position 422, A at position 424, N at position 426, L, T, P, Y, F, I, A, K, H, or W at position 428, S at position 434, L, V, H, F, P, R, or W at position 436, F or W at position 438, L, P, E, N, V, A, I, or D at position 440, P at position 441, and A, V, M, Q, F, P, L, Y, K, R, H, or M at position 442 The set of amino acid substitutions may include at least 11, 12, 13, 14, or 15 substitutions, or may include at least one of the following: S or V at position 378; D, M, N, P, F, or H at position 380; R, Y, F, S, W, Y, K, or N at position 382; T at position 383; L, Y, A, S, or F at position 384; F, K, D, M, I, N, Y, L, or H at position 385; T, P, E, K, A, V, D, T, or F at position 386; N, L, Y, R, F, G, S, D, oror T, T, Y, or F at position 389, D, E, or Q at position 421, I, K, L, R, T, F, or H at position 422, V, W, G, L, I, P, or Y at position 424, D, A, Q, W, L, or P at position 426, L or Y at position 428, S at position 434, F at position 436, I, V, F, N, P, or S at position 438, and K, T, P, I, or F at position 440, and Q or M at position 442, with at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitutions selected from the set of amino acid substitutions consisting of:

[0340] For example, the first Fc polypeptide from dimer pair K of Table 2D (i.e., SEQ ID NO: 11) is further modified to include an L at position 380, an N at position 382, ​​an R at position 384, an F at position 385, a V at position 386, an L at position 387, an E at position 421, an I at position 422, an A at position 424, an N at position 426, a Y at position 428, an F at position 438, an N at position 440, and an A at position 442.

[0341] In one embodiment, a monovalent Fc dimer that specifically binds to CD98hc described herein comprises a first and second Fc polypeptide pair from Table 2D, wherein the first polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a sequence from the first Fc polypeptide sequence from Table 2D. , the second polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a second Fc polypeptide sequence from Table 2B, and the first Fc polypeptide is further modified to contain a CD98hc binding site within a modified CH3 domain comprising a set of modifications selected from Table 2B.

[0342] Dimers for modification of X.TFR binding sites In some embodiments, a polypeptide (e.g., an Fc polypeptide) that binds to TfR can form a dimer (e.g., an Fc dimer) comprising two polypeptides (e.g., Fc polypeptides). The dimer can be a heterodimer or a homodimer.

[0343] Dimers that bind bivalently to TfR In some embodiments, the dimer is an Fc dimer comprising two polypeptides (e.g., Fc polypeptides) that each contain a TfR-binding site, i.e., that bind bivalently to TfR. In an Fc dimer that bivalently binds to TfR, the first and second Fc polypeptides may contain the same CH3 domain. In other embodiments, the second Fc polypeptide may contain a CH3 domain that is different from that in the first Fc polypeptide to provide a second TfR-binding site.

[0344] In some embodiments, a bivalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2C, wherein (i) each of the first and second Fc polypeptides is further modified to contain a CD98hc-binding site in a modified CH3 domain described herein, or (ii) the first and second Fc polypeptides contain a CD98hc-binding site in a modified CH3 domain described herein. In other embodiments, a bivalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2C, wherein the first Fc polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to the first Fc polypeptide sequence from Table 2C. The second Fc polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a second Fc polypeptide sequence from Table 2C, and each of the first and second Fc polypeptides is further modified to contain a TfR binding site within a modified CH3 domain as described herein.

[0345] In one embodiment, a bivalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2C, wherein the first Fc polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to the first Fc polypeptide sequence from Table 2C, and the second Fc polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to the second Fc polypeptide sequence from Table 2C. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to the first and second Fc polypeptides, wherein each of the first and second Fc polypeptides is further modified to contain a TfR binding site within a modified CH3 domain comprising 3, 4, 5, 6, 7, or 8 amino acid substitutions and / or 1 or 2 amino acid deletions at the set of amino acid positions including 380 and 382-389, and 5, 6, or 7 amino acid substitutions at the set of amino acid positions including 422, 424, 426, 433, 434, 438, and 440, wherein the positions are determined according to EU numbering. In some embodiments, the substitution and / or deletion is selected from E, N, F, or Y at position 380, F at position 382, ​​Y, S, A, or an amino acid deletion at position 383, G, D, E, or N at position 384, D, G, N, or A at position 385, Q, S, G, A, or N at position 386, K, I, R, or G at position 387, E, L, D, or Q at position 388, N, T, S, or R at position 389, L at position 422, A at position 424, E at position 426, H or E at position 433, N or G at position 434, Y at position 438, and L at position 440.

[0346] In one embodiment, a bivalent Fc dimer that specifically binds to TfR described herein comprises a pair of first and second Fc polypeptides from Table 2C, wherein the first Fc polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to the first Fc polypeptide sequence from Table 2C, and the second Fc polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to the second Fc polypeptide sequence from Table 2C, and wherein the first and Each of the two Fc polypeptides is further modified to contain a TfR-binding site within a modified CH3 domain comprising 3, 4, 5, 6, 7, or 8 amino acid substitutions at amino acid positions 380 and 382-389 inclusive (F at position 382, ​​Y or S at position 383, G, D, or E at position 384, D, G, N, or A at position 385, Q, S, or A at position 386, K at position 387, E or L at position 388, and N, T, or S at position 389), and 5, 6, or 7 amino acid substitutions at amino acid positions 422, 424, 426, 433, 434, 438, and 440 inclusive (e.g., L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440), where positions are determined according to EU numbering.

[0347] Dimers that bind monovalently to TfR In some embodiments, the dimer is a monovalent Fc dimer comprising two Fc polypeptides, wherein only one of the two Fc polypeptides in the monovalent Fc dimer comprises a TfR-binding site, and the other Fc polypeptide does not bind to TfR. In addition, the Fc polypeptides may contain modifications to promote heterodimerization of the Fc dimer (e.g., T366W, and T366S, L368A, and Y407V). In some embodiments, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2D, wherein the first Fc polypeptide is further modified to contain a TfR-binding site within a modified CH3 domain described herein. In some embodiments, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2D, wherein the second Fc polypeptide is further modified to contain a TfR-binding site within a modified CH3 domain described herein.

[0348] In other embodiments, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2D, wherein the first polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a sequence from the first Fc polypeptide sequence from Table 2D. and the second polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a second Fc polypeptide sequence from Table 2D, and the first Fc polypeptide is further modified to contain a TfR binding site within a modified CH3 domain as described herein.

[0349] In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2D, wherein the first polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to the first Fc polypeptide sequence from Table 2D, and the second polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, and a TfR-binding site within the modified CH3 domain comprising 3, 4, 5, 6, 7, or 8 amino acid substitutions and / or 1 or 2 amino acid deletions at amino acid positions 380 and 382-389 inclusive, and 5, 6, or 7 amino acid substitutions at amino acid positions 422, 424, 426, 433, 434, 438, and 440 inclusive, wherein the positions are determined according to EU numbering. In some embodiments, the substitution and / or deletion is selected from E, N, F, or Y at position 380, F at position 382, ​​Y, S, A, or an amino acid deletion at position 383, G, D, E, or N at position 384, D, G, N, or A at position 385, Q, S, G, A, or N at position 386, K, I, R, or G at position 387, E, L, D, or Q at position 388, N, T, S, or R at position 389, L at position 422, A at position 424, E at position 426, H or E at position 433, N or G at position 434, Y at position 438, and L at position 440.

