CN118591553A

CN118591553A - Polypeptide engineering, libraries and engineering CD98 heavy chains and transferrin receptor binding polypeptides

Info

Publication number: CN118591553A
Application number: CN202280087521.8A
Authority: CN
Inventors: 帕德玛·阿卡佩迪; 杰拉尔德·麦克斯韦·切尔夫; 凯利·S·丘; 马克·S·丹尼斯; 谷振宇; 米哈利斯·S·卡里奥利斯; 海·L·特兰; 罗伯特·C·威尔斯; 乔伊·余·祖切罗
Original assignee: Denali Therapeutics Inc
Current assignee: Denali Therapeutics Inc
Priority date: 2021-12-17
Filing date: 2022-12-16
Publication date: 2024-09-03

Abstract

The present disclosure includes engineering methods and libraries of polypeptides that can be used to introduce non-native binding sites into polypeptides. Also provided herein are polypeptides that bind to CD98hc or transferrin receptor (TfR) proteins, methods of producing such polypeptides, and methods of using the polypeptides to target compositions across the blood-brain barrier or to CD98 hc-expressing cells or TfR-expressing cells.

Description

Polypeptide engineering, libraries and engineering CD98 heavy chains and transferrin receptor binding polypeptides

Cross Reference to Related Applications

The present application claims priority from U.S. provisional patent application No. 63/291,161, filed on 12 months 17, 2021, and U.S. provisional patent application No. 63/423,418, filed on 11 months 7, 2022, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

Background

Various techniques have been developed to engineer proteins to bind to targets that would not normally bind. For example, libraries can be generated to screen for engineered proteins having a desired binding or enzymatic activity.

Disclosure of Invention

We have developed several methods to find polypeptides with novel binding sites, particularly those comprising the β -sheet portion of the binding site. These methods include the development of β -sheet libraries, and the use of "limited reliability" methods to reduce the frequency of amino acids that can produce proteins with undesirable characteristics. We have used these types of libraries to find polypeptides that bind to targets such as CD98 heavy chain (CD 98 hc) and transferrin receptor (TfR), as detailed below. We have also developed delivery methods using CD98hc polypeptides (e.g., across the blood brain barrier), particularly extracellular targets in the brain.

In one aspect, the present disclosure provides a method of engineering a non-native binding site into a polypeptide, the method comprising:

(a) Generating a library of polypeptides, wherein at least a portion of the polypeptides comprise at least seven randomized positions, wherein 10% -60% of the randomized positions have one or more of the following amino acids deleted: cys, trp, met, arg or Gly, but includes a diversity of 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; and

(D) Isolating the selected library members, thereby engineering non-native binding sites into the polypeptide.

In some embodiments, the method comprises repeating steps (b) - (d) using library members isolated from the first step (d). 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 comprises positions of limited diversity separated by positions of no limited diversity.

In some embodiments, each polypeptide comprises a β -sheet and at least three of the randomized positions are present in a single β -sheet. In certain embodiments, at least three of the randomized positions are present within at least two β -strands forming a β -sheet. In certain embodiments, at least three of the randomized positions are present within at least one β -strand forming a β -sheet. In some embodiments, at least three of the randomized positions form a surface on one side of the β -sheet. In certain embodiments, at least three of the randomized positions are surface exposed. In some embodiments, the β -sheet comprises at least one position with limited diversity. In certain embodiments, the β -sheet comprises at least two positions with limited diversity. In certain embodiments, at least two locations with limited diversity are separated by a location that does not have limited diversity. In some embodiments, the β -sheet comprises at least two positions that do not have limited diversity. In certain embodiments, at least two locations that do not have limited diversity are separated by a location that has limited diversity. In particular embodiments, the separation is relative to the primary amino acid sequence of the polypeptide or relative to the spatial three-dimensional localization of the amino acids in the protein structure.

In some embodiments, positions with limited diversity are encoded by degenerate codons. In certain embodiments, at least one of the degenerate codons is NHK. In some embodiments, positions that do not have limited diversity are encoded by degenerate codon NNK.

In some embodiments, the polypeptide comprises an immunoglobulin-like fold. In certain embodiments, the polypeptide comprises an immunoglobulin (IgG) domain. In certain embodiments, the IgG domain is from IgG, igA, igE, igM or IgD families. In certain embodiments, the IgG domain is selected from an IgG1, igG2, igG3, or IgG4 molecule. In particular embodiments, the IgG domain comprises a VH, CH1, CH2, CH3, VL, or CL domain. In some embodiments, the randomized positions are surface accessible. In a particular embodiment, 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.

In another aspect, the present disclosure provides a library of polypeptides, wherein at least a portion of the polypeptides comprise at least seven randomized positions, wherein 10% -60% of the randomized positions have one or more of the following amino acids that are limited to being excluded: cys, trp, met, arg or Gly, but includes a diversity of at least eight amino acids at each position.

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 comprises positions of limited diversity separated by positions of no limited diversity.

In some embodiments, each polypeptide comprises a β -sheet and at least three of the randomized positions are present in a single β -sheet. In certain embodiments, at least three of the randomized positions are present within at least two β -strands forming a β -sheet. In certain embodiments, at least three of the randomized positions are present within at least one β -strand forming a β -sheet. In some embodiments, at least three of the randomized positions form a surface on one side of the β -sheet. In certain embodiments, at least three of the randomized positions are surface exposed. In some embodiments, the β -sheet comprises at least one position with limited diversity. In certain embodiments, the β -sheet comprises at least two positions with limited diversity. In certain embodiments, at least two locations with limited diversity are separated by a location that does not have limited diversity. In particular embodiments, the separation is relative to the primary sequence of the polypeptide or relative to the spatial three-dimensional localization of the amino acids in the protein structure. In some embodiments, the β -sheet comprises at least two positions that do not have limited diversity. In certain embodiments, at least two locations that do not have limited diversity are separated by a location that has limited diversity. In particular embodiments, the separation is relative to the primary sequence of the polypeptide or relative to the spatial three-dimensional localization of the amino acids in the protein structure.

In another aspect, the present disclosure provides a polypeptide comprising a constant domain or a non-CDR portion of a variable domain of an immunoglobulin having at least three modified positions in a β -sheet, wherein:

(i) The modified positions are located in at least two β -strands forming the β -sheet;

(ii) The modified position forms at least a portion of a binding site capable of binding to an antigen; and

(Iii) The β -sheet does not bind to antigens that do not have modified positions.

In some embodiments, the constant domain comprises an Fc polypeptide. In some embodiments, at least two β -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, wherein positions are determined according to EU numbering. In certain embodiments, the location is surface accessible. In certain embodiments, the positions are selected from those listed in table 1B.

In some embodiments, the modified positions form a continuous surface on the β -sheet. In some embodiments, the modified position is a surface accessible residue. 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, wherein 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, wherein the positions are determined according to EU numbering.

In some embodiments, the modified positions comprise three, four, five, six, or seven amino acid substitutions in a set of amino acid positions comprising 380, 382, 383, 424, 426, 438, and 440, wherein the positions are determined according to EU numbering. In some embodiments, the modified positions comprise three, four, five, six, seven, eight, nine, ten, or eleven amino acid substitutions in a set of amino acid positions comprising 378, 380, 382, 383, 422, 424, 426, 428, 438, 440, and 442, wherein the positions are determined according to EU numbering.

In some embodiments, the binding site comprises one or more modified positions in at least one loop region. In certain embodiments, one or more modified positions in at least one loop region are selected from the group consisting of: amino acid positions 387 and 422, where positions are determined according to EU numbering. In certain embodiments, the loop region connects two β -strands.

In another aspect, the present disclosure provides a method of introducing a non-natural binding site into a non-CDR region of a constant domain or variable domain of an immunoglobulin, the method comprising:

(a) Generating a library of polynucleotides encoding immunoglobulin sequences having at least three modified positions in a β -sheet, wherein the library is randomized at codons encoding amino acids at the modified positions, wherein the modified positions are located in at least two β -strands forming the β -sheet;

(b) Expressing the library to produce a library of sequence variants;

(c) Contacting the sequence variant with a target protein; and

(D) Sequence variants that bind to the target protein are isolated, thereby introducing a non-native binding site into the constant domain or non-CDR regions of the variable domain of the immunoglobulin.

In some embodiments, the immunoglobulin sequence comprises an Fc polypeptide. In some embodiments, at least two β -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.

In another aspect, the present disclosure provides a library of immunoglobulin variants comprising at least ten members, wherein the variants each comprise at least three modified positions in a β -sheet forming part of a constant domain or a non-CDR variable domain of an immunoglobulin, wherein the modified positions are located in at least two β -strands forming the β -sheet.

In some embodiments, the library of immunoglobulin variants comprises at least 10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹² or more members. In some embodiments, the library is generated from a set of encoding polynucleotides encoding at least seven randomized amino acid positions, wherein 10% -60% of the randomized positions have one or more of the following amino acids deleted: cys, trp, met, arg or Gly, but includes a diversity of at least eight amino acids at each position. In certain embodiments, at least one of the diversity-limited positions does not encode tryptophan or cysteine. In certain embodiments, at least two diversity-limited positions do not encode tryptophan or cysteine. In certain embodiments, at least two diversity-limited positions do not encode tryptophan, cysteine, or arginine.

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

In some embodiments, the disclosure provides a polynucleotide library encoding immunoglobulin variants from the libraries described herein.

In another aspect, the present disclosure provides a method of engineering a non-native binding site of a transferrin receptor (TfR) or CD98hc protein into a polypeptide, the method comprising:

(a) Generating a library of polypeptides, wherein at least a portion of the polypeptides comprise at least seven randomized positions, wherein 10% -60% of the randomized positions have one or more of the following amino acids restricted to exclusion: cys, trp, met, arg or Gly, but includes a diversity of 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; and

(D) The selected library members are isolated, thereby engineering the non-native binding site of TfR or CD98hc into the polypeptide.

In some embodiments of this aspect, the method comprises repeating steps (b) - (d) using library members isolated from the first step (d).

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 comprises positions of limited diversity separated by positions of no limited diversity. In certain embodiments, each polypeptide comprises a β -sheet and at least three of the randomized positions are present in a single β -sheet. In certain embodiments, at least three of the randomized positions are present within at least two β -strands forming a β -sheet. In certain embodiments, at least three of the randomized positions are present within at least one β -strand forming a β -sheet. In certain embodiments, at least three of the randomized positions form a surface on one side of the β -sheet. In certain embodiments, at least three of the randomized positions are surface exposed.

In some embodiments of this aspect, the β -sheet comprises at least one position with limited diversity. In some embodiments, the β -sheet comprises at least two positions with limited diversity. In some embodiments, at least two locations with limited diversity are separated by a location that does not have limited diversity.

In some embodiments, the β -sheet comprises at least two positions that do not have limited diversity. In certain embodiments, at least two locations that do not have limited diversity are separated by a location that has limited diversity.

In some embodiments, the separation is relative to the primary amino acid sequence of the polypeptide or relative to the spatial three-dimensional localization of the amino acids in the protein structure.

In some embodiments, positions with limited diversity are encoded by degenerate codons. In certain embodiments, at least one of the degenerate codons is NHK. In certain embodiments, positions that do not have limited diversity are encoded by degenerate codon NNK.

In some embodiments of this aspect, the polypeptide comprises an immunoglobulin-like fold. In some embodiments, the polypeptide comprises an immunoglobulin (IgG) domain. In certain embodiments, the IgG domain is from IgG, igA, igE, igM or IgD families. In certain embodiments, the IgG domain is selected from IgG1, and IgG2, and IgG3 or IgG4 molecules. 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.

In another aspect, the present disclosure provides a polypeptide having at least three modified positions in the β -sheet portion, wherein:

(i) The modified positions are located in at least two β -strands forming a β -sheet;

(ii) The modified position forms at least a portion of a binding site capable of binding to CD98 hc; and

In some embodiments of this aspect, the polypeptide comprises at least 4 or 5 modified positions in the β -sheet. In some embodiments, the polypeptide comprises at least seven modified positions that form at least a portion of a binding site capable of binding CD98 hc. In some embodiments, the polypeptide comprises an immunoglobulin-like fold. In certain embodiments, the polypeptide comprises an immunoglobulin (IgG) domain. In certain embodiments, the IgG domain is from IgG, igA, igE, igM or IgD families. In particular embodiments, the IgG domain is selected from IgG1, and IgG2, and IgG3 or IgG4 molecules. In certain embodiments, the IgG domain comprises a VH, CH1, CH2, CH3, VL, or CL domain.

In some embodiments, the modified location is surface accessible. In some embodiments, the modified 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.

(ii) The modified position forms at least a portion of a binding site capable of binding to a TfR or CD98hc protein; and

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

In some embodiments, at least two β -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, wherein positions are determined according to EU numbering. In some embodiments, the location is surface accessible. In certain embodiments, the positions are selected from those listed in table 1B.