[0350] In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2D, wherein the first polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a first Fc polypeptide sequence from Table 2D, and the second polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a second Fc polypeptide sequence from Table 2D, and The c polypeptide is further modified to contain a TfR binding site within a modified CH3 domain comprising 3, 4, 5, 6, 7, or 8 amino acid substitutions at amino acid positions 380 and 382-389 inclusive (F at position 382, ​​Y or S at position 383, G, D, or E at position 384, D, G, N, or A at position 385, Q, S, or A at position 386, K at position 387, E or L at position 388, and N, T, or S at position 389), and 5, 6, or 7 amino acid substitutions at amino acid positions 422, 424, 426, 433, 434, 438, and 440 inclusive (e.g., L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440), where positions are determined according to EU numbering.

[0351] In other embodiments, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2D, wherein the first polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a sequence from the first Fc polypeptide sequence from Table 2D. and the second polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a second Fc polypeptide sequence from Table 2D, wherein the second Fc polypeptide is further modified to contain a TfR binding site within a modified CH3 domain as described herein.

[0352] In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2D, wherein the first polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to the first Fc polypeptide sequence from Table 2D, and the second polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, and a TfR binding site within the modified CH3 domain comprising 3, 4, 5, 6, 7, or 8 amino acid substitutions and / or 1 or 2 amino acid deletions at amino acid positions 380 and 382-389 inclusive, and 5, 6, or 7 amino acid substitutions at amino acid positions 422, 424, 426, 433, 434, 438, and 440 inclusive, wherein the positions are determined according to EU numbering. In some embodiments, the substitution and / or deletion is selected from E, N, F, or Y at position 380, F at position 382, ​​Y, S, A, or an amino acid deletion at position 383, G, D, E, or N at position 384, D, G, N, or A at position 385, Q, S, G, A, or N at position 386, K, I, R, or G at position 387, E, L, D, or Q at position 388, N, T, S, or R at position 389, L at position 422, A at position 424, E at position 426, H or E at position 433, N or G at position 434, Y at position 438, and L at position 440.

[0353] In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2D, wherein the first polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a first Fc polypeptide sequence from Table 2D, the second polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to a second Fc polypeptide sequence from Table 2D, and The c polypeptide is further modified to contain a TfR binding site within a modified CH3 domain comprising 3, 4, 5, 6, 7, or 8 amino acid substitutions at amino acid positions 380 and 382-389 inclusive (F at position 382, ​​Y or S at position 383, G, D, or E at position 384, D, G, N, or A at position 385, Q, S, or A at position 386, K at position 387, E or L at position 388, and N, T, or S at position 389), and 5, 6, or 7 amino acid substitutions at amino acid positions 422, 424, 426, 433, 434, 438, and 440 inclusive (e.g., L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440), where positions are determined according to EU numbering.

[0354] For example, the first Fc polypeptide from dimer pair K of Table 2D (i.e., SEQ ID NO: 11) is further modified to include 3, 4, 5, 6, 7, or 8 amino acid substitutions at the set of amino acid positions including 380 and 382-389 (e.g., F at position 382, ​​Y or S at position 383, G, D, or E at position 384, D, G, N, or A at position 385, Q, S, or A at position 386, K at position 387, E or L at position 388, and N, T, or S at position 389), and 5, 6, or 7 amino acid substitutions at the set of amino acid positions including 422, 424, 426, 433, 434, 438, and 440 (e.g., L at position 422, A at position 424, E at position 426, Y at position 438, and L at position 440), wherein the positions are determined according to EU numbering.

[0355] In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2D, wherein the first Fc polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to the first Fc polypeptide sequence from Table 2D, and the second Fc polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity, and wherein the first Fc polypeptide is further modified to contain a TfR binding site within a modified CH3 domain comprising a set of modifications selected from a row of Table 29 (e.g., the TfR binding site modifications from clone 42.2.19, 42.2.3-1H, 42.8.196, 42.8.80, 42.8.15, or 42.8.17 of Table 29). In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2D, wherein the first Fc polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to the first Fc polypeptide sequence from Table 2D, and the second Fc polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity, and the second Fc polypeptide is further modified to contain a TfR binding site within a modified CH3 domain comprising a set of modifications selected from a row of Table 29 (e.g., the TfR binding site modifications from clone 42.2.19, 42.2.3-1H, 42.8.196, 42.8.80, 42.8.15, or 42.8.17 of Table 29).

[0356] In another aspect, the present disclosure provides an Fc polypeptide dimer having the sequences of a first and second Fc polypeptide listed in Tables 2C and 2D below.

[0357] Table 2C. Dimer combinations for bivalent CD98hc or TfR binding site modification. TIFF2025503437000007.tif62165

[0358] Table 2D. Knob-hole dimer combinations for monovalent CD98hc or TfR binding site modification TIFF2025503437000008.tif134165

[0359] XI. Conjugates In some embodiments, a polypeptide described herein (e.g., an Fc polypeptide) is linked via a linker to an agent for internalization into a cell and / or for transcytosis across an endothelium, such as the BBB. The linker may be any linker suitable for attaching an agent to a polypeptide. In some embodiments, the linkage is enzymatically cleavable. In certain embodiments, the linkage is cleavable by an enzyme present in the central nervous system.

[0360] In some embodiments, the linker is a peptide linker. The peptide linker may allow rotation of the drug and the polypeptide relative to each other and / or be resistant to digestion by proteases. In some embodiments, the linker may be a flexible linker containing amino acids such as Gly, Asn, Ser, Thr, Ala, etc. Such linkers are designed using known parameters. For example, the linker may have repeats such as Gly-Ser repeats.

[0361] In various embodiments, conjugates can be produced using well-known chemical cross-linking reagents and protocols. For example, the cross-linking agent is a heterobifunctional cross-linker that can be used to link molecules in a stepwise manner. Heterobifunctional cross-linkers allow for the design of more specific coupling methods for protein conjugation, thereby reducing the occurrence of undesired side reactions such as homoprotein polymers. A wide variety of heterobifunctional cross-linkers are known in the art, including N-hydroxysuccinimide (NHS) or its water-soluble analog, N-hydroxysulfosuccinimide (sulfo-NHS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), Examples include succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene (SMPT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and succinimidyl 6-[3-(2-pyridyldithio)propionate]hexanoate (LC-SPDP). These crosslinkers containing an N-hydroxysuccinimide moiety can be obtained as N-hydroxysulfosuccinimide analogs, which generally have higher water solubility. Furthermore, these crosslinkers containing disulfide bridges within the linking chain can be synthesized as alkyl derivatives instead, to reduce the amount of linker cleavage in vivo. In addition to heterobifunctional crosslinkers, numerous other crosslinkers exist, including homobifunctional and photoreactive crosslinkers.Disuccinimidyl suberate (DSS), bismaleimidohexane (BMH), and dimethylpimelimidate·2HCl (DMP) are examples of useful homobifunctional crosslinkers, and bis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) and N-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) are examples of useful photoreactive crosslinkers.

[0362] Agents of interest can be therapeutic agents including cytotoxic agents, DNA or RNA molecules, antisense oligonucleotides, chemical moieties, etc. In some embodiments, the agent is a peptide or small molecule therapeutic agent or imaging agent. In some embodiments, the small molecule is less than 1000 Da, less than 750 Da, or less than 500 Da.

[0363] The target drug is a drug that inhibits the binding of a CD98hc-binding polypeptide to CD98hc or CD98 heterodimers, i.e., the binding of CD98hc and CD98 light chains (LAT1 (SLC7A5), LAT2 (SLC7A8), y + LAT1(SLC7A7), y + The binding may be to the N-terminal or C-terminal region of the CD98hc-binding polypeptide, or to any region of the polypeptide, as long as it does not interfere with binding to CD98hc in complex with LAT2 (SLC7A6), Asc-1 (SLC7A10), or xCT (SLC7A11).

[0364] The agent of interest may be linked to the N-terminal or C-terminal region of the TfR-binding polypeptide, or may be attached to any region of the polypeptide, so long as the agent does not interfere with binding of the TfR-binding polypeptide to the TfR.

[0365] XII. Methods of Engineering Polypeptides to Bind to CD98HC or TFR In a further aspect, methods are provided for engineering a modified CH3 domain to bind to CD98hc. In some embodiments, the modification of the CH3 domain comprises substituting different amino acids relative to the sequence of SEQ ID NO: 3 or relative to amino acids 111-217 of the sequence of SEQ ID NO: 1. In some embodiments, the method comprises modifying a polynucleotide encoding the modified CH3 domain polypeptide to incorporate amino acid changes relative to the sequence of SEQ ID NO: 3 or relative to amino acids 111-217 of the sequence of SEQ ID NO: 1.

[0366] In some embodiments of engineering a polypeptide to bind to CD98hc, the method comprises modifying a polynucleotide encoding a modified CH3 domain to comprise a sequence having: (i) a first sequence comprising at least one substitution or deletion relative to the sequence of EWESNGQP (SEQ ID NO:52, to position 387 of the Fc polypeptide (e.g., SEQ ID NO:1), EU numbering); (ii) a second sequence comprising at least one substitution relative to the sequence of NVFSCSVM (SEQ ID NO:53, to position 428 of the Fc polypeptide (e.g., SEQ ID NO:1), EU numbering); and (iii) a third sequence comprising at least one substitution relative to the sequence of YTQKSLS (SEQ ID NO:53, to position 442 of the Fc polypeptide (e.g., SEQ ID NO:1), EU numbering). In some embodiments, the method further comprises expressing and recovering the polypeptide comprising the modified CH3 domain and determining whether the polypeptide binds to CD98hc.

[0367] In some embodiments of engineering a polypeptide to bind to TfR, the method comprises modifying a polynucleotide encoding a modified CH3 domain to comprise a sequence having (i) a first sequence comprising at least one amino acid substitution and / or deletion relative to the sequence of AVEWESNGQPENN (SEQ ID NO:56), and (ii) a second sequence comprising at least one amino acid substitution in the sequence of VFSCSVMHEALHNHYTQKS (SEQ ID NO:57), wherein the sequence of SEQ ID NO:56 is located at positions 378-390 of the Fc polypeptide (e.g., SEQ ID NO:1) and the sequence of SEQ ID NO:57 is located at positions 422-440 of the Fc polypeptide (e.g., SEQ ID NO:1), the positions being determined according to EU numbering. In some embodiments, the method further comprises expressing and recovering the polypeptide comprising the modified CH3 domain and determining whether the polypeptide binds to TfR.

[0368] The amino acids introduced at desired positions can be generated by randomization or partial randomization to generate a library of CH3 domain polypeptides with amino acid substitutions at various positions as described herein. In some embodiments, the modified CH3 domain polypeptides are mutated in the context of an Fc region, which may or may not contain part or all of a complete hinge region.

[0369] Polypeptides comprising modified CH3 domains may be expressed using any number of systems. For example, in some embodiments, the polypeptides are expressed in a display system. In other exemplary embodiments, the mutant polypeptides are expressed as soluble polypeptides secreted from host cells. In some embodiments, the expression system is a display system, e.g., a cell surface display system such as a viral display system, a yeast display system, an mRNA display system, or a polysome display system. The library is screened using known methodologies to identify CD98hc binders, which can be further characterized to determine binding kinetics. Additional mutations may then be introduced into selected clones.

[0370] CD98hc-binding polypeptides of the present disclosure can have a wide range of binding affinities, depending, for example, on the format of the polypeptide. For example, in some embodiments, polypeptides comprising modified CH3 domains have CD98hc binding affinities anywhere from 1 pM to 10 μM. In some embodiments, the polypeptides bind to human CD98hc with an affinity of 15 nM to 5 μM (e.g., 15 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM). In another embodiment, the polypeptide binds to cynomolgus CD98hc with an affinity of 80 nM to 5 μM (e.g., 80 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM). In some embodiments, affinity may be measured in a monovalent format. In other embodiments, affinity may be measured in a bivalent format.

[0371] TfR-binding polypeptides of the present disclosure can have a wide range of binding affinities, depending, for example, on the format of the polypeptide. For example, in some embodiments, polypeptides comprising modified CH3 domains have TfR binding affinities anywhere from 1 pM to 10 μM. In some embodiments, the polypeptides bind to human TfR with an affinity of 15 nM to 10 μM (e.g., 15 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, or 10 μM). In another embodiment, the polypeptide binds to cynomolgus monkey TfR with an affinity of 80 nM to 5 μM (e.g., 80 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM). In some embodiments, affinity may be measured in a monovalent format. In other embodiments, affinity may be measured in a bivalent format.

[0372] Methods for analyzing binding affinity, binding kinetics, and cross-reactivity are known in the art. These methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore™ (GE Healthcare, Piscataway, NJ)), kinetic exclusion assays (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), BioLayer interferometry (e.g., Octet® (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to determine binding affinity and / or cross-reactivity. Methods for performing ELISA assays are known in the art and are also described in the Examples section below. In some embodiments, surface plasmon resonance (SPR) is used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, equilibrium binding exclusion methods are used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, BioLayer interferometry assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity.

[0373] XIII. Nucleic Acids, Vectors, and Host Cells The CD98hc-binding and TfR-binding polypeptides described herein are typically prepared using recombinant methods. Thus, in some aspects, the disclosure provides an isolated nucleic acid comprising a nucleic acid sequence encoding any of the polypeptides described herein and a host cell into which nucleic acid used to replicate the nucleic acid encoding the polypeptide and / or express the polypeptide is introduced. In some embodiments, the host cell is a eukaryotic, e.g., a human cell.

[0374] In another aspect, a polynucleotide is provided that comprises a nucleotide sequence encoding a polypeptide described herein. The polynucleotide can be single-stranded or double-stranded. In some embodiments, the polynucleotide is DNA (e.g., cDNA). In some embodiments, the polynucleotide is RNA.

[0375] In some embodiments, the polynucleotide is comprised within a nucleic acid construct. In some embodiments, the construct is a replicable vector. In some embodiments, the vector is selected from a plasmid, a viral vector, a phagemid, a yeast chromosomal vector, and a non-episomal mammalian vector.

[0376] In some embodiments, the polynucleotide is operably linked to one or more regulatory nucleotide sequences in an expression construct. In one set of embodiments, the nucleic acid expression construct is adapted for use as a surface expression library (e.g., yeast or phage). In another set of embodiments, the nucleic acid expression construct is adapted for expression of a polypeptide in a system capable of isolating the polypeptide in milligram or gram quantities. In some embodiments, the system is a mammalian cell or yeast cell expression system.

[0377] Expression vehicles for the production of recombinant polypeptides include plasmids and other vectors. Any suitable plasmid or vector can be used for this purpose, including those suitable for transient expression of polypeptides in eukaryotic cells. In some embodiments, it may be desirable to express the recombinant polypeptide by using a baculovirus expression system with an appropriate vector. Additional expression systems include adenovirus, adeno-associated virus, and other viral expression systems.