In some embodiments, the modified positions form a continuous surface on the β -sheet.

In some embodiments, the modified position is a surface accessible residue. 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, wherein 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, wherein the positions are determined according to EU numbering.

In some embodiments, the modified positions comprise three, four, five, six, or seven amino acid substitutions in a set of amino acid positions comprising 380, 382, 383, 424, 426, 438, and 440, wherein the positions are determined according to EU numbering. In some embodiments, the modified positions comprise three, four, five, six, seven, eight, nine, ten, or eleven amino acid substitutions in a set of amino acid positions comprising 378, 380, 382, 383, 422, 424, 426, 428, 438, 440, and 442, wherein the positions are determined according to EU numbering. In certain embodiments, the binding site comprises one or more modified positions in at least one loop region. In certain embodiments, one or more modified positions in at least one loop region are selected from the group consisting of: amino acid positions 387 and 422, where positions are determined according to EU numbering. In certain embodiments, the loop region connects two β -strands.

In another aspect, the present disclosure provides a method of introducing a non-native binding site of a TfR or CD98hc protein into a non-CDR region of a constant domain or variable domain of an immunoglobulin, the method comprising:

(b) Expressing the library to produce a library of sequence variants;

(c) Contacting the sequence variant with a TfR or CD98hc protein; and

(D) Sequence variants that bind to the TfR or CD98hc protein are isolated, thereby introducing a non-native binding site into the non-CDR regions of the constant or variable domains of the immunoglobulin.

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

In some embodiments, at least two β -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.

In some embodiments, the binding site comprises one or more modified positions in at least one loop region. In some embodiments, the one or more modified positions in the at least one loop region are selected from the group consisting of: amino acid positions 387 and 422, where positions are determined according to EU numbering.

In another aspect, the present disclosure provides a method of introducing a CD98hc binding site into a polypeptide comprising a β -sheet, the method comprising:

(a) Generating a library of polynucleotides encoding a polypeptide sequence having at least three modified positions in a β -sheet, wherein the library is randomized at codons encoding amino acids at the modified positions, wherein the modified positions are located in at least two β -strands forming the β -sheet;

(b) Expressing the library to produce a library of sequence variants;

(c) Contacting the sequence variant with at least a portion of a CD98hc protein; and

(D) Sequence variants were isolated that bound to Cd98hc protein.

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 β -sheet. In some embodiments, the polypeptide has at least 7 modified positions in the β -sheet. In some embodiments, the polypeptide has at least 10 modified positions in the β -sheet.

In certain embodiments, the binding site comprises one or more modified positions in at least one loop region.

In some embodiments, the binding site comprises one or more β -sheet 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 in the β -sheet and loop regions.

In another aspect, the 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 CD98hc protein. In some embodiments, the modified CH3 domain is part of an Fc polypeptide. In a particular embodiment, the CD98hc protein is human CD98hc protein. In particular embodiments, the CD98hc protein forms a complex with LAT1 (SLC 7 A5), LAT2 (SLC 7 A8), y+lat1 (SLC 7 A7), y+lat2 (SLC 7 A6), asc-1 (SLC 7a 10), or xCT (SLC 7a 11). In certain embodiments, the CD98hc protein forms a complex with LAT1 (SLC 7 A5).

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

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 five, six, seven, eight, or nine substitutions in a set of amino acid positions consisting of 382, 384, 385, 387, 422, 424, 426, 438, 440; and wherein the position is determined with reference to EU numbering. In another aspect, the substitution is determined with reference to SEQ ID NO. 1.

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 eleven, twelve, thirteen, fourteen or fifteen substitutions in a set of amino acid positions consisting of 380, 382, 384, 385, 386, 387, 421, 422, 424, 426, 428, 436, 438, 440 and 442; and wherein the position is determined according to EU numbering. In another aspect, the substitution is determined with reference to SEQ ID NO. 1.

In some embodiments of this aspect, the modified constant domain (e.g., modified CH3 domain) comprises a sequence having at least 85%, 90%, or 95% sequence identity to amino acids 111-217 of the sequence of any of SEQ ID NOs 28-43, wherein the modified constant domain comprises at least eleven, twelve, thirteen, fourteen, or fifteen substitutions in a 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 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. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO. 28.

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, N at position 440, and a at position 442. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO. 29.

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, 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. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:30.

In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises 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. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO. 31.

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, 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. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO. 32.

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, 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. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO. 33.

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.

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.

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.

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.

In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises 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. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:38.

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.

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, 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. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO:40.

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.

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.

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, 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. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO. 43.

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:

(i) A first amino acid sequence LX ₁NX₂X₃X₄X₅ L (SEQ ID NO: 46), wherein 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;

(ii) A second amino acid sequence X ₁X₂X₃AX₄X₅X₆X₇ (SEQ ID NO: 47), wherein X ₁ is E, N, Q or A, X ₂ is I, V, T or P, X ₃ and X ₄ are any amino acids, X ₅ is N or S, X ₆ is any amino acid, and X ₇ is Y or W; and

(Iii) A third amino acid sequence X ₁X₂X₃X₄NX₅X₆ (SEQ ID NO: 48), wherein X ₁ is Y, R or W, X ₂ is any amino acid, X ₃ is F or W, X ₄ and X ₅ are any amino acids, and X ₆ is A, Q, K, R, H, M or S.

In some embodiments, the polypeptide binds human CD98hc with an affinity of 15nM to 5 μm (e.g., 15nM、50nM、100nM、200nM、300nM、400nM、500nM、600nM、700nM、800nM、900nM、1μM、1.5μM、2μM、2.5μM、3μM、3.5μM、4μM、4.5μM or 5 μm). In some embodiments, the polypeptide has cynomolgus monkey (cyno) cross-reactivity. In particular embodiments, the polypeptide binds to cyno CD98hc with an affinity of 80nM to 5 μm (e.g., 80nM, 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, 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 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 eight, nine, ten, eleven, twelve, or thirteen substitutions in a set of amino acid positions consisting of 380, 382, 384, 385, 386, 387, 422, 424, 426, 428, 434, 438, and 440; and wherein the substitution is determined with reference to SEQ ID NO.1 and the position is 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.

In another aspect, the disclosure features a polypeptide comprising a modified constant domain that specifically binds to a CD98hc protein (e.g., a modified CH3 domain), wherein the modified constant domain comprises at least eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen substitutions in a 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; and wherein the substitution is determined with reference to SEQ ID NO. 1 and the position is determined according to EU numbering. In some embodiments, the modified constant domain (e.g., 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.

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 eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen substitutions in a set of amino acid positions consisting of 382, 383, 384, 385, 386, 387, 389, 421, 422, 424, 426, 428, 436, 438 and 440; and wherein the substitution is determined with reference to SEQ ID NO. 1 and the position is determined according to EU numbering. In some embodiments, the modified constant domain (e.g., modified CH3 domain) comprises a sequence having at least 85%, 90%, or 95% sequence identity to amino acids 111-217 of the sequence of any one of SEQ ID NOs 44-45, wherein the modified constant domain comprises at least eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen substitutions in a set of amino acid positions consisting of: r at location 382, T at location 383, Y at location 384, K at location 385, P at location 386, Y at location 387, T at location 389, D at location 421, I at location 422, V at location 424, D at location 426, L at location 428, F at location 436, I at location 438, and K at location 440.

In some embodiments of this aspect, the modified constant domain (e.g., modified CH3 domain) comprises 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. In a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO 44.

In some embodiments of this aspect, the modified constant domain (e.g., modified CH3 domain) comprises 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 a particular embodiment, the modified constant domain (e.g., modified CH3 domain) comprises SEQ ID NO. 45.

(i) A first amino acid sequence X ₁X₂YKPYX₃ T (SEQ ID NO: 49), wherein X ₁ is E or R, X ₂ is S or T, and X ₃ is any amino acid;

(ii) A second amino acid sequence X ₁X₂X₃VX₄DX₅X₆ (SEQ ID NO: 50), wherein X ₁ is N or D, X ₂ is V or I, X ₃、X₄ and X ₅ are any amino acids, X ₆ is M or L; and

(Iii) The third amino acid sequence X ₁X₂IX₃X₄ (SEQ ID NO: 51), wherein X ₁ is Y or F, X ₂ and X ₃ are any amino acids, and X ₄ is S or K.

In another aspect, the disclosure features a polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises five, six, or seven amino acid substitutions in a set of amino acid positions comprising positions 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 a combination of G at position 437, F at position 438, and D at position 440, and wherein the positions are determined according to EU numbering.

In another aspect, the present disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises three, four, five, six, seven or eight amino acid substitutions and/or one or two amino acid deletions in a set of amino acid positions comprising positions 380 and 382-389 of an Fc polypeptide (e.g., SEQ ID NO: 1); and five, six or seven amino acid substitutions in a set of amino acid positions comprising positions 422, 424, 426, 433, 434, 438 and 440 of an Fc polypeptide (e.g., SEQ ID NO: 1), wherein the positions are determined according to EU numbering.

In another aspect, the disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises a sequence comprising at least one (e.g., one, two, three, four, five, six, or seven) amino acid substitution in sequence VFSCSVMHEALHNHYTQKS (SEQ ID NO: 57), wherein sequence SEQ ID NO:57 is from position 422 to position 440 of the Fc polypeptide (e.g., SEQ ID NO: 1), the sequence does not have a combination of G at position 437, F at position 438, and D at position 440, and the positions are determined according to EU numbering. In some embodiments of this aspect, the sequence comprises five, six, or seven amino acid substitutions in a set of amino acid positions comprising 422, 424, 426, 433, 434, 438, and 440.

In another aspect, the disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises a first sequence comprising at least one amino acid substitution (e.g., one, two, three, four, five, six, seven, or eight amino acid substitutions) and/or a deletion in sequence AVEWESNGQPENN (SEQ ID NO: 56), and a second sequence comprising at least one (e.g., one, two, three, four, five, six, or seven) amino acid substitution in sequence VFSCSVMHEALHNHYTQKS (SEQ ID NO: 57), wherein sequence SEQ ID NO:56 is from position 378 to position 390 of the Fc polypeptide (e.g., SEQ ID NO: 1), sequence SEQ ID NO:57 is from position 422 to position 440 of the Fc polypeptide (e.g., SEQ ID NO: 1), and position is determined according to EU numbering. In some embodiments of this aspect, the modified CH3 domain comprises three, four, five, six, seven or eight amino acid substitutions in a set of amino acid positions comprising 380 and 382-389. In some embodiments, the modified CH3 domain comprises five, six, or seven amino acid substitutions in a set of amino acid positions comprising 422, 424, 426, 433, 434, 438, and 440. In a particular embodiment, the modified CH3 domain comprises a deletion of one or two amino acids of sequence SEQ ID NO: 56.

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

In some embodiments, the modified CH3 domain comprises F at position 382.

In some embodiments, the modified CH3 domain comprises an a or polar amino acid (e.g., Y or S) at position 383.

In some embodiments, the modified CH3 domain comprises G, N or an acidic amino acid (e.g., D or E) at position 384.

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

In some embodiments, the modified CH3 domain comprises at least one amino acid substitution at a β -sheet position relative to sequence SEQ ID NO: 56.

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

In certain embodiments, the modified CH3 domain comprises an amino acid substitution at β -sheet position 380 relative to sequence SEQ ID NO. 56. In particular embodiments, the modified CH3 domain comprises E, N, F or Y (e.g., E) at position 380.

In some embodiments, the modified CH3 domain comprises an amino acid substitution at β -sheet position 382 relative to sequence SEQ ID NO. 56. In a particular embodiment, the modified CH3 domain comprises F at position 382.

In some embodiments, the modified CH3 domain comprises an amino acid substitution or amino acid deletion at position 383 relative to the β -sheet of sequence SEQ ID NO: 56. In particular embodiments, the modified CH3 domain comprises Y or a (e.g., Y) at position 383.

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

In some embodiments, the modified CH3 domain comprises an amino acid substitution at β -sheet position 424 relative to sequence SEQ ID NO. 57. In a particular embodiment, the modified CH3 domain comprises a at position 424.

In some embodiments, the modified CH3 domain comprises an amino acid substitution at beta-sheet position 426 relative to sequence SEQ ID NO. 57. In a particular embodiment, the modified CH3 domain comprises E at position 426.

In some embodiments, the modified CH3 domain comprises an amino acid substitution at β -sheet position 438 relative to sequence SEQ ID NO: 57. In a particular embodiment, the modified CH3 domain comprises Y at position 438.

In some embodiments, the modified CH3 domain comprises an amino acid substitution at β -sheet position 440 relative to sequence SEQ ID NO. 57. In a particular embodiment, the modified CH3 domain comprises L at position 440.

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

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

In some embodiments, the modified CH3 domain comprises at least one position selected from the group consisting of: e, N, F or Y at position 380, F at position 382, Y, S, A or 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 particular embodiments, the modified CH3 domain comprises five, six, seven or eight positions selected from the group consisting of: 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, N, T or S at position 389.

In some embodiments, the modified CH3 domain comprises at least one position selected from the group consisting of: 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 a particular embodiment, the modified CH3 domain comprises five positions selected from the group consisting of: l at position 422, a at position 424, E at position 426, Y at position 438, and L at position 440.

In another aspect, the present disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: (i) Sequence AVX₁WFX₂X₃X₄X₅X₆X₇X₈N(SEQ ID NO:65), wherein X ₁ is E, N, F or Y; x ₂ is Y, S, A or absent; x ₃ is G, D, E or N; x ₄ is D, G, N or A; x ₅ is Q, S, G, A or N; x ₆ is K, I, R or G; x ₇ is E, L, D or Q; and X ₈ is N, T, S or R; and (ii) sequence LFACEVMHEALX ₁X₂ HYTYKL (SEQ ID NO: 67), wherein X ₁ is H or E; and X ₂ is N or G.

In some embodiments of the above five aspects, the modified CH3 domain comprises 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 sequence LFACEVMHEA LHNHYTYKL (SEQ ID NO: 64).

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

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

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 of SEQ ID NOs 72-77.

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.

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

In another aspect, the present disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to a 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, L at position 440, wherein positions are determined according to EU numbering.

In another aspect, the present disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to a 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, L at position 440, wherein positions are determined according to EU numbering.

In another aspect, the present disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to a 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, L at position 440, wherein positions are determined according to EU numbering.

In another aspect, the present disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to a 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, L at position 440, wherein positions are determined according to EU numbering.

In another aspect, the present disclosure provides a polypeptide comprising a modified CH3 domain that specifically binds to a 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, L at position 440, wherein positions are determined according to EU numbering.

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

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

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

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

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

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

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 that is at least 85% (e.g., at least 90%, 91%, 93%, 95%, 97%, 98%, or 99%) identical to amino acids 111-217 of sequence SEQ ID NO:137, wherein the modified CH3 domain comprises Ala, asp, his, tyr or Phe at position 378; ala, asp, phe, leu, gln, glu or Lys at position 380; gly at position 382; leu, ala, or Glu at position 384; val at location 385; gln or Ala at position 386; val, ile, phe or Leu at position 422; ser, ala or Pro at position 424; thr or Ile at position 426; ile or Tyr at position 438; and Gly, ser, thr or Val at location 440. In some embodiments, the modified CH3 domain has Met or Leu at position 428.

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 that is at least 85% (e.g., at least 90%, 91%, 93%, 95%, 97%, 98%, or 99%) identical to amino acids 111-217 of sequence SEQ ID NO:137, wherein the modified CH3 domain comprises any of the set of substitutions provided by any of the clones in table 32B-1, table 32C, table 32D, table 32E, table 32F, table 323G, table 32H-1, table 32J, and table 32K, or comprises the possible amino acids shown in table 32I.

In some embodiments of both aspects above, the modified CH3 domain comprises Ala or His at position 378 according to EU numbering; asp or Glu at position 380; gly at position 382; leu at position 384; val at location 385; gln or Ala at position 386; ile or Val at location 422; ala or Pro at position 424; thr or Ile at position 426; ile at position 438; and Gly or Thr at position 440. The modified CH3 domain may also include Met or Leu at position 428.

In some embodiments, the modified CH3 domain comprises His at position 378 according to EU numbering; glu at position 380; gly at position 382; leu at position 384; val at location 385; gln at position 386; ile at location 422; pro at position 424; ile at location 426; ile at position 438; and Thr at location 440. The modified CH3 domain may also include Met or Leu at position 428.

In some embodiments, the modified CH3 domain comprises His at position 378 according to EU numbering; glu at position 380; gly at position 382; leu at position 384; val at location 385; gln at position 386; ile at location 422; pro at position 424; ile at location 426; leu at location 428; ile at position 438; and Thr at location 440.

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 that is at least 85% (e.g., at least 90%, 91%, 93%, 95%, 97%, 98%, or 99%) identical to amino acids 111-217 of sequence SEQ ID NO:137, wherein the modified CH3 domain comprises His at position 378 according to EU numbering; glu at position 380; gly at position 382; leu at position 384; val at location 385; gln at position 386; ile at location 422; pro at position 424; ile at location 426; ile at position 438; and Thr at location 440. The modified CH3 domain may also include Met or Leu at position 428.

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 that is at least 85% (e.g., at least 90%, 91%, 93%, 95%, 97%,98%, or 99%) identical to amino acids 111-217 of sequence SEQ ID NO:138, wherein the modified CH3 domain comprises His at position 378 according to EU numbering; glu at position 380; gly at position 382; leu at position 384; val at location 385; gln at position 386; ile at location 422; pro at position 424; ile at location 426; leu at location 428; ile at position 438; and Thr at location 440. In some embodiments of the disclosure provided herein, the modified constant domain (e.g., modified CH3 domain) further comprises at least one modification that promotes heterodimerization. In particular embodiments, the modified constant domain (e.g., modified CH3 domain) further comprises a T366W substitution according to EU numbering. In particular embodiments, the modified constant domain (e.g., modified CH3 domain) further comprises T366S, L a and Y407V substitutions according to EU numbering.

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

In some embodiments, the CH2 domain is a human IgG1, igG2, igG3, or IgG4 CH2 domain.

In some embodiments of any aspect described herein, the polypeptide is part of a dimer. In some embodiments, the dimer is an Fc dimer. In some embodiments, the polypeptide further binds to Fab.

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

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

In some embodiments of any of the aspects described herein, the C-terminal lysine of the polypeptide is removed.

In another aspect, the present disclosure provides a nucleic acid comprising a sequence encoding a polypeptide described herein.

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

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

In another aspect, the present disclosure provides a method for producing a polypeptide comprising a modified constant domain (e.g., a modified CH3 domain), the method comprising culturing a host cell under conditions that express a polypeptide encoded by a polynucleotide described herein.

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

In another aspect, the present disclosure provides a method of endocytosis of a therapeutic agent across endothelial cells. In some embodiments, the methods comprise contacting endothelial cells with a composition comprising a polypeptide dimer capable of binding CD98hc (e.g., a polypeptide dimer described herein) fused to a therapeutic agent. In some embodiments, the methods comprise contacting the endothelial cells with a composition comprising a polypeptide dimer capable of binding TfR (e.g., a polypeptide dimer described herein) fused to a therapeutic agent. In some embodiments, the endothelial cell is the BBB.

In another aspect, the present disclosure provides a method for engineering a polypeptide comprising a modified CH3 domain 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 sequence EWESNGQP (SEQ ID NO: 52); (ii) A second sequence comprising at least one substitution relative to sequence NVFSCSVM (SEQ ID NO: 53); and (iii) a third sequence comprising at least one substitution relative to sequence YTQKSLS (SEQ ID NO: 54);

(b) Expressing and recovering a polypeptide comprising a modified CH3 domain; and

(C) Determining whether the polypeptide binds to the CD98hc protein,

Wherein sequence SEQ ID NO:52 is from position 380 to position 387 of the Fc polypeptide (e.g., SEQ ID NO: 1), sequence SEQ ID NO:53 is from position 421 to position 428 of the Fc polypeptide (e.g., SEQ ID NO: 1), sequence SEQ ID NO:54 is from position 436 to position 442 of the Fc polypeptide (e.g., SEQ ID NO: 1), and positions are determined according to EU numbering.

In another aspect, the disclosure provides a method for engineering a polypeptide comprising a modified CH3 domain 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 sequence AVEWESNGQPENN (SEQ ID NO: 56); and (ii) a second sequence comprising at least one amino acid substitution in sequence VFSCSVMHEALHNHYTQKS (SEQ ID NO: 57);

(C) Determining whether the polypeptide binds to a TfR protein,

Wherein sequence SEQ ID NO:56 is from position 378 to position 390 of the Fc polypeptide (e.g., SEQ ID NO: 1) and sequence SEQ ID NO:57 is from position 422 to position 440 of the Fc polypeptide (e.g., SEQ ID NO: 1) and positions are determined according to EU numbering.

In some embodiments of this aspect, the steps of expressing a polypeptide comprising a modified CH3 domain and determining whether the modified CH3 domain binds to CD98hc or TfR are performed using a display system. In particular 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.

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

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

In some embodiments of both aspects, one polypeptide of the polypeptide dimers comprises at least eleven, twelve, thirteen, fourteen or fifteen substitutions in a 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.

In some embodiments of both aspects, one polypeptide of the polypeptide dimers comprises at least eight, nine, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen or nineteen substitutions in a 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.

In some embodiments, neither polypeptide of the polypeptide dimers has the substitution L234A, L235A nor P329G.

In another embodiment, the present disclosure provides a method of delivering across the BBB to a biological target in the brain, the method comprising: (a) a CD98hc binding polypeptide described herein; and (b) means for binding to a biological target in the brain.

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

In some embodiments, the biological target is a cell surface target on a hematologic cancer cell. In 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.

In some embodiments, the target is located 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 (CD 66E), EGFR viii, ETBR, FGFR (1-4), folate receptor alpha, GAL-3BP (galectin binding protein), GD2, GD3, globohexacyl ceramide (globohexasylceramide)), gp100, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC (15 containing leucine rich repeats), MET, naPi2B, PDL1, PMEL17, PRAME, PSMA, PTK (CCK 4; colon cancer kinase), RON, ROR1, TF (tissue factor) and TROP2.

In some embodiments, the target may comprise α -synuclein or a derivative or fragment thereof, β -amyloid peptide or a derivative or 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 another aspect, the present disclosure provides a method of targeting an extracellular target in the brain by a CD98hc binding polypeptide, the method comprising administering the CD98hc binding polypeptide to a patient, wherein the polypeptide is transported across the BBB and into the parenchyma, without endocytosis into cells within the brain. In some embodiments, the extracellular target is on or near an astrocyte, microglial cell, oligodendrocyte, or cancer cell. 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 the therapeutic agent to an extracellular target.

In another aspect, the present disclosure provides a method of delivering a therapeutic agent across the BBB to astrocytes, the method comprising contacting the BBB with a composition comprising a polypeptide dimer described herein fused to the therapeutic agent. In some embodiments, both polypeptides in the polypeptide dimer comprise at least eleven, twelve, thirteen, fourteen or fifteen substitutions in a 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.

In another aspect, the present disclosure provides a method of delivering a therapeutic agent to a peripheral CD98 hc-expressing organ, the method comprising administering to a subject a composition comprising a polypeptide dimer described herein fused to a therapeutic agent. In certain embodiments, the peripheral CD98 hc-expressing organ is a kidney, testis, bone marrow, spleen, or pancreas.

In another aspect, the present 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 at positions selected from the group consisting of: 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, the polypeptide, when bound to human CD98hc, additionally binds to at least 1 additional residue at a position selected from the group consisting of: 229, 231, 232, 236, 235, 488, 495 and 496 of SEQ ID NO. 134. In certain embodiments, the polypeptide, when bound to human CD98hc, additionally binds to at least 1 additional residue at a position selected from the group consisting of: 312, 315, 348, 381, 439, 444, 443, 485, 484, 476, 475 and 442 of SEQ ID NO. 134.

In another aspect, the present 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 of residues at positions selected from the group consisting of: 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 another aspect, the present 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 of the residues at positions selected from the group consisting of: 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 CD98hc protein.

In another aspect, the present disclosure provides a method of increasing exposure of a subject's brain to a therapeutic agent 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 20nM to about 550nM, wherein the molecule is linked to the therapeutic agent, and wherein the reference molecule comprises the therapeutic agent but does not comprise a CD98hc binding moiety.

In another aspect, the present disclosure provides a method of increasing exposure of a subject's brain to a therapeutic agent 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 275nM to about 2100nM, wherein the molecule is linked to the therapeutic agent, and wherein the reference molecule comprises the therapeutic agent but does not comprise a CD98hc binding moiety.

In another aspect, the present disclosure provides a composition for delivery across the BBB to a biological target in the brain, the composition comprising: (a) A CD98hc binding polypeptide as described herein, and (b) a device for binding a biological target in the brain.

In some embodiments, the target is located 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 (CD 66E), EGFR viii, ETBR, FGFR (1-4), folate receptor alpha, GAL-3BP (galectin binding protein), GD2, GD3, globohexacyl ceramide, gp100, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (15 containing leucine rich repeats), MET, naPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK 4; colon cancer kinase), RON, ROR1, TF (tissue factor) and TROP2.