[0378] The vector can be transformed into any suitable host cell. In some embodiments, host cells, such as bacterial or yeast cells, can be adapted for use as a surface expression library. In certain cells, the vector is expressed in the host cell, resulting in the expression of relatively large amounts of the polypeptide. Such host cells include mammalian cells, yeast cells, insect cells, and prokaryotic cells. In some embodiments, the cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a baby hamster kidney (BHK) cell, an NS0 cell, a YO cell, an HEK293 cell, a COS cell, a Vero cell, or a HeLa cell.

[0379] Host cells transfected with an expression vector encoding a CD98hc-binding or TfR-binding polypeptide can be cultured under appropriate conditions to allow expression of the polypeptide. The polypeptide can be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, the polypeptide can be retained in the cytoplasm or a membrane fraction, and the cells can be harvested and lysed using a desired method, and the polypeptide can be isolated.

[0380] XIV. Methods of Delivery, Targeting, and Treatment The polypeptides described herein according to the present disclosure can be used therapeutically in many indications. In some embodiments, the polypeptides are used to deliver therapeutic agents to target cell types that express CD98hc or TfR. In some embodiments, the polypeptides may be used to transport therapeutic moieties across endothelia, e.g., the BBB, to be taken up by the brain. Thus, the polypeptides of the present disclosure can be conjugated to therapeutic agents and used to deliver therapeutic agents to treat neurological disorders, such as brain or central nervous system (CNS) diseases, to treat cancer, to treat autoimmune or inflammatory diseases, or to treat cardiovascular diseases, for example.

[0381] In some embodiments, provided herein are methods for targeting extracellular targets in the brain using polypeptides of the present disclosure. In some embodiments, the polypeptides of the present disclosure are transported across the BBB into the parenchyma without being transcytosed into cells in the brain. In some embodiments, the method includes delivering a therapeutic agent across the BBB to an extracellular target on or near astrocytes, microglia, oligodendrocytes, or cancer cells. In other embodiments, the extracellular target is an antigen in the brain, such as a plaque, tangle, or other non-cellular target. In some embodiments, the targeted delivery is to an extracellular target on microglia. In some embodiments, the targeted delivery is to an extracellular target on cancer cells.

[0382] In some embodiments, provided herein are methods for treating a disease in a patient's brain, the method comprising delivering a therapeutic agent to an extracellular target in the brain using a polypeptide of the present disclosure. In some embodiments, the method comprises delivering the therapeutic agent across the BBB to the parenchyma without transcytosis into cells in the brain. In some embodiments, the method comprises delivering the therapeutic agent across the BBB to an extracellular target on or near an astrocyte, microglia, oligodendrocyte, or cancer cell. In other embodiments, the extracellular target is an antigen in the brain, e.g., plaque, tangle, or other non-cellular target (e.g., Abeta, Tau, or alpha-synuclein). In some embodiments, the brain disease being treated is selected from the group consisting of frontotemporal dementia, amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease. In some embodiments, the cancer is glioblastoma or metastatic cancer in the brain.

[0383] The polypeptides of the present disclosure are administered to a subject in a therapeutically effective amount or dose. The dose can vary according to several factors, including the selected route of administration, the formulation of the composition, the patient's response, the severity of the condition, the subject's weight, and the prescribing physician's judgment. The dose can be increased or decreased over time as needed by an individual patient. In some embodiments, the patient is initially administered a low dose, which is then increased to an effective dosage that the patient can tolerate. Determining an effective amount is well within the capabilities of one skilled in the art.

[0384] In various embodiments, polypeptides of the present disclosure are administered parenterally (e.g., subcutaneously, subcutaneously, intradermally, or intramuscularly). In some embodiments, the polypeptides are administered intravenously. Intravenous administration can be by infusion or as an intravenous bolus. A combination of infusion and bolus administration can also be used. In other embodiments, the polypeptides can be administered orally, pulmonary, intranasally, intraocularly, or topically. Pulmonary administration can also be used, for example, by use of an inhaler or nebulizer and formulation with an aerosolizing agent.

[0385] XV. Pharmaceutical Compositions and Kits In another aspect, pharmaceutical compositions and kits comprising the polypeptides according to the present disclosure are provided.

[0386] Pharmaceutical Composition Guidance for preparing the formulations used in this disclosure can be found in any number of handbooks for pharmaceutical preparation and formulation known to those skilled in the art.

[0387] In some embodiments, pharmaceutical compositions comprise a polypeptide described herein and further comprise one or more pharmaceutically acceptable carriers and / or excipients. Pharmaceutically acceptable carriers include any solvent, dispersion medium, or coating that is physiologically compatible and preferably does not interfere with or otherwise inhibit the activity of the active agent. A variety of pharmaceutically acceptable excipients are well known.

[0388] In some embodiments, the carrier is suitable for intravenous, intrathecal, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compounds, for example, to stabilize the composition or to increase or decrease the absorption of polypeptides. Physiologically acceptable compounds can include, for example, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins, compositions that reduce the clearance or hydrolysis of active substances, or excipients or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and their formulations are also available in the art.

[0389] The pharmaceutical compositions described herein can be manufactured in a manner known to those skilled in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0390] Typically, pharmaceutical compositions for use in in vivo administration are sterile. Sterilization can be achieved by methods known in the art, for example, heat sterilization, steam sterilization, sterile filtration, or irradiation.

[0391] Dosages and desired drug concentrations of the pharmaceutical compositions of the present disclosure may vary depending on the particular use envisioned. Determination of the appropriate dosage or route of administration can be determined by one skilled in the art.

[0392] kit In some embodiments, kits are provided that include the polypeptides described herein, hi some embodiments, the kits are used to prevent or treat neurological disorders, such as diseases of the brain or central nervous system (CNS).

[0393] In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit comprises a polypeptide described herein and further comprises one or more additional therapeutic agents for use in treating a neurological disorder. In some embodiments, the kit further comprises instructional materials containing instructions (i.e., protocols) for practicing the methods described herein (e.g., instructions for using the kit to administer a composition across the BBB). The instructional materials typically include, but are not limited to, written or printed materials. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), and the like. Such media may include the address of an internet site providing such instructional materials. [Example]

[0394] XVI. Working Examples The present disclosure will be explained in more detail by specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the present disclosure in any way. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially the same results. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation may exist. The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the skill of the art. Such techniques are fully explained in the literature. Furthermore, it will be apparent to those skilled in the art that methods for manipulation applied to a particular library can also be applied to the other libraries described herein.

[0395] Example 1. Generation of a limited liability library and selection of CD98HC-binding clones for manipulation We developed a beta-sheet library that was used to discover polypeptides capable of binding to CD98hc. The library included randomization at human Fc residues 380, 382, ​​384-387, 422, 424, 426, 438, and 440 (EU numbering). We observed that large libraries (e.g., more than nine residue positions) often have numerous unfavorable residues at adjacent positions, resulting in clones that are nonspecific and / or perform poorly. To reduce the frequency of residues that can cause such burden (particularly cysteine, arginine, tryptophan, and glycine, as described below), we engineered the library using codon bias to restrict the frequency and location of these residues. Libraries implementing this technique are referred to as "limited liability" libraries. These unfavorable residues include arginine and tryptophan, which can increase interaction affinity but also increase nonspecificity (entropic interactions), cysteine, which is an oxidative burden, and glycine, which can increase protein structural flexibility and destabilize secondary structure. To avoid these four residues at defined positions, we introduced the NHK codon into the library. Alternating NNK (which allows all 20 amino acids) and NHK codons restricts these four amino acids to adjacent positions, which can overly limit the diversity of the library. This technique improved the quality of the polypeptides identified in the library.