In another aspect, the present disclosure provides a method for delivering across the BBB to 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 device for binding a biological target; and

(B) Administering the composition of step (a) to the periphery of the subject.

In another aspect, the present 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 device for binding a biological target;

(b) Peripherally administering the composition of step (a) to a subject;

Wherein the composition binds to a biological target in the brain of the subject.

Unless otherwise indicated or apparent from context, all position numbers (e.g., "position x") of Fc, CH2, or CH3 polypeptides throughout this document are based on the EU numbering system.

Drawings

FIG. 1 shows the plasma pharmacokinetics of LLB2 and LLB1 CD98hc binding molecules in C57/B6 (WT) mice.

FIG. 2 shows the plasma pharmacokinetics of additional LLB2 and LLB1 CD98hc binding molecules in C57/B6 (WT) mice.

FIG. 3 shows the plasma pharmacokinetics of affinity matured LLB2 CD98hc binding molecules in C57/B6 (WT) mice.

FIG. 4 shows the plasma pharmacokinetics of the affinity-depleted mature LLB2 CD98hc binding molecule in C57/B6 (WT) mice.

FIGS. 5A-5C show brain absorption of LLB2 and LLB1 CD98hc binding molecules in CD98hc ^mu/hu KI mice. huIgG in plasma 48 hours after the (a) dose. (B) huIgG in whole brain lysate. (C) ratio of huIgG in brain to plasma.

FIG. 6 shows capillary depletion indicating that CD98hc binding molecules cross the BBB into the brain parenchyma of CD98hc ^mu/hu KI mice.

FIG. 7 shows CNS biodistribution of LLB2 and LLB1 variants in CD98hc ^mu/hu KI mice.

FIG. 8 shows the cell-specific biodistribution of CD98hc binding molecules IBA1 (microglial cells) in CD98hc ^mu/hu KI mice by immunohistochemistry.

FIG. 9 shows the cell-specific biodistribution of CD98hc binding molecule AQPN (astrocytes) in CD98hc ^mu/hu KI mice by immunohistochemistry.

FIGS. 10A and 10B show brain uptake of additional LLB2 and LLB1 variants in CD98hc ^mu/hu KI mice. huIgG in plasma 48 hours after the (a) dose. (B) huIgG in whole brain lysate.

FIGS. 11A-11F show peripheral tissue localization of LLB2 and LLB1 variants in CD98hc ^mu/hu KI mice.

FIGS. 12A and 12B show the brain absorption time course of monovalent and bivalent LLB2 variants in CD98hc ^mu/hu KI mice. huIgG PK in plasma 10 days after dose (a). (B) huIgG PK in whole brain lysate.

Figure 13 shows capillary depletion indicating that monovalent and bivalent LLB2 variants cross BBB into brain parenchyma of CD98hc ^mu/hu KI mice.

Fig. 14A-14H show huIgG Pharmacokinetics (PK) in peripheral tissues of monovalent and bivalent LLB2 variants in CD98hc ^mu/hu KI mice.

FIG. 15 shows the time course of the biodistribution of monovalent and divalent LLB2-10-8 by immunohistochemistry of huIgG.

FIG. 16 shows the time course of the biodistribution of monovalent and divalent LLB2-10-8 by immunohistochemistry of huIgG and Iba1 (microglial cells).

Fig. 17A and 17B show (a) plasma and (B) brain exposure after repeated administration of monovalent LLB2 variants.

FIG. 18 shows the biodistribution time course after repeated administration of monovalent LLB2-10-8 variants.

FIGS. 19A and 19B show yeast display library surfaces modeled on a wild-type IgG1 Fc backbone (PDB 1 hzh).

FIGS. 20A-20C show the concentrations of clones and controls in plasma (FIG. 20A) and whole blood (FIG. 20B) of chimeric huTfR ^{Top end} gene knock-in mice 24 hours after administration of the clones or controls to mice at a dose of 50 mg/kg. Fig. 20C shows a decrease in aβ40 levels in the mouse brain.

FIGS. 21A-21G show cloned and control plasma PK at a dose of 10mg/kg in wild-type mice (FIG. 21A). Chimeric huTfR ^{Top end} Gene knock-in mice at a dose of 50mg/kg, monovalent clone 6.5.11.5.42.2, bivalent clone 6.5.11.5.42.2, and control brain PK (FIG. 21B), brain PD (FIG. 21C), and plasma PK (FIG. 21D). Figures 3E and 3F show safety data indicating the percentage of Ter119 ⁺ red blood cells (figure 21E) or CD71 ⁺ bone marrow reticulocytes (figure 21F) in the total plasma cell population for the anti-BACE 1 control or bivalent clone 6.5.11.5.42.2. Fig. 21G shows the levels of whole brain TfR at 24 hours post-treatment compared to the load control GAPDH.

Figure 22 shows Size Exclusion Chromatography (SEC) analysis of clone 6.5.11.5.42.2 under low pH and control conditions.

FIGS. 23A-23C show the structure of clone 6.5.11.5.42 with the top domain of the human TfR cycle alignment, where the library residues are in the form of rods (FIG. 23A). The structure of clone 6.5.11.5.42 with TfR top domain and modeled full-length human TfR domain (fig. 23B). The scale of fig. 23B shows the conflict between human TfR domains (fig. 23C).

Fig. 24A and 24B show the concentrations of monovalent and bivalent clone 42.2.1.2, monovalent and bivalent clone 6.5.11.5.42.2, and controls in whole brain (fig. 24A) and plasma (fig. 24B) of chimeric huTfR ^{Top end} gene knocked-in mice 24 hours after administration of clones or controls to mice at a dose of 50 mg/kg.

Figure 25 shows safety data indicating the levels of monovalent and divalent clones 42.2.1.2, monovalent and divalent clone 6.5.11.5.42.2, and control reticulocytes.

FIGS. 26A-26D show 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 control plasma PK (FIGS. 26A and 26B) and brain PK (FIGS. 26C and 26D) at a dose of 50mg/kg in chimeric huTfR ^{Top end} gene knock-in mice.

FIGS. 27A and 27B show (A) plasma and (B) brain exposure in CD98hc ^mu/hu KI mice following repeated administration of the bivalent LLB2 variants.

FIGS. 28A and 28B show the crystal structure of a bivalent CD98hc binding molecule having a CD98 hc: (A) LLB2-10-6 dimer (B) LLB1-3-16 dimer.

Fig. 29A and 29B show a co-complex with CD98hc and a crystal structure of a CD98hc binding molecule modeled with two FcRn-B2M: (A) LLB2-10-6 dimer (B) LLB1-3-16 dimer.

FIGS. 30A-30C show the orientation of the divalent CD98hc binding molecule crystal structure relative to modeled CD98hc-LAT1 complexes: (A) a divalent LLB2-10-6 dimer, (B) a divalent LLB1-3-16 dimer, and (C) a monovalent LLB2-10-6 dimer.

Fig. 31A-31F show plasma and brain PK and capillary depletion results in CD98hc ^mu/hu KI mice following dosing with monovalent LLB2 variants: (a) plasma PK, (B) brain PK, (C) a substantial fraction, (D) a vasculature fraction, (E) a cell associated fraction, and (F) a non-cell associated fraction.

Fig. 32A-32F show plasma and brain PK and capillary depletion results in CD98hc ^mu/hu KI mice following dose of bivalent LLB2 variant: (a) plasma PK, (B) brain PK, (C) a substantial fraction, (D) a vasculature fraction, (E) a cell associated fraction, and (F) a non-cell associated fraction.

Fig. 33A and 33B show huIgG PK in (a) plasma and (B) whole brain lysates in affinity-matched LLB1 and LLB2 variants 21 days post dose.

Figures 34A-34F show plasma and brain PK and capillary depletion results in CD98hc ^mu/hu KI mice following dosing with LLB2-10-8 variants: (a) plasma PK, (B) brain PK, (C) a substantial fraction, (D) a vasculature fraction, (E) a cell associated fraction, and (F) a non-cell associated fraction.

FIG. 35 shows immunohistochemistry of huIgG on brain sections from CD98hc ^mu/hu KI mice 1, 7, 14, and 21 days after 50mpk dose of LLB2-10-8 variant.

FIGS. 36A-36C show immunohistochemistry of huIgG and CNS cell type markers on brain sections from CD98hc ^mu/hu KI 7 days after 50mpk dose of LLB2-10-8 variant: iba1 for microglial cells, (B) AQP4 for astrocyte processes and (C) NeuN for neurons.

Fig. 37A-37C show (a) plasma PK, (B) brain PK, and (C) Abeta reduction (PD), with CD98hc TV having BACE1 Fab.

FIGS. 38A and 38B show immunohistochemistry for huIgG, neuN (neurons), and LAMP2 (lysosomes) on brain sections from CD98hc ^mu/hu KI 7 days after CD98hc TV dose with BACE1 Fab.

Fig. 39A-39D show huIgG PK in plasma and whole blood lysates in mice dosed at 15 mpk: (a) plasma PK for monovalent variants, (B) brain PK for monovalent variants, (C) plasma PK for divalent variants, and (D) brain PK for divalent variants.

Fig. 40A to 40F show plasma and brain exposure in NHP and capillary depletion results in NHP brain tissue: (a) plasma exposure, (B) brain exposure, (C) brain parenchymal fraction, (D) vasculature fraction, (E) cell associated fraction, and (F) non-cell associated fraction.

Fig. 41A-41C show immunohistochemistry for huIgG and CNS cell type markers on NHP brain sections: iba1 for microglial cells, (B) AQP4 for astrocyte processes and (C) NeuN for neurons.

Fig. 42A and 42B show binding epitopes of CD98hc binding molecules: (A) LLB2 family and (B) LLB1 family.

Fig. 43A and 43B show quantification of cell uptake in clone 1 with LALA, clone 3 with LALA and controls in HEK293T human TfR positive cells (fig. 43A) and Chinese Hamster Ovary (CHO) cells (fig. 43B) that ectopically express cynomolgus monkey TfR at 37 ℃.

FIG. 44 shows plasma PK for clone 1 with LALA and clone 3 with LALA.

FIGS. 45A and 45B show the concentrations of chimeric huTfR top gene knockin monovalent clone 1-112_L, monovalent clone 1-112_LS, monovalent clone 1-292, monovalent clone 1-321, and controls in whole brain (FIG. 45A) and plasma (FIG. 45B) of mice 24 hours after administration of the clone or control to mice at a dose of 50 mg/kg.

FIG. 46 shows a multi-dose study of clones and controls dosed at 50mg/kg on day 0, day 3 and day 5 in chimeric huTfR-top gene knock-in mice 24 hours after the final dose, which shows brain Abeta 40 levels.

Fig. 47A-47C show the structure of clone 6.5.11.5.42 with TfR top domain and modeled full-length human TfR domain (fig. 47A); the scale of fig. 47A shows the conflicts between human TfR domains (fig. 47B); the structure of clones 1-112 with LALA and M428L with TfR top domain and modeled full-length human TfR domain (fig. 47C).

Detailed Description

I. Introduction to the invention

We have developed a number of methods for generating non-natural binding sites in polypeptides by screening libraries of polypeptides for novel conjugates. One challenge of introducing non-natural binding sites is that such libraries typically contain a large number of sequences with undesirable properties (e.g., non-specific binding or lack of developability). As described below, the "limited reliability" approach may reduce the frequency of amino acids associated with these undesirable characteristics, thereby allowing the library to produce more useful sequences. Such limited reliability methods can be used in a variety of protein frameworks including immunoglobulins (i.e., in CDR and non-CDR portions) and other frameworks such as fibronectin or any other protein frameworks described herein to enhance and accelerate the discovery of novel polypeptide conjugates. In certain instances, it may be used in a library, wherein the engineered portion of the polypeptide comprises the exposed side of the β -sheet within the polypeptide as described below.

We have also developed libraries of immunoglobulins that have been engineered with modifications in the beta-sheet surface. These libraries have been used to generate novel binding sites in the non-CDR portions of immunoglobulins, and in particular have been used to generate novel molecules that bind to the CD98 heavy chain (CD 98 hc) and transferrin receptor (TfR). The present disclosure is based in part on the following findings: certain amino acids (particularly those at the β -sheet position in the CH3 domain of an Fc polypeptide) may be substituted to generate a modified CH3 domain containing a novel binding site specific for CD98hc (e.g., a CD98hc binding site). The β -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 the EU numbering, and the other β -sheet residues in the constant domain are described herein, e.g., in Table 1B. Substitution of amino acids at the β -sheet position may provide several advantages in the generation of immunoglobulin domains containing non-natural binding sites. First, the β -sheet surface in the domain is stable and allows for diversity of amino acid substitutions at the surface without disrupting the domain structural sheet. In some embodiments, the amino acid substitution is located on the solvent exposed side of the β -sheet surface of the domain. Second, amino acid substitutions at the β -sheet positions avoid altering the flexible loop regions in the domain, which may, in some cases, introduce undesired conformational flexibility. Furthermore, the concave surface of the β -sheet structure in the domain is well suited for forming protein-protein interactions, and the β -sheet structure is also different from FcRn and fcγr binding sites in the CH3 domain.

The engineering methods described herein have been used to find specific polypeptides that bind to CD98hc or TfR. These polypeptides are transported across the blood brain barrier in mammals as described herein via endocytosis. CD98 is highly expressed on brain endothelial cells and is therefore a promising target for receptor-mediated endocytic transport (RMT). CD98 is a heterodimer formed between CD98hc (4F 2 heavy chain) and CD98 light chain. Six CD98 light chains have been identified to date, namely LAT1 (SLC 7A5,4F2 light chain), LAT2 (SLC 7 A8), y ⁺LAT1(SLC7A7)、y⁺ LAT2 (SLC 7 A6), asc-1 (SLC 7a 10) or xCT (SLC 7a 11). In complex cases, the CD98 heavy chain transports the light chain to the cell surface where it functions as a larger neutral amino acid transporter that preferentially transports branched (valine, leucine, isoleucine) and aromatic (tryptophan, tyrosine, phenylalanine) amino acids. Polypeptides containing the CD98hc binding sites described herein are useful for transporting therapeutic agents across the BBB using CD98 receptor mediated endocytic transport pathways. Such a method may significantly improve the uptake of therapeutic agents by the brain and is therefore highly useful for treating conditions and diseases where brain delivery is beneficial. In addition, such methods can be used to provide brain uptake and delivery for specific extracellular or neurooncology targets in the brain. For example, if desired, the CD98hc binding polypeptides provided herein can be used to target such extracellular targets or neurooncology targets while retaining wild-type effector function. In addition, such CD98hc binding polypeptides provided herein can be used to target such extracellular targets (e.g., targets that are antigens or plaques, such as Abeta, tau, or a-synuclein) where neuronal uptake is not desirable. The CD98hc binding polypeptides provided herein have unique kinetics, biodistribution, and safety profiles that can provide a BBB transport platform for protein-based therapeutics that is optimized and suitable for the purpose.

Also described herein are polypeptides that bind to transferrin receptor (TfR). TfR is highly expressed on the Blood Brain Barrier (BBB) and naturally moves transferrin from the blood into the brain. With these advantages that TfR has provided, polypeptides containing the TfR binding sites described herein can be used to transport therapeutic agents across the BBB. Such a method may significantly improve the uptake of therapeutic agents by the brain and is therefore highly useful for treating conditions and diseases where brain delivery is beneficial.

Also provided herein are methods of producing a polypeptide comprising a modified CH3 domain that binds to CD98hc or TfR. CD98hc binding or TfR binding of polypeptides comprising modified CH3 domains described herein can be analyzed and further mutated as described herein to enhance binding.

In another aspect, provided herein are methods of treatment and methods of using a CD98 hc-binding or TfR-binding polypeptide to target a composition to a CD98 hc-expressing cell or TfR-expressing cell (e.g., to deliver the composition to a cell, or to deliver the composition across an endothelial cell such as the BBB).

II. Definition of

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

As used herein, the terms "about" and "approximately" when used to modify an amount specified in a numerical value or range indicates that the numerical value as well as a reasonable deviation (e.g., ±20%, ±10% or ±5%) from the value as known to one of skill in the art is within the intended meaning of the recited value.

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 UNIPAT accession number P08195. CD98hc sequences from other species are also known (e.g., mouse, UNIPOT accession number P10852 and cynomolgus monkey, UNIPOT accession number G8F3Z 0).

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, 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 a dual gene variant of an exemplary reference sequence (e.g., human sequence) encoded by a gene at the transferrin receptor protein 1 chromosomal locus. Full length transferrin receptor proteins include short N-terminal intracellular regions, transmembrane regions, and large extracellular domains. The extracellular domain is characterized by three domains: protease-like domains, helical domains and apical domains.

As used herein, the term "CH3 domain" and "CH2 domain" refer to immunoglobulin constant region polypeptides. For the purposes of the present application, a CH3 domain polypeptide refers to an amino acid segment from about position 341 to about position 447 as numbered according to the EU numbering scheme, and a CH2 domain polypeptide refers to an amino acid segment from about position 231 to about position 340 as numbered according to the EU numbering scheme and does not include a hinge region sequence. CH2 and CH3 domain polypeptides may also be numbered by the IMGT (ImMunoGeneTics) numbering scheme, wherein CH2 domain numbers are 1-110 and CH3 domain numbers are 1-107 according to the IMGT SCIENTIFIC chart numbering (IMGT website). The CH2 and CH3 domains are part of the Fc region of an immunoglobulin. An Fc region refers to an amino acid segment numbered from about position 231 to about position 447 as per the EU numbering scheme, but as used herein may include at least a portion of an antibody hinge region. An illustrative hinge region sequence is the human IgG1 hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO: 4).

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

As used herein, the term "mutant" as used in reference to a mutant polypeptide or mutant polynucleotide is used interchangeably with "variant". Variants with respect to a given wild-type CH3 or CH2 domain reference sequence may include naturally occurring dual gene variants. "non-naturally occurring CH3 or CH2 domain refers to a variant or mutant domain that does not occur naturally in a cell and is created by genetic engineering techniques or mutation-inducing techniques, for example, by genetic engineering of a native CH3 domain or CH2 domain polynucleotide or polypeptide. "variant" includes any domain comprising at least one amino acid mutation relative to the wild type. Mutations may include substitutions, insertions, and deletions.

As used herein, the term "amino acid" refers to naturally occurring amino acids as well as synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, and those amino acids which are subsequently modified, such as hydroxyproline, gamma-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 (gin), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of naturally occurring alpha-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 having the same basic chemical structure (i.e., an alpha carbon to which hydrogen, carboxyl, amino, and R groups are bound) as a naturally occurring amino acid, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. These 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 mimetic" refers to a compound that differs in structure 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 their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB biochemical nomenclature committee (Biochemical Nomenclature Commission).

As used herein, "limited diversity" refers to codons or positions that are limited to allow for fewer than all 20 naturally occurring amino acids in the context of randomized codons within a polynucleotide library or any amino acid position within a polypeptide library described herein.

As used herein, in the context of a polypeptide, a "β -sheet position" means an amino acid that falls within a portion of the polypeptide whose structure is predominantly β -sheet.

As used herein, the term "immunoglobulin-like fold" refers to a protein domain of about 80-150 amino acid residues, comprising two layers of antiparallel β -sheets, and wherein the flat hydrophobic faces of the two β -sheets are opposite each other.

As used herein, the terms "polypeptide" and "peptide" are used interchangeably to refer to a polymer of amino acid residues that are single-stranded. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The amino acid polymer may comprise complete L-amino acids, complete D-amino acids, or a mixture of L and D amino acids.

As used herein, the term "constant domain" refers to a domain in a constant region of an immunoglobulin molecule (e.g., CH1, CH2, CH3, CH4, ck, cλ).

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

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 in the domain. The Fc polypeptide contains a constant region sequence comprising at least a CH2 domain and/or a CH3 domain, and may contain at least a portion of a hinge region, but no variable region.

As used herein, the term "protein" refers to a polypeptide or a dimer (i.e., two) or a multimer (i.e., three or more) of single-chain polypeptides. Single-chain polypeptides of proteins may be joined by covalent bonds (e.g., disulfide bonds) or non-covalent interactions.

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 the same or have a specified percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more) of identical amino acid residues over a specified region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection, when compared and aligned for maximum correspondence over a comparison window or specified region.

For sequence comparison of polypeptides, typically an amino acid sequence is used as a reference sequence to which candidate sequences are compared. Alignment may be performed using various methods available to those skilled in the art, such as visual alignment or maximum alignment using publicly available software using known algorithms. Such programs include BLAST programs, ALIGN-2 (Genntech, south San Francisco, calif.) or Megalign (DNASTAR). The parameters for alignment to achieve maximum alignment can be determined by those skilled in the art. For the purposes of the present application, BLASTP algorithm standard protein BLAST for aligning two protein sequences with preset parameters is used for sequence comparison of polypeptide sequences.

As used herein, the term "binding affinity" refers to the strength of a non-covalent interaction between two molecules (e.g., between a Fab or scFv and an antigen or between a polypeptide (or target binding portion thereof) described herein and a target). Thus, for example, unless indicated otherwise or apparent from context, the term may refer to a 1:1 interaction between a Fab or scFv and an antigen or between a polypeptide (or target binding portion thereof) and a target as described herein. Binding affinity can be quantified by measuring the equilibrium dissociation constant (K _D), which refers to the dissociation rate constant (K _d, time ^-1) divided by the association rate constant (K _a), time ^-1M^-1).K_D can be determined by measuring the kinetics of complex formation and dissociation, e.g., using Surface Plasmon Resonance (SPR) methods, e.g., biacore ^TM system, kinetic exclusion assays (kinetic exclusion assay), such asBiological layer interference (e.g. usingOctet platform). As used herein, "binding affinity" includes not only formal binding affinities, such as those that reflect a 1:1 interaction between Fab or scFv and an antigen or between a polypeptide (or target binding portion thereof) described herein and a target, but also K _D is calculated to reflect the apparent affinity of affinity binding.

As used herein, the term "specifically binds" refers to a molecule that binds to an epitope or target (e.g., a Fab, scFv, or polypeptide described herein (or target binding portion thereof)) to the 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, the Fab, scFv, or polypeptide described herein (or target binding portion thereof) that specifically binds to an epitope or target is the following Fab, scFv, or polypeptide described herein (or target binding portion thereof): its binding affinity to the epitope or target is at least 5-fold, 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 more, than to other epitopes or non-target compounds. As used herein, the term "specifically binds to", "specifically binds to" or "is specific for" a particular epitope or target may be revealed, for example, by a molecular equilibrium dissociation constant K _D for the epitope or target to which it binds being, for example, 10 ^-4 M or less (e.g., 10 ^-5M、10^-6M、10^-7M、10^-8M、10^-9M、10^-10M、10^-11 M or 10 ^-12 M). Those skilled in the art will recognize that Fab or scFv that specifically bind to a target from one species may also specifically bind to a heterologous homolog of the target.

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.

As used herein, the term "treatment" and similar terms generally mean obtaining a desired pharmacological and/or physiological effect. "treatment" may refer to any indication of success in treating or ameliorating a neurodegenerative disease, such as Alzheimer's disease or another neurodegenerative disease described herein, including any objective or subjective parameter, such as alleviation, remission, improvement in patient survival, increased survival time or survival rate, reduction in symptoms, or making a patient more tolerant of the disease, slowing the rate of regression or regression, or improving the physical or mental health of a patient. Treatment or amelioration of symptoms can be based on objective or subjective parameters. The therapeutic effect can be compared to an individual or group of individuals who did not receive the treatment, or to the same patient at a different time prior to or during the treatment.

As used herein, the term "pharmaceutically acceptable excipient" refers to an inactive pharmaceutical ingredient that is biologically or pharmacologically suitable for use in humans or animals, such as, but not limited to, a buffer, carrier, or preservative.