[0396] Using the techniques described above, libraries were designed to restrict the inclusion of culpable residues at adjacent sites or throughout, as shown in Table 2E. Libraries were generated using Kunkel mutagenesis with the mixture 6 oligos using the WT Fc gene in a phage display vector. Phage libraries, designated "LLB" for restricted culpable B, were screened for binding to human CD98hc. Screening of these libraries yielded five clones (shown in Table A1) that weakly bound to human CD98hc, designated LLB1, LLB2, and LLB3, LLB6, and LLB7, which were selected for further manipulation. LLB2 and LLB3 were found to be part of the same family (designated LLB2). Clones LLB1, LLB6, and LLB7 were found to be part of the same family (designated LLB1).

[0397] (Table 2E) Oligos were mixed together and pooled to yield 9 different library variants. TIFF2025503437000009.tif50165

[0398] (Table A1) Clones of the LLB1 and LLB2 families TIFF2025503437000010.tif19165

[0399] Example 2. Generation of LLB2 family CD98HC binders LLB2 family affinity maturation (AM1-AM4) Two affinity maturation libraries (LLB2-AM1 and LLB2-AM2, Table 3) were designed for the LLB2 and LLB3 clones using Kunkel mutagenesis as follows. Codons were selected to include residues from the LLB2 and LLB3 hits to increase the library positions screened based on residues useful for affinity maturation of these libraries. The libraries were screened against human CD98hc using phage display, resulting in 13 unique clones from these libraries with affinity for human and cynomolgus CD98hc (measured by surface plasmon resonance (SPR) using a Biacore™ machine). The affinities of the clones are shown in Table 9A, and the sequences of the clones are shown in Table A2. The affinities of these clones were measured in a bivalent format (i.e., without knob or hole mutations) using an anti-BACE1 Fab with full effector function (effector+) (i.e., without modifications to modulate effector function, such as LALA or PG / PS). The cell binding of the clones was also tested on HeLa cells for huCD98hc binding, CHO:cyCD98hc cells for cynomolgus binding, and CHO cells as a negative control.

[0400] Table 3: Libraries LLB2-AM1 and LLB2-AM2 TIFF2025503437000011.tif67160

[0401] Affinity for human and cynomolgus CD98hc was further engineered and improved for the LLB2 family using two additional libraries generated using LLB2-10 as the background to further explore sequence space (Table 4). Twenty-one clones were selected from these libraries using phage display on human CD98hc. The sequences of the selected clones are shown in Table A3, and their affinities for human and cynomolgus CD98hc (measured by surface plasmon resonance (SPR) using a Biacore™ machine) are shown in Table 9A. The affinity of the clones was measured with an anti-BACE1 Fab with intact effector function and in a bivalent format (i.e., without knob and hole mutations). Cell binding of the clones was also tested using HeLa cells for huCD98hc binding, CHO:cyCD98hc cells for cynomolgus binding, and CHO cells as a negative control.

[0402] Table 4: Libraries LLB2-AM3 and LLB2-AM4 TIFF2025503437000012.tif70167

[0403] Concurrent with the selection using LLB2-AM3 and LLB2-AM4, rational design modifications were made using the clone LLB2-10 background, including residues predicted to further improve affinity for human and cynomolgus monkey CD98hc. The clone sequences are shown in Table A4, and the clone affinities (measured by surface plasmon resonance (SPR) using a Biacore™ machine) are shown in Table 9A. The affinities of the clones were measured with an anti-BACE1 Fab in a bivalent format (i.e., without knob or hole mutations) with full effector function. Clones LLB2-10-5, 2-10-6, and 2-10-8 were converted to a monovalent format and tested for affinity to CD98hc by SPR using a Biacore™ machine with an anti-BACE1 Fab with full effector function on both sides and a CD98hc-binding site on the knob side (and no CD98hc-binding site on the hole side). The valency of the CD98hc binding molecules did not significantly affect the affinity for CD98hc, i.e., the difference was less than a factor of 2. Cell binding of the clones was also tested on HeLa cells for huCD98hc binding, CHO:cyCD98hc cells for cynomolgus binding, and CHO cells as a negative control.

[0404] Yeast display LLB2 soft library Using yeast display, we exploited the ability to select well-behaving clones using higher surface expression levels to improve the properties of the LLB2 family. To improve the properties of this family, we generated a soft-mutagenized library (70:10:10:10 oligo bias) (Table 5). To this end, we used a desired codon that mixed 70% of the original base and 10% of each of the other three bases. This resulted in approximately 50% of the original amino acids, with the remainder being a mixture of other amino acids. The original LLB2 clone backbone was used for soft mutagenesis, except that residue L380 was soft-mutated to a wild-type Glu residue and M428 was soft-mutated to a Leu residue. The library was assembled by two-step PCR and yeast homologous recombination. This library was then displayed on the yeast surface, and the highest 20% of expressing clones that also strongly bound to human CD98hc were selected. This screen yielded nine clones that were tested for binding to CD98hc by surface plasmon resonance (SPR) using a Biacore™ machine. See Table A5. The affinity of the clones was measured by surface plasmon resonance (SPR) using a Biacore™ machine with a non-binding Fab with intact effector function and in a bivalent format (i.e., without knob and hole mutations). Cell binding of the clones was also tested on HeLa cells for huCD98hc binding, CHO:cyCD98hc cells for cynomolgus binding, and CHO cells as a negative control.

[0405] Table 5. LLB2 soft mutagenesis library TIFF2025503437000013.tif41165

[0406] Rational design to improve PK of the LLB2 family The M428L mutation in the various LLB2 backbones appeared to contribute to poor HIC profiles and faster clearance in wild-type mice. This may be due to instability, lower specificity, and / or different interactions with mouse FcRn compared to wild-type IgG. Therefore, the clones in Table A6 were designed using previous clones that further engineered the M428Y mutation in the LLB2 background mutation. In addition, these clones also carry the E380L mutation. These clones were generated with full effector function and in a monovalent format (i.e., the CD98hc-binding site on the knob side and the CD98hc-binding site on the hole side).

[0407] LLB2-10-8 patch library for affinity maturation of binding to CD98hc The LLB2-10-8 lead clone was affinity matured using mutagenesis of four to five positions for every amino acid (NNK) within the patch around the structure. This was done to individually optimize each region and build a consensus for the best sequence. The 10 libraries are shown in Table 6 below, where NNK indicates the residue mutated in the background of the LL2-10-8 clone. The libraries were assembled by two-step PCR and yeast homologous recombination. The top 24 clones were selected for affinity measurement by surface plasmon resonance (SPR) using a Biacore™ machine. The affinities of the clones are shown in Table 9A, and the sequences of the clones are listed in Table A7. Clones were generated with intact effector function and in a monovalent format (i.e., the CD98hc binding site is on the knob side and not on the hole side). The cell binding of the clones was also tested on HeLa cells for huCD98hc binding, CHO:cyCD98hc cells for cynomolgus binding, and CHO cells as a negative control.