As used herein, the term "therapeutic agent" refers to any molecule, drug, or agent that is used to treat and/or prevent a disease. The therapeutic agent may be a small organic molecule or a compound, polypeptide, protein, nucleic acid, and/or any combination of the foregoing. In some embodiments, the therapeutic agent may be a known molecule, drug, or agent. In some embodiments, the therapeutic agent is an antigen binding domain-containing polypeptide, such as an antibody variable domain polypeptide having one or more Complementarity Determining Regions (CDRs), or an antigen binding fragment thereof. In particular embodiments, the therapeutic agent may be a Fab (e.g., a Fab that binds to a target that is not TfR or CD98 hc). In some embodiments, depending on the disease to be treated, the therapeutic agent may bind to a target (e.g., a biological target, a therapeutic target, a target that is not TfR or CD98 hc) to treat and/or prevent the disease. Such targets may include cell surface targets in the brain, such as 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 α -synuclein or a derivative or fragment thereof, β -amyloid peptide or a derivative or 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 located on a tumor cell and is selected from the group consisting of: ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD 66E), 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, LRRC (containing leucine rich repeat 15), MET, naPi2B, PDL1, PMEL17, PRAME, PSMA, PTK (CCK 4; 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 treating cancer include, for example, loratidine (lorlatinib), crizotinib (crizotinib), cabozantine, basiliximab, daclizumab, bivalizumab, pra Luo Mishan antibody (promiximab), lox Wo Tuozhu mab (lorvotuzumab), polotouzumab (polatuzumab), tercetuximab (tusamitamab), sunitinib, cetuximab, panitumumab, nimotuzumab (nimotuzumab), rituximab (necitumumab), rindopepimut (CDX-110), E Mo Tuo mab (amivantamab), pemetrexed (pemigatinib), erdastinib (erdafitinib), STRO-002, bevacizumab, natalizumab (naxitamab), ipilimumab (ipilimumab), tibetafos (tebentafusp), geobatuzumab (glembatumumab), MAGetuximab-cmkb, enhertu, qu Tuo mab (trastuzumab), pertuzumab, pertuzumab (patritumab), sirtuin (seribantumab), lu Tuozhu mab (lumretuzumab), eformizumab (elgemtumab), U3-1402, AV-203, KTN3379, AVE1642, MK-0646, cetuximab (cixutumumab), trastuzumab (ladiratuzumab), gemtuzumab (gemtuzumab), pambolizumab (pembrolizumab), Cetuximab (sacituzumab), sha Matuo mab (samrotamab), elstuzumab Mo Tuo-vmjw, TEPMETKO, rituximab (lifastuzumab), ¹⁷⁷ lutetium-PSMA-617, colfetuzumab (cofetuzumab), zt/g4-MMAE, VLS-101, brexucabtagene, CS5001, ticatuzumab (tisotumab), and combinations thereof, Cetuximab, territuximab (teclistamab), atuzumab, avistuzumab (avelumab), ke Xili mab (cosibelimab), divaruzumab (durvalumab), bei Lantuo mab (belantamab), benralizumab (benralizumab), tafatitaxeumab (tafasitamab), rituximab (loncastuximab), oxuzumab (obinutuzumab), ofatumumab, Rituximab, MEN1309/OBT076, oxtuzumab (inotuzumab) and bentuximab (brentuximab).

Additional known targets in the brain and agents that bind to such targets are described in the following references, which are hereby incorporated by reference ：WO 2016/023019;WO 2017/062672;WO 2018/195506;WO 2019/118513;WO 2019/023292;WO 2019/079529;WO 2019/180224;US2019/0040130;US2019/0174730;WO 2020/069050;US2017/0137518;US2012/0258110;WO 2019/126472;US 8,691,227;WO 2019/152715;US2007/026425;WO 2019/028283;US2018/016066,US 9,079,958;WO 2020/069050;WO 2022/258841;J Immunol 2000 165:1197-1209;Translational Neuro degeneration,11,18(2022).

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

As used herein, the term "administering" refers to a method of delivering an agent, compound, or composition to a desired site of biological action. Such methods include, but are not limited to, topical, oral, parenteral, intravenous, intradermal, intramuscular, intrathecal, colonic, rectal, or intraperitoneal delivery. In one embodiment, the protein as described herein is administered intravenously.

III engineering of polypeptides

We have developed a "limited reliability" design approach for libraries of polypeptides, as well as libraries in polypeptides (especially immunoglobulin molecules) that include a large number of β -sheet components. These methods are described in detail in the following sections. Engineering methods are also described that can be used with these libraries and library design methods to generate polypeptides having non-native binding sites, including, for example, sites that bind CD98hc or TfR.

Library with limited reliability

We have observed that large (. Gtoreq.9 positions) combinatorial libraries in polypeptides, when used to screen potential targets, produce large amounts of non-specifically bound polypeptides (e.g., through hydrophobic interactions) or have a propensity to make them difficult to use (e.g., poorly expressed, excessively hydrophobic, poorly stable). To reduce but not eliminate the occurrence of amino residues associated with these properties in our library, we have adopted what we call "limited reliability". Such methods involve reducing the frequency of occurrence of certain amino acids (e.g., cys, trp, met, arg and Gly) in libraries, but do not completely eliminate their presence, while also maintaining variability at these limited positions, e.g., allowing for at least 8,10, 12, 14, 15, or 16 amino acids to be present at these positions. In particular, this involves reducing the occurrence of at least one of these amino acids at 10% -60% (e.g., 20% -60%, 30% -60% or 40% -60%) of the randomized positions in the library, particularly where some or even all other randomized positions allow for all twenty naturally occurring amino acids. In certain cases, positions with limited diversity alternate with positions that allow all twenty amino acids. This can avoid having too many very close amino acids that can lead to undesirable properties. In some cases, the alternating sequence is positioned relative to the primary sequence of the polypeptide. Where the protein structure is known (e.g., if the crystal structure has been resolved), placement of the limited reliability locations may be spaced apart relative to locations in three-dimensional space that have greater or complete diversity. As explained in the examples below, this approach has led to the discovery of specific CD98hc binding polypeptides described herein.

Any known peptide library development method can be used to generate a limited reliability library. The libraries described in the examples herein are generated from a library of polynucleotides encoding a polypeptide of interest using degenerate codons, particularly NNK codons interspersed with codons of limited reliability (which allow all 20 amino acids), such as NHK (which do not allow Arg, cys, trp or Gly). The present invention also contemplates the use of other codons that provide limited reliability advantages. Possible codons may be selected from any known codon that provides "limited reliability", such as those shown below described in Mena et al, protein ENG DES SEL18:559-61, 2005. Table II in Mena et al is shown in Table 1A below.

Table 1A: degenerate codons calculated by LibDesign at each position from maximum inclusion to minimum inclusion

* Residues E "are shown in bold in the sequence set, underlined are the desired wild-type amino acids.

In addition to codon-based techniques for generating degenerate codon-based libraries, trinucleotide mutation-inducing techniques can also be used to generate limited reliability libraries. These methods involve high throughput techniques whereby a specific proportion of trinucleotide base pairs (each encoding a single amino acid) can be added to the library to precisely control the amino acid ratio at a given position. Techniques using such methods 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 libraries of polypeptides suitable for screening targets, including TfR and CD98 hc.

Beta-sheet library

Libraries comprising randomized amino acids within the β -sheet secondary structure of a polypeptide are also described herein. Generally, these libraries use exposed portions of the β -sheet, wherein the randomized amino acids together form a surface capable of creating an antigen binding site. In addition to the β -sheet surface, antigen binding sites may also include residues from adjacent regions on the polypeptide, for example in loop regions connecting β -strands or in other structural features of proteins that are in close proximity in three dimensions.

The use of β -sheet regions has certain advantages, including those described herein, including greater structural stability of the antigen binding site (as compared to the loop region or other lower structural portions of the polypeptide), and in some cases, the formation of a different surface topology (e.g., a flat extended concave surface) is well suited for the formation of some protein-protein interactions.

Specific examples of beta-sheet libraries include those generated from beta-sheet portions of immunoglobulins. In some examples, the β -sheet library is generated in a constant domain of an immunoglobulin, e.g., in a CH1, CH2, CH3, CH4, or CL domain. Other examples include the β -sheet portion of the variable domain, which may include the non-CDR portion of the variable region.

For the constant domains of human IgG1 molecules, the positions shown in table 1B are suitable for generating β -sheet libraries.

TABLE 1B surface and beta-sheet position in the constant domain of IgG1 heavy chain

Based on these positions in the constant domain of the IgG1 heavy chain, the corresponding positions can be identified in different domains (e.g., variable region and light chain), different subtypes (e.g., igG2, igG3, igG 4), different species (e.g., mouse, rat, cynomolgus monkey), and other Ig types (e.g., igA, igM, igE). As an example, alignment of primary amino acid sequences from different domains with corresponding domains in the IgG1 heavy chain constant region can be used to determine similar positions suitable for generating a β -sheet library in additional domains. Alternatively, structural alignment of the domain with one or more Ig domain structures in the IgG1 heavy chain constant region can be used to determine the presence of structural information or potential β -sheet library positions in the predicted domain. The residues identified are surface exposed and suitable for inclusion in libraries, or buried at the protein-protein interface (e.g., CH3-CH3 interface, VH-VL interface), can similarly be determined using structural information about the specific domain, which can be found in databases such as Protein Data Bank (Berman et al Nucleic Acids Res,28:235-242,2000) or based on predictions such as AlphaFold Protein Structure Database (Jumper et al Nature,596:583-589,2021).

Protein backbone for libraries

The limited reliability methods for library design and diversity, and libraries comprising the β -sheet secondary structures described herein, can be used to generate libraries on any suitable polypeptide scaffold. These polypeptide backbones can encompass any polypeptide having a beta-sheet secondary structure, including immunoglobulins, fibronectin type III domains, anti-cargo proteins, kunitz domains, nanofitin, centyrin, affimer, and lipocalins, as well as many other proteins having such typical beta-sheet structures.

Generation of binding proteins from polypeptide libraries

As described below, we have used a limited library concept, possibly in some cases, to find a library of β -sheet polypeptides that have been engineered to bind to polypeptides of a target protein (particularly CD98hc or TfR).

In general terms, a library of polypeptides is expressed (e.g., on the cell surface) and interrogated for binding to a target protein. This may be accomplished in any suitable manner, and various aspects of the screening method are described in Kariolis et al, SCI TRANSL MED (545): eaay1359,2020. In one method, the polypeptide library is expressed as a surface display library (e.g., phage display or yeast display) and incubated with a 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, the conjugate is separated from the non-conjugate, and the process is repeated to enrich the library of desired polypeptide clones that interact with the target antigen.

After the initial binders are identified from the polypeptide library, various improvements in biochemical and biophysical properties can be further engineered. Some of these improvements may include, but are not limited to, stronger binding to antigens, specificity (e.g., binding to cynomolgus monkey and human forms of antigens), or increased structural (e.g., thermal) stability. To this end, mature libraries (e.g., as described herein) can be designed and screened to isolate variants with desired improved properties. Methods for designing these libraries may include: the epitope is extended by mutating amino acid positions near the original library position, the sequence of the original conjugate is randomized using a method that favors retention of some portion of the original sequence, or mutations throughout the domain are randomly pooled using error-prone PCR to explore additional sequence space within and near the binding epitope. These libraries were then screened using the methods described above to isolate clones with a set of desired properties.

CD98 heavy chain binding polypeptide

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). These polypeptides are capable of transporting across the Blood Brain Barrier (BBB).

The polypeptides provided herein may comprise a modified CH3 domain that specifically binds to CD98hc protein. As described herein, when describing polypeptides (e.g., fc polypeptides) comprising a modified CH3 domain comprising amino acids 111-217 of one or more SEQ ID NOs, or a modified CH3 domain comprising amino acid substitutions or deletions relative to amino acids 111-217 of one or more SEQ ID NOs, or a modified CH3 domain comprising a sequence having a percent identity to amino acids 111-217 of one or more SEQ ID NOs, such descriptions are directed to the sequence of the modified CH3 domain and should not be construed as limiting the polypeptide to contain amino acids 1-110 of the one or more SEQ ID NOs.

It will be appreciated by those skilled in the art that the CH3 domains of other immunoglobulin isoforms (e.g., igM, igA, igE, igD, etc.) can be similarly modified by identifying amino acids in those domains that correspond to the amino acid substitutions at the positions described herein. Modifications may also be made to the corresponding domains of immunoglobulins from other species (e.g., non-human primate, monkey, mouse, rat, or other non-human mammal).

CD98hc binding site modification

In one embodiment, provided herein is a modified polypeptide comprising a modified constant domain that specifically binds to a CD98hc protein (e.g., a modified CH3 domain), wherein the modified constant domain comprises at least five, six, seven, eight, or nine substitutions in a set of amino acid positions consisting of 382, 384, 385, 387, 422, 424, 426, 438, 440; and wherein the substitution is determined with reference to SEQ ID NO. 1 and the position is determined according to EU numbering.

In some embodiments, the polypeptide that binds to CD98hc is from the LLB2 family. In some embodiments, the polypeptide comprises at least eleven, twelve, thirteen, fourteen or fifteen substitutions in a 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 substitution is selected from 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 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.

In some embodiments, the polypeptide that binds to CD98hc is from the LLB1 family. In some embodiments, the polypeptide comprises at least eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen substitutions in a 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, 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 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.

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 the sequence of any of SEQ ID NOs 28-45.

Transferrin receptor binding polypeptides

This section describes the production of polypeptides according to the present disclosure that bind to transferrin receptor (TfR), i.e., polypeptides having a TfR binding site. These polypeptides are capable of transporting across the Blood Brain Barrier (BBB).

The polypeptides provided herein may comprise a modified CH3 domain that specifically binds to TfR. As described herein, when describing polypeptides (e.g., fc polypeptides) comprising a modified CH3 domain comprising amino acids 111-217 of a certain SEQ ID NO, or a modified CH3 domain comprising amino acid substitutions and/or deletions relative to amino acids 111-217 of a certain SEQ ID NO, or a modified CH3 domain comprising a sequence having a percent identity to amino acids 111-217 of a certain SEQ ID NO, such descriptions are directed to the sequence of the CH3 domain and should not be construed as limiting the polypeptide to contain amino acids 1-113 of the certain SEQ ID NO.