[0408] Table 6. LLB2 affinity matured / rational designed library TIFF2025503437000014.tif160165

[0409] LLB2-10-8 Patch Library To obtain affinity variants that bind CD98hc with weaker affinity than the LLB2-10-8 clone, single, double, or triple amino acid mutations were made at residues previously observed to bind CD98hc with weaker affinity (Table 7, Table A8). The variants were cloned, expressed, purified, and tested for their affinity to human and cynomolgus CD98hc by surface plasmon resonance (SPR) using a Biacore™ machine. The affinities of the clones are shown in Table 9A. Combining these mutations allowed the development of variants with a larger affinity range. Together, the LLB2 family exhibited K values ​​ranging from 15 nM to 5 μM for human CD98hc and 80 nM to 5 μM for cynomolgus CD98hc. d The clones were also tested for cell binding on HeLa cells for huCD98hc binding, CHO:cyCD98hc cells for cynomolgus binding, and CHO cells as a negative control.

[0410] Table 7. Diversity used in the rational design of LLB2 TIFF2025503437000015.tif41160

[0411] To obtain additional variants within the affinity range of 600-5000 nM, a second dematuration round was performed using the LLB2-10-8-d6, LLB2-10-8-d12, and LLB2-10-8-d18 clones as templates, except for the following changes, which, based on previous data, were predicted to reduce affinity for human CD98hc: position 382 was either maintained as N or changed to S; position 385 alternated between Y and F; position 386 alternated between V, E, and Q; and position 387 was either maintained as L or alternated to P. Variants were generated in combinations not previously tested. Variants binding with measurable affinity from this round of engineering are listed in Table A12, and the corresponding human binding affinities measured by surface plasmon resonance (SPR) using a Biacore™ machine are listed in Table 9B. Cell binding of the clones was also tested on HeLa cells for huCD98hc binding and CHO cells as a negative control.

[0412] LLB2 family amino acid consensus Table 8 below lists residues in the LLB2 family that enable binding to CD98hc.

[0413] Table 8. Amino acids tolerated in the LLB2 family TIFF2025503437000016.tif56165

[0414] To understand the nature of the interaction between the CD98hc-binding agents described herein and CD98hc, the bivalent LLB2-10-6 CD98hc-binding agent and the bivalent LLB1-3-16 CD98hc-binding agent (neither of which had any Fab attached) were co-crystallized with the human CD98hc extracellular domain (ECD) at 2.25 Å resolution. The structures show that the CD98hc-binding agents bind to CD98hc on an engineered surface, to epitopes on CD98hc in the structured loop region adjacent to the alpha / beta barrel structure (Figures 28A and 28B). The CD98hc epitope for LLB2 is shown in Figure 42A, and the CD98hc epitope for LLB1 is shown in Figure 42B. Furthermore, we used the crystal structure to generate a model of how FcRn binds to CD98hc-binding agents in the presence and absence of CD98hc, suggesting that FcRn can bind in the absence of CD98hc but not in the presence of CD98hc (Figures 29A and 29B). We also generated a model of the interaction between CD98hc-binding agents and CD98hc in complex with LAT1 at the membrane. A model of a monovalent CD98hc-binding agent bound to the CD98hc / LAT1 complex indicates that the CD98hc-binding agent can readily bind to CD98hc on the surface (Figure 30C). Furthermore, the model suggests that one bivalent TV can bind to two CD98hc / LAT1 complexes, albeit at extreme angles on opposing membranes (Figures 30A and 30B).

[0415] Table 9A. Affinity measurements of LLB2 variants TIFF2025503437000017.tif184165TIFF2025503437000018.tif221165TIFF2025503437000019.tif221165TIFF2025503437000020.tif213165

[0416] Table 9B. Affinity measurements of additional LLB2 variants TIFF2025503437000021.tif96165

[0417] (Table A2) Hits from the engineering rounds of LLB2-AM1 and LLB-AM2 TIFF2025503437000022.tif46165

[0418] (Table A3) Hits from the engineering rounds of LLB2-AM3 and LLB-AM4 TIFF2025503437000023.tif68165

[0419] Table A4: Hits from the rational design engineering round of LLB2-10-X TIFF2025503437000024.tif53165

[0420] Table A5. Hits from soft mutagenesis of LLB2 by yeast display TIFF2025503437000025.tif36165

[0421] (Table A6) Hits from rational design engineering rounds of M428Y or E380+M428Y TIFF2025503437000026.tif37165

[0422] Table A7: Patch library for LLB2 design screened by yeast display TIFF2025503437000027.tif87165

[0423] Table A8: LLB2 library for affinity non-maturation engineering rounds using rational design TIFF2025503437000028.tif90165

[0424] Table A12: LLB2 library for additional rounds of affinity non-maturation engineering TIFF2025503437000029.tif154165

[0425] Example 3. Generation of LLB1 family CD98HC binders LLB1 family affinity maturation (LLB1-AM1 and LLB1-AM2) Two phage display libraries (LLB1-AM1 and LLB1-AM2) were generated using residues found in the LLB1 family of clones (i.e., LLB1, LLB6, and LLB7) to increase affinity for CD98hc (Table 10). LLB1-AM1 did not expand the number of positions in the library. LLB1-AM2 expanded to residues 428 and 434. These libraries were generated using Kunkel mutagenesis and screened against human CD98hc using phage display. Sixteen clones were selected for recombinant expression that possessed anti-BACE1 Fabs with intact effector function and in a bivalent format (i.e., without knob and hole mutations). The sequences of the clones are shown in Table A9. These clones were shown to bind to human CD98hc but not to cynomolgus monkey CD98hc. The lead clone was reformatted in a monovalent format, designated LLB1-3 monovalent, with an anti-BACE1 Fab with intact effector function and a knob-only CD98hc binding site, and its affinity for human CD98hc was measured by surface plasmon resonance (SPR) using a Biacore™ machine. The valency of the CD98hc-binding molecule did not significantly affect affinity for CD98hc, i.e., there was less than a two-fold difference. Cell binding of the clones was also tested on HeLa cells for huCD98hc binding, CHO:cyCD98hc cells for cynomolgus monkey binding, and CHO cells as a negative control.

[0426] Table 10. LLB1-AM1 and LLB2-AM2 affinity maturation libraries TIFF2025503437000030.tif98165

[0427] Table A9: LLB1 affinity maturation libraries AM1 and AM2 TIFF2025503437000031.tif54165

[0428] LLB1 family affinity maturation (LLB1-AM3 and LLB1-AM4) A second round of affinity maturation using phage display was performed to increase the affinity of the LLB1 family for CD98hc (Table 11). The backbone of the library was clone LLB1-3. Library LLB1-AM3 expanded the library positions from the original clone using NHK or ARY. Library LLB1-AM4 expanded the library to include a mixture of residues previously found at those positions within the LLB1 family. The library was assembled using Kunkel mutagenesis and screened against human CD98hc. Twenty clones were selected, and affinity was measured by surface plasmon resonance (SPR) using a Biacore™ machine. The sequences of the clones are shown in Table A10, and the affinities of the clones are shown in Table 12A. The format was an anti-BACE1 Fab with full effector function and in a bivalent format (i.e., without knob and hole mutations). The cell binding of the clones was also tested on HeLa cells for huCD98hc binding, CHO:cyCD98hc cells for cynomolgus binding, and CHO cells as a negative control.

[0429] Table 11. LLB1-AM3 and LLB2-AM4 affinity maturation libraries TIFF2025503437000032.tif110165

[0430] Table A10: LLB1 library for AM3 and AM4 engineering rounds TIFF2025503437000033.tif58165

[0431] LLB1 Rational Design and PK Fix Clone 1-3-16 was generated using all beneficial mutations for binding from the previous library and reverting E380 to wild type to improve PK. Additional clones were generated with E380 reduced to wild type. The sequences and affinities of these clones are shown in Table A11 and Table 12A. Cell binding of the clones was also tested on HeLa cells for huCD98hc binding, CHO:cyCD98hc cells for cynomolgus binding, and CHO cells as a negative control.

[0432] Engineering LLB1 for cynomolgus cross-reactivity To engineer the LLB1 family for cynomolgus monkey cross-reactivity, new libraries were designed using the LLB1-3-16 clone as a template. In some libraries, right-register position 382 was retained as R or NNK; position 383 was primarily retained as S or T, occasionally altered to NNK; position 384 was primarily altered to NNK, with a few cases restricted to Y; position 385 was fixed to NNK; position 386 was retained as P, occasionally altered to NNK; position 387 was randomized to NNK; position 388 was unchanged or fixed to E; and position 389 was switched between N and T. Left-register position 424 was mostly fixed to V, with some alteration to NNK; and position 440 was fixed to K, occasionally altered only to NNK. Library sizes were kept small, down to 1e6, and they were screened using yeast display technology. The affinities of the resulting clones are shown in Table 12B, and the sequences of the clones are shown in Table A13. The affinity of these clones was measured in a bivalent format (i.e., without knob or hole mutations) using a non-binding Fab with full effector function (effector+) (i.e., without modifications to modulate effector function, such as LALA or PG / PS). Cell binding of the clones was also tested in HeLa cells for huCD98hc binding and CHO cells as a negative control.

[0433] Table 12A. Affinity measurements of LLB1 variants TIFF2025503437000034.tif151165TIFF2025503437000035.tif194165

[0434] Table 12B. Affinity measurements of additional LLB2 variants TIFF2025503437000036.tif97165

[0435] Table A11: Rational design and PK fix engineering rounds for LLB1 TIFF2025503437000037.tif44165

[0436] Table A13: LLB1 library engineered for cynomolgus cross-reactivity TIFF2025503437000038.tif218165

[0437] Example 4. Plasma PK of LLB2 and LLB1 CD98HC binders To characterize the CD98hc-binding molecules, pharmacokinetics (PK) was evaluated in wild-type mice to demonstrate in vivo stability in a model lacking CD98hc-mediated clearance, since these CD98hc-binding molecules bind only to human CD98hc but not to mouse CD98hc. The study design is shown in Table 13 below. Six- to eight-week-old C57B16 (WT) mice were intravenously administered, and in-life bleeding was collected via submandibular bleed at the time points indicated in Table 13 below. Blood was collected into EDTA plasma tubes and centrifuged at 14,000 rpm for 5 minutes, after which plasma was separated for subsequent analysis.

[0438] (Table 13) Study design TIFF2025503437000039.tif1...

Claims

1. An Fc polypeptide comprising a modified CH3 domain that specifically binds to human CD98hc protein and can be transported across the blood-brain barrier (BBB).

2. The modified CH3 domain (a) containing at least 11, 12, 13, 14, or 15 substitutions at the set of amino acid positions consisting of 380, 382, ​​384, 385, 386, 387, 421, 422, 424, 426, 428, 436, 438, 440, and 442 according to EU numbering; (b) containing at least 8, 9, 10, 11, 12, or 13 substitutions at the set of amino acid positions consisting of 380, 382, ​​384, 385, 386, 387, 422, 424, 426, 428, 434, 438, and 440 according to EU numbering; (c) containing at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitutions at the set of amino acid positions consisting of 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 434, 436, 438, 440, and 442 according to EU numbering; (d) containing at least 8, 9, 10, 11, 12, 13, 14, or 15 substitutions at the set of amino acid positions consisting of 382, ​​383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 436, 438, and 440 according to EU numbering; The Fc polypeptide of claim 1.

3. 3. The Fc polypeptide of claim 2, wherein the modified CH3 domain comprises a sequence having at least 85%, 90%, or 95% sequence identity to amino acids 111-217 of any one of SEQ ID NOs: 28-43, and the modified CH3 domain comprises at least 11, 12, 13, 14, or 15 substitutions at the set of amino acid positions consisting of: L at position 380, N at position 382, ​​R, H, or Q at position 384, F or Y at position 385, V, L, I, F, Y, or E at position 386, L at position 387, E, Q, or A at position 421, I, T, or P at position 422, A at position 424, N at position 426, Y or W at position 428, R or W at position 436, F or W at position 438, N at position 440, and A, Q, K, R, H, or M at position 442.

4. the modified CH3 domain is (a) containing L at position 380, N at position 382, ​​H at position 384, Y at position 385, E at position 386, L at position 387, I at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and A at position 442; (b) containing L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, E at position 421, I at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and A at position 442; (c) containing L at position 380, N at position 382, ​​H at position 384, Y at position 385, E at position 386, L at position 387, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and A at position 442; (d) containing L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and A at position 442; (e) containing L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, I at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and R at position 442; (f) containing L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, I at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and H at position 442; (g) containing L at position 380, N at position 382, ​​H at position 384, Y at position 385, E at position 386, L at position 387, E at position 421, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and A at position 442; (h) containing L at position 380, N at position 382, ​​Q at position 384, Y at position 385, E at position 386, L at position 387, E at position 421, I at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and A at position 442; (i) containing L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, I at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and A at position 442; (j) containing L at position 380, N at position 382, ​​Q at position 384, Y at position 385, E at position 386, L at position 387, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and A at position 442; (k) containing L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, I at position 422, A at position 424, N at position 426, Y at position 428, R at position 436, F at position 438, N at position 440, and R at position 442; (l) containing L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, I at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and K at position 442; (m) containing L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, I at position 422, A at position 424, N at position 426, Y at position 428, W at position 436, F at position 438, N at position 440, and R at position 442; (n) containing L at position 380, N at position 382, ​​Q at position 384, Y at position 385, L at position 386, L at position 387, E at position 421, I at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and A at position 442; (o) containing L at position 380, S at position 382, ​​R at position 384, Y at position 385, V at position 386, L at position 387, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and A at position 442; (p) containing L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, E at position 421, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and A at position 442; (q) containing L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, E at position 421, I at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, and N at position 440; (r) containing L at position 380, N at position 382, ​​Q at position 384, F at position 385, H at position 386, L at position 387, I at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and L at position 442; (s) containing L at position 380, N at position 382, ​​R at position 384, F at position 385, V at position 386, L at position 387, T at position 422, A at position 424, N at position 426, Y at position 428, F at position 438, N at position 440, and A at position 442; The Fc polypeptide of claim 2.

5. 3. The Fc polypeptide of claim 2, wherein the modified CH3 domain comprises any of the sequences set forth in SEQ ID NOs: 38, 29, 31, 32, 35, 36, 28, 30, 37, 41, 42, 43, 33, 34, 39, and 40.

6. The modified CH3 domain, (a) comprising D, M, N, P, F, or H at position 380, R, Y, F, S, W, Y, K, or N at position 382, ​​L, Y, A, S, or F at position 384, F, K, D, M, I, N, Y, L, or H at position 385, T, P, E, K, A, V, D, T, or F at position 386, N, L, Y, R, G, S, D, or T at position 387, I, K, R, T, F, or H at position 422, V, W, G, L, I, P, or Y at position 424, D, A, Q, W, L, or P at position 426, L at position 428, S at position 434, I, F, N, P, or S at position 438, and K, T, I, or F at position 440; (b) containing S or V at position 378, D at position 380, R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T, Y, or F at position 389, D, E, or Q at position 421, I at position 422, V at position 424, D at position 426, L or Y at position 428, S at position 434, F at position 436, I or V at position 438, K at position 440, and Q or M at position 442; (c) a sequence having at least 85%, 90%, or 95% sequence identity to amino acids 111-217 of any one of SEQ ID NOs: 44-45, wherein the modified CH3 domain comprises at least 8, 9, 10, 11, 12, 13, 14, or 15 substitutions at the following amino acid positions: R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T at position 389, D at position 421, I at position 422, V at position 424, D at position 426, L at position 428, F at position 436, I at position 438, and K at position 440; (d) containing R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T at position 389, D at position 421, I at position 422, V at position 424, D at position 426, F at position 436, I at position 438, and K at position 440; (e) containing R at position 382, ​​T at position 383, Y at position 384, K at position 385, P at position 386, Y at position 387, T at position 389, D at position 421, I at position 422, V at position 424, D at position 426, L at position 428, F at position 436, I at position 438, and K at position 440; or (f) SEQ ID NO: 44 or 45; The Fc polypeptide of claim 2.

7. The modified CH3 domain of claim 1, wherein: (a) (i) LX at amino acid positions 382-389 １ NX ２ X ３ X ４ X ５ L (SEQ ID NO:46), X １ is any amino acid, X ２ is R, H, or Q; X ３ is F or Y, X ４ is V, L, I, F, Y, or E, and X ５ is any amino acid, the first amino acid sequence; (ii) X at amino acid positions 421-428 １ X ２ X ３ AX ４ X ５ X ６ X ７ (SEQ ID NO: 47), X １ is E, N, Q, or A; X ２ is I, V, T, or P; X ３ and X ４ is any amino acid, X ５ is N or S, X ６ is any amino acid, and X ７ is Y or W; the second amino acid sequence; and (iii) X at amino acid positions 436-442 １ X ２ X ３ X ４ NX ５ X ６ (SEQ ID NO: 48), X １ is Y, R, or W; X ２ is any amino acid, X ３ is F or W, X ４ and X ５ is any amino acid, and X ６ is A, Q, K, R, H, M, or S; the third amino acid sequence; and Contains, or (b)(i) a first amino acid sequence that is X 1 X 2 YKPYX 3 T (SEQ ID NO:49) at amino acid positions 382-389; X 1 is E or R; X 2 is S or T, and X3 is any amino acid; the first amino acid sequence; (ii) a second amino acid sequence at amino acid positions 421-428 that is X 1 X 2 X 3 VX 4 DX 5 X 6 (SEQ ID NO: 50), X 1 is N or D; X 2 is V or I; X 3 , X 4 , and X 5 are any amino acid; and X 6 is M or L; the second amino acid sequence; and (iii) a third amino acid sequence at amino acid positions 436-440 which is X 1 X 2 IX 3 X 4 (SEQ ID NO: 51), X 1 is Y or F; X 2 and X 3 are any amino acid, and X 4 is S or K; the third amino acid sequence; and Including, The Fc polypeptide of claim 1.

8. The Fc polypeptide of claim 1, wherein: (a) the Fc polypeptide binds to human CD98hc protein with an affinity of 15 nM to 5 μM; (b) the Fc polypeptide has cynomolgus monkey CD98hc cross-reactivity; and / or (c) The Fc polypeptide binds to cynomolgus CD98hc with an affinity of 80 nM to 5 μM.

9. 3. The Fc polypeptide of claim 2, wherein the modified CH3 domain further comprises at least one modification that promotes heterodimerization.

10. the at least one modification that promotes heterodimerization is (a) T366W substitution according to EU numbering; or (b) T366S, L368A, and Y407V substitutions according to EU numbering 10. The Fc polypeptide of claim 9, comprising:

11. 2. The Fc polypeptide of claim 1, comprising L at position 428 and S at position 434.

12. The Fc polypeptide of claim 1, wherein the Fc polypeptide further comprises: (a) one or more modifications that reduce effector function; (b) Ala at position 234 and Ala at position 235 according to EU numbering; (c) Gly or Ser at position 329 according to EU numbering; (d) Ala at position 234, Ala at position 235, and Gly at position 329 according to EU numbering; or (e) Ala at position 234, Ala at position 235, and Ser at position 329 according to EU numbering.

13. An Fc polypeptide described in claim 1, wherein the Fc polypeptide forms a dimer with a second Fc polypeptide.

14. The second Fc polypeptide, (a) comprises a modified CH3 domain that specifically binds to human CD98hc protein such that the dimer is bivalent with respect to CD98hc binding; or (b) does not contain a modified CH3 domain that specifically binds to human CD98hc protein such that the dimer is bivalent with respect to CD98hc binding; 14. The Fc polypeptide of claim 13.

15. The Fc polypeptide of claim 1 further linked to a Fab.

16. 2. The Fc polypeptide of claim 1, wherein the C-terminal lysine of the polypeptide is absent or removed.

17. The Fc polypeptide of claim 1, wherein the Fc polypeptide is conjugated to a therapeutic agent.

18. A polynucleotide or vector comprising a nucleic acid sequence encoding the Fc polypeptide of any one of claims 1 to 17.

19. A host cell comprising the polynucleotide or vector of claim 18.

20. A pharmaceutical composition comprising the Fc polypeptide of any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

21. The Fc polypeptide of claim 17 for use in: (a) increasing transcellular transport of the therapeutic agent across the endothelium or the BBB; (b) delivering said therapeutic agent to the brain parenchyma; or (c) delivering the therapeutic agent to a biological target in the brain.

22. The Fc polypeptide for use in claim 21, wherein the biological target comprises: (a) a cell surface target selected from the group consisting of TREM2, PILRA, CD33, CR1, ABCA1, ABCA7, MS4A4A, MS4A6A, MS4A4E, HLA-DR5, HLA-DR1, IL1RAP, TREML2, IL-34, SORL1, ADAM17, and Siglec11; (b) cell surface targets on hematological cancer cells; (c) a cell surface target on a hematological cancer cell selected from the group consisting of B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b; (d) tumor cells; (e) ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, a cell surface target in tumor cells selected from the group consisting of ETBR, FGFR(1-4), folate receptor alpha, GAL-3BP (galectin-binding protein), GD2, GD3, GloboH (globohexacylceramide), gp100, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine-rich repeat-containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4, colon cancer kinase), RON, ROR1, TF (tissue factor), and TROP2; (f) alpha-synuclein or a derivative or fragment thereof, a derivative of amyloid-beta peptide or a fragment thereof, Tau or a derivative or fragment thereof, pTau, huntingtin, transthyretin, or TAR DNA binding protein 43 (TDP-43) or a derivative or fragment thereof; (g) extracellular targets in the brain; (h) astrocytes, microglia, oligodendrocytes, plaques, tangles, or non-neuronal targets.

23. An Fc polypeptide as described in claim 17 for use in delivering a therapeutic agent to a peripheral CD98hc-expressing organ, wherein the peripheral CD98hc-expressing organ is the kidney, testis, bone marrow, spleen, or pancreas.

24. A monovalent molecule that binds to CD98hc with a binding affinity of about 20 nM to about 550 nM, or a bivalent molecule that binds to CD98hc with a binding affinity of about 275 nM to about 2100 nM, conjugated to a therapeutic agent for use in increasing brain exposure to the therapeutic agent.

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