TfR binding site modification

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

Also provided herein is a polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises three, four, five, six, seven or eight amino acid substitutions and/or one or two amino acid deletions in a set of amino acid positions comprising 380 and 382-389; and five, six or seven amino acid substitutions in a set of amino acid positions comprising 422, 424, 426, 433, 434, 438 and 440, wherein the positions are determined according to EU numbering.

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

Also provided herein is a polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises a first sequence comprising at least one amino acid substitution in sequence AVEWESNGQPENN (SEQ ID NO: 56), and a second sequence comprising at least one amino acid substitution in sequence VFSCSVMHEALHNHYTQKS (SEQ ID NO: 57), wherein sequence SEQ ID NO:56 is from position 378 to position 390 of an Fc polypeptide (e.g., SEQ ID NO: 1), sequence SEQ ID NO:57 is from position 422 to position 440 of an Fc polypeptide (e.g., SEQ ID NO: 1), and positions are determined according to EU numbering. In some embodiments, the modified CH3 domain comprises three, four, five, six, seven or eight amino acid substitutions in a set of amino acid positions comprising 380 and 382-389. In certain embodiments, the modified CH3 domain comprises five, six, or seven amino acid substitutions in a set of amino acid positions comprising 422, 424, 426, 433, 434, 438, and 440.

Modifications at positions 380 and 382-389

Provided herein are polypeptides comprising a modified CH3 domain having at least one (e.g., one, two, three, four, five, six, seven, or eight (e.g., three, four, five, six, seven, or eight)) amino acid substitution and/or at least one (e.g., one or two) amino acid deletion in a set of amino acid positions comprising 380 and 382-389 according to EU numbering. The modified CH3 domain may comprise a sequence comprising at least one (e.g., one, two, three, four, five, six, seven, or eight (e.g., three, four, five, six, seven, or eight)) amino acid substitution and/or at least one (e.g., one or two) amino acid deletion in sequence AVEWESNGQPENN (SEQ ID NO: 56), sequence SEQ ID NO:56 being from position 378 to position 390 of an Fc polypeptide (e.g., SEQ ID NO: 1). In some embodiments, the modified CH3 domain may comprise the following sequence: comprising at least one (e.g., one, two, three, four, five, six, seven or eight (e.g., three, four, five, six, seven or eight)) amino acid substitution and/or at least one (e.g., one or two) amino acid deletion(s) in a set of amino acid positions 380 and 382-389 relative to the sequence of SEQ ID No. 56, wherein the positions are numbered according to EU numbering.

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

In certain embodiments, at least one of the amino acid substitutions in the set of amino acid positions comprising 380 and 382-389 is at a β -sheet position relative to sequence SEQ ID NO. 56. In some embodiments, the modified CH3 domain comprises one, two, or three amino acid substitutions at the β -sheet position relative to sequence SEQ ID NO: 56. In particular embodiments, the β -sheet position is 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, such as E, N, F or Y, at position 380 relative to the sequence SEQ ID NO. 56. In a particular embodiment, 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 sequence SEQ ID NO: 56. In certain embodiments, the modified CH3 domain comprises an amino acid substitution, such as Y or A, at position 383 relative to sequence SEQ ID NO: 56. In a particular embodiment, the amino acid substitution at position 383 is Y.

In some embodiments of the polypeptides described herein, the polypeptide may comprise a modified CH3 domain comprising at least one position selected from the group consisting of: e, N, F or Y at position 380, F at position 382, Y, S, A or 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, wherein positions are numbered according to EU numbering. In particular embodiments, the modified CH3 domain may comprise five, six, seven or eight positions selected from the group consisting of: 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, N, T or S at position 389. In particular embodiments, the modified CH3 domain may comprise the following five positions: f at location 382, E at location 384, S at location 386, K at location 387, and T at location 389. In particular embodiments, the modified CH3 domain may comprise the following five positions: f at location 382, G at location 384, a at location 385, K at location 387, and S at location 389. In particular embodiments, the modified CH3 domain may 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 particular embodiments, the modified CH3 domain may comprise the following six positions: f at location 382, Y at location 383, E at location 384, a at location 385, K at location 387, and L at location 388. In particular embodiments, the modified CH3 domain may comprise the following seven positions: f at location 382, Y at location 383, G at location 384, N at location 385, a at location 386, K at location 387, and T at location 389. In particular embodiments, the modified CH3 domain may comprise the following eight positions: f at location 382, Y at location 383, D at location 384, D at location 385, S at location 386, K at location 387, L at location 388, and T at location 389.

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., one, two, three, four, five, six, or seven (e.g., five, six, or seven)) amino acid substitution in a set of amino acid positions comprising 422, 424, 426, 433, 434, 438, and 440 according to EU numbering. The modified CH3 domain may comprise a sequence comprising at least one (e.g., one, two, three, four, five, six, or seven (e.g., five, six, or seven)) amino acid substitutions in sequence VFSCSVMHEALHNHYTQKS (SEQ ID NO: 57), sequence SEQ ID NO:57 being from position 422 to position 440 of the Fc polypeptide (e.g., SEQ ID NO: 1). In some embodiments, the modified CH3 domain may comprise the following sequence: comprises at least one (e.g., one, two, three, four, five, six or seven (e.g., five, six or seven)) amino acid substitution(s) in a set of amino acid positions comprising 422, 424, 426, 433, 434, 438 and 440 relative to the sequence SEQ ID NO:57, wherein the positions are numbered according to EU numbering. The modified CH3 domain does not have a combination of G at position 437, F at position 438, and D at position 440, wherein positions are determined according to EU numbering.

In some embodiments of the modified CH3 domain in the polypeptide, at least one of the amino acid substitutions in the set of amino acid positions comprising 422, 424, 426, 433, 434, 438 and 440 is at a β -sheet position relative to the sequence SEQ ID NO 57. In some embodiments, the modified CH3 domain comprises one, two, three, or four amino acid substitutions at β -sheet positions relative to sequence SEQ ID NO: 57. In particular embodiments, the β -sheet position 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 β -sheet position 424 relative to sequence SEQ ID NO. 57. The amino acid substitution at β -sheet position 424 in the modified CH3 domain may be a. In certain embodiments, the modified CH3 domain comprises an amino acid substitution at beta-sheet position 426 relative to sequence SEQ ID NO. 57. The amino acid substitution at β -sheet position 426 may be E. In certain embodiments, the modified CH3 domain comprises an amino acid substitution at β -sheet position 438 relative to sequence SEQ ID NO: 57. The amino acid substitution at β -sheet position 438 may be Y. In some embodiments, the modified CH3 domain comprises an amino acid substitution at β -sheet position 440 relative to sequence SEQ ID NO. 57. The amino acid substitution at β -sheet position 440 may be L.

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

In some embodiments of the polypeptides described herein, the polypeptide may comprise a modified CH3 domain comprising at least one position selected from the group consisting of: 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 may comprise five positions selected from: l at position 422, a at position 424, E at position 426, Y at position 438, and L at position 440.

TfR binding polypeptides

The present disclosure provides polypeptides comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: (i) Sequence AVX₁WFX₂X₃X₄X₅X₆X₇X₈N(SEQ ID NO:65), wherein X ₁ is E, N, F or Y; x ₂ is Y, S, A or absent; X ₃ is G, D, E or N; x ₄ is D, G, N or A; x ₅ is Q, S, G, A or N; x ₆ is K, I, R or G; X ₇ is E, L, D or Q; and X ₈ is N, T, S or R; and (ii) sequence LFACEVMHEALX ₁X₂ HYTYKL (SEQ ID NO: 67), wherein X ₁ is H or E; And X ₂ is N or G. The present disclosure provides polypeptides comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: (i) Sequence AVEWFX ₁X₂X₃X₄KX₅X₆ N (SEQ ID NO: 66), wherein X ₁ is Y or S; x ₂ is G, D or E; x ₃ is D, G, N or A; x ₄ is Q, S or A; x ₅ is E or L; And X ₆ is N, T or S; and (ii) sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64).

In some embodiments, the modified CH3 domain comprises 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 sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64).

The modified CH3 domain in the polypeptides described herein may comprise sequence AVEWFYD DSKLTN (SEQ ID NO: 58) and sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64). The modified CH3 domain in the polypeptides described herein may comprise sequence AVEWFYG NAKETN (SEQ ID NO: 59) and sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64). The modified CH3 domain in the polypeptides described herein may comprise sequence AVEWFYE AQKLNN (SEQ ID NO: 60) and sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64). The modified CH3 domain in the polypeptides described herein may comprise sequence AVEWFSE GSKETN (SEQ ID NO: 61) and sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64). The modified CH3 domain in the polypeptides described herein may comprise sequence AVEWFSG AQKESN (SEQ ID NO: 62) and sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64). The modified CH3 domain in the polypeptides described herein may comprise sequence AVEWFSG AQKLTN (SEQ ID NO: 63) and sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64).

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, 442, and 443, wherein the positions are determined according to EU numbering. In particular embodiments, the modified CH3 domain comprises Q or P at position 419, G or R at position 420, N or G at position 421, S or G at position 442, and/or L or E at position 443. In certain embodiments, the modified CH3 domain comprises P at position 419, R at position 420, G at position 421, G at position 442, and E at position 443.

The present disclosure provides a polypeptide comprising sequence APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVX₁WF X₂X₃X₄X₅X₆X₇X₈NYKTTPPVLDSDGSFFLYSKLTVDKSRWQX₉X₁ ₀X₁₁LFACEVMHEALX₁₂X₁₃HYTYKLLX₁₄X₁₅SPGK(SEQ ID NO:68), wherein X ₁ is E, N, F or Y; x ₂ is Y, S, A or absent; x ₃ is G, D, E or N; x ₄ is D, G, N or A; x ₅ is Q, S, G, A or N; x ₆ is K, I, R or G; x ₇ is E, L, D or Q; x ₈ is N, T, S or R; x ₉ is Q or P; x ₁₀ is G or R; x ₁₁ is N or G; x ₁₂ is H or E; x ₁₃ is N or G; x ₁₄ is S or G; and X ₁₅ is L or E.

In some embodiments, the disclosure provides a polypeptide comprising the sequence:

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVX₁WFX₂X₃X₄X₅X₆X₇X₈NYKTTPPVLD SDGSFFLYSKLTVDKSRWQX₉X₁₀X₁₁LFACEVMHEALHNHYTYKLLX₁₂X₁₃SPGK(SEQ ID NO:69), Wherein X ₁ is E, N, F or Y; x ₂ is Y, S, A or absent; x ₃ is G, D, E or N; x ₄ is D, G, N or A; x ₅ is Q, S, G, A or N; x ₆ is K, I, R or G; x ₇ is E, L, D or Q; x ₈ is N, T, S or R; x ₉ is Q or P; x ₁₀ is G or R; x ₁₁ is N or G; x ₁₂ is S or G; and X ₁₃ is L or E.

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVX₁WFX₂X₃X₄X₅X₆X₇X₈NYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNLFACEVMHEALHNHYTYKLLSLSPGK(SEQ IDNO:70), Wherein X ₁ is E, N, F or Y; x ₂ is Y, S, A or absent; x ₃ is G, D, E or N; x ₄ is D, G, N or A; x ₅ is Q, S, G, A or N; x ₆ is K, I, R or G; x ₇ is E, L, D or Q; and X ₈ is N, T, S or R.

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWFX₁X₂X₃X₄KX₅X₆NYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNLFACEVMHEALHNHYTYKLLSLSPGK(SEQ ID NO:71), Wherein X ₁ is Y or S; x ₂ is G, D or E; x ₃ is D, G, N or A; x ₄ is Q, S or A; x ₅ is E or L; and X ₆ is N, T or S.

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 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 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 in each of SEQ ID NOs 72-77 are unchanged. In certain embodiments, the modified CH3 domain comprises amino acids 111-217 of any of SEQ ID NOs 72-77.

In some embodiments, polypeptides (e.g., fc polypeptides) comprising a modified CH3 domain described herein comprise 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 the sequence of any 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 the sequence of any 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 in each of SEQ ID NOs 72-77 are unchanged. In certain embodiments, a polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein comprises the sequence of any of SEQ ID NOs 72-77.

A polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein can comprise: f at location 382, Y at location 383, D at location 384, D at location 385, S at location 386, K at location 387, L at location 388, T at location 389, P at location 419, R at location 420, G at location 421, L at location 422, a at location 424, E at location 426, Y at location 438, L at location 440, G at location 442, and E at location 443, wherein location is determined according to EU numbering.

A polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein can comprise: 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, L at position 440, wherein positions are determined according to EU numbering.

A polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein can comprise: 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, L at position 440, wherein positions are determined according to EU numbering.

A polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein can comprise: 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, L at position 440, wherein positions are determined according to EU numbering.

A polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein can comprise: 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, L at position 440, wherein positions are determined according to EU numbering.

A polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein can comprise: 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, L at position 440, wherein positions are determined according to EU numbering.

In some embodiments, the polypeptide described above may further comprise W at position 366. In some embodiments, the polypeptides described above may further comprise S at position 366, a at position 368, and V at position 407. In certain embodiments, the polypeptides described above may further comprise a at position 234 and a at position 235. In certain embodiments, the polypeptide described above may further comprise Gly or Ser at position 329. In some embodiments, the polypeptide described above may further comprise L at position 428 and S at position 434. The position is determined according to EU numbering.

VI additional polypeptide modifications

Polypeptides (e.g., fc polypeptides) comprising a modified CH3 domain as provided herein may also comprise additional modifications, e.g., to provide for pestle and mortar heterodimerization of the polypeptide, to modulate effector function, to extend serum half-life, to affect glycosylation, and/or to reduce immunogenicity in humans.

Polypeptide modification for heterodimerization

In some embodiments, polypeptides (e.g., fc polypeptides) comprising modified CH3 domains described herein include mutations to promote heterodimer formation and to hinder homodimer formation. These modifications are useful, for example, where only one of the polypeptides for which a dimer is desired has a CD98hc or TfR binding site (i.e., a monovalent CD98hc or TfR conjugate).

The knob-and-socket approach generally involves introducing a protrusion ("knob") at the interface of a polypeptide (e.g., an Fc polypeptide) and a corresponding cavity ("socket") in the interface of a second polypeptide (e.g., an Fc polypeptide), such that the protrusion can be positioned in the cavity to promote heterodimer formation and thereby hinder homodimer formation. The protrusions are constructed by replacing smaller amino acid side chains from the interface of the first polypeptide (e.g., fc polypeptide) with larger side chains (e.g., tyr or Trp). Compensatory cavities of the same or similar size as the protrusions are created in the interface of a second polypeptide (e.g., an Fc polypeptide) by replacing a larger amino acid side chain with a smaller amino acid side chain (e.g., ala or Thr). In some embodiments, such additional mutations are at positions in the polypeptide (e.g., fc polypeptide) that do not have a negative effect on binding of the polypeptide to CD98hc or TfR.

In one illustrative embodiment of the pestle and mortar method for dimerization, position 366 of one of the polypeptides (e.g., fc polypeptides) comprises Trp in place of native Thr. Another polypeptide in the dimer has Val in place of native Tyr at position 407. Another polypeptide (e.g., an Fc polypeptide) may further comprise a substitution wherein native Thr at position 366 is substituted with Ser and native Leu at position 368 is substituted with Ala. Thus, one of the polypeptides (e.g., fc polypeptide) has a T366W knob mutation and the other polypeptide (e.g., fc polypeptide) has a Y407V knob mutation, which is typically accompanied by T366S and L368A knob mutations. As described above, all positions are numbered according to EU numbering.

In some embodiments, one or both polypeptides (e.g., fc polypeptides) present in a polypeptide dimer (e.g., fc polypeptide dimer) may also be engineered to contain other modifications for heterodimerization, such as electrostatic engineering of contact residues within the CH3-CH3 interface that are modified as naturally charged or hydrophobic patches.

The knob and socket method (e.g., T366W knob substitution on one polypeptide (e.g., fc polypeptide) and T366S, L a and Y407V knob substitution on another polypeptide (e.g., fc polypeptide)) can be used with any of the polypeptides described herein (e.g., CD98hc binding polypeptide having the sequence of any of SEQ ID NOs 28-45, or TfR binding polypeptide having the sequence of any of SEQ ID NOs 72-77, or the sequence of any of the clones shown in table 29).

In some embodiments, only one of the two polypeptides (e.g., an Fc polypeptide) comprises a CD98hc binding site (e.g., a CD98hc binding polypeptide having the sequence of any of SEQ ID NOS: 28-45), while the other polypeptide (e.g., an Fc polypeptide) does not contain a CD98hc binding site. In particular embodiments, one of the polypeptides (e.g., fc polypeptides) is a CD98 hc-binding polypeptide and contains a knob mutation (e.g., T366W), while the other polypeptide (e.g., fc polypeptide) does not bind CD98hc and contains a knob mutation (e.g., T366S, L368A and Y407V). In other embodiments, one of the polypeptides (e.g., fc polypeptide) is a CD98 hc-binding polypeptide and contains a mortar mutation (e.g., T366S, L a and Y407V), while the other polypeptide (e.g., fc polypeptide) does not bind CD98hc and contains a mortar mutation (e.g., T366W).

In some embodiments, only one of the two polypeptides (e.g., an Fc polypeptide) comprises 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 shown in table 29), while the other polypeptide (e.g., an Fc polypeptide) does not contain a TfR binding site. In particular 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 knob 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 a mortar mutation (e.g., T366S, L a and Y407V), while the other polypeptide (e.g., fc polypeptide) does not bind to TfR and contains a mortar mutation (e.g., T366W).

In particular embodiments, a polypeptide dimer that specifically binds to CD98hc (e.g., an Fc polypeptide dimer) can have 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 any of SEQ ID NOs 28-45, and a second polypeptide (e.g., an Fc polypeptide) that has a T366S, L a and Y407V 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. In other embodiments, a polypeptide dimer that specifically binds CD98hc (e.g., an Fc polypeptide dimer) can have 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 T366S, L a and Y407V knob 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 any of SEQ ID nos. 28-45. In other embodiments, a polypeptide dimer that specifically binds to CD98hc (e.g., an Fc polypeptide dimer) can have 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. 28-45, and a second polypeptide (e.g., an Fc polypeptide) that has a T366S, L a and Y407V 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 any of SEQ ID nos. 28-45.

In particular embodiments, a polypeptide dimer that specifically binds to TfR (e.g., an Fc polypeptide dimer) can have 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 the sequence of any of SEQ ID NOs 72-77 or the sequence of any of the clones set forth in table 29, and a second polypeptide (e.g., an Fc polypeptide) that has a T366S, L a and Y407V 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. In other embodiments, a polypeptide dimer that specifically binds to TfR (e.g., an Fc polypeptide dimer) can have 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, L a and Y407V 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 the sequence of any of the clones set forth in table 29 or the sequence of any of SEQ ID nos. 72-77. In other embodiments, an Fc polypeptide dimer that specifically binds to TfR (e.g., an Fc polypeptide dimer) can have 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 the sequence of any of SEQ ID NOs 72-77 or the sequence of any of the clones set forth in table 29, and a second polypeptide (e.g., an Fc polypeptide) that has a T366S, L a and Y407V 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 the sequence of any of the clones set forth in table 29.

Polypeptide modifications for modulating effector function

In some embodiments, the polypeptide dimers described herein are Fc polypeptide dimers comprising two Fc polypeptides. In some embodiments, two Fc polypeptides in an Fc polypeptide dimer may comprise modifications that reduce or eliminate effector function, i.e., have the ability to induce a reduction in certain biological functions when bound to Fc receptors expressed on effector cells that mediate 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.

Illustrative 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 (also referred to herein as "LALA") at positions 234 and 235. 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, the two Fc polypeptides comprise Ala residues at positions 234 and 235, and a Gly residue at position 329 (also referred to herein as "LALA PG"). In some embodiments, the two Fc polypeptides comprise Ala residues at positions 234 and 235, and Ser residue at position 329 (also referred to herein as "LALA PS").

Additional Fc polypeptide mutations that modulate effector function include, but are not limited to, the following: position 329 may have a mutation wherein Pro is substituted with Gly, ala, ser or Arg or an amino acid residue large enough to disrupt the Fc/Fc gamma receptor interface formed between proline 329 of Fc and Trp residues Trp87 and Trp110 of Fc gamma RIII. Additional illustrative substitutions include S228P, E233P, L235E, N297A, N297D and P331S according to the EU numbering scheme. Multiple substitutions may also be present, for example, L234A, L a and P329G of human IgG1 according to the EU numbering scheme; S228P and L235E of human IgG 4; L234A and G237A of human IgG 1; L234A, L A and G237A of human IgG 1; V234A and G237A of human IgG 2; L235A, G a and E318A of human IgG 4; and S228P and L236E of human IgG 4.

In some embodiments, a polypeptide that specifically binds to CD98hc (e.g., an Fc polypeptide) comprises LALA substitutions and a sequence 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 of SEQ ID NOs 28-45.

In some embodiments, polypeptides that specifically bind CD98hc (e.g., fc polypeptides) comprise LALA and P329G or P329S substitutions, 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 of SEQ ID NOs 28-45.

In some embodiments, a polypeptide that specifically binds to TfR (e.g., an Fc polypeptide) comprises LALA substitutions and sequences that are 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 the sequence of any one of SEQ ID NOs 72-77 or the sequence of any one of the clones shown in table 29.

In some embodiments, a polypeptide that specifically binds to TfR (e.g., an Fc polypeptide) comprises LALA and P329G or P329S substitutions, and a sequence 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 the sequence of any one of SEQ ID NOs 72-77 or the sequence of any one of the clones set forth in table 29.

Polypeptide modifications for extending serum half-life

In some embodiments, modifications that enhance serum half-life may be incorporated into any of the polypeptides described herein. For example, in some embodiments, the polypeptide dimers described herein are Fc polypeptide dimers comprising two Fc polypeptides. In some embodiments, two Fc polypeptides in an Fc polypeptide dimer may comprise M428L and N434S substitutions (also referred to as LS substitutions) as numbered according to the EU numbering scheme. Alternatively, both Fc polypeptides in the Fc polypeptide dimer may have N434S or N434A substitution. Alternatively, both Fc polypeptides in the Fc polypeptide dimer may have an M428L substitution. In other embodiments, two Fc polypeptides in an Fc polypeptide dimer may comprise M252Y, S254T and T256E substitutions.

In any of the embodiments described herein, the polypeptide that specifically binds to CD98hc (e.g., an Fc polypeptide) may further comprise an LS substitution. For example, in some embodiments, a polypeptide that specifically binds to CD98hc (e.g., an Fc polypeptide) comprises an LS substitution and a sequence 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 of SEQ ID NOs 28-45.

In any of the embodiments described herein, the polypeptide that specifically binds to TfR (e.g., an Fc polypeptide) can further comprise an LS substitution. For example, in some embodiments, a polypeptide that specifically binds to TfR (e.g., an Fc polypeptide) comprises an LS substitution and a sequence 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 the sequence of any one of SEQ ID NOs 72-77 or the sequence of any one of the clones shown in table 29.

Polypeptides for removing C-terminal lysine residues

In some embodiments, the C-terminal lysine (e.g., lys residue at position 447 of the Fc polypeptide according to EU numbering) of one or both of the polypeptides (e.g., fc polypeptides) is removable. The C-terminal lysine residues are highly conserved among immunoglobulins spanning many species and can be removed, either completely or partially, during protein production by cellular mechanisms. In some embodiments, removal of the C-terminal lysine in the Fc polypeptide may improve the stability of the protein.

VI I. illustrative Polypeptides that bind CD98HC

The modified CH3 domains of the disclosure may be joined to a CH2 domain, which CH2 domain may be a naturally occurring CH2 domain or a variant CH2 domain, typically located at the C-terminus of the CH2 domain to form a polypeptide (e.g., an Fc polypeptide) that binds CD98 hc. In some embodiments, the polypeptide (e.g., fc polypeptide) further comprises a portion or all of the hinge region of an antibody that is conjugated to the N-terminus of the CH2 domain. The hinge region may be from any immunoglobulin subclass or isotype. An illustrative immunoglobulin hinge is an IgG hinge region, such as an IgG1 hinge region, e.g., the human IgG1 hinge amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 4).

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 of the residues at positions selected from the group consisting of: 477, 478, 479, 480, 481, 482, 483, 486, 499, 497, 498, 500, 501 and 502 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, or 14 of the residues at positions selected from the group consisting of: 477, 478, 479, 480, 481, 482, 483, 486, 499, 497, 498, 500, 501 and 502 of SEQ ID NO. 134 and additionally with at least 1, 2, 3, 4, 5, 6, at a position selected from the group consisting of, 7 or 8 additional residues: 229, 231, 232, 236, 235, 488, 495 and 496 of SEQ ID No. 134, or at least 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 additional residues at positions selected from the group consisting of: 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 that, when bound to human CD98hc, bind 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, 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 of residues at positions selected from the group consisting of: 229, 231, 232, 236, 235, 486, 488, 495, 496, 498, 500, 499, 497, 482, 481, 483, 477 of SEQ ID NO. 134, 480. 501, 502, 478, and 479. In some embodiments, provided herein are CD98hc binding polypeptides that, when bound to human CD98hc, bind to residues at 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 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 of residues at positions selected from the group consisting of: 312, 315, 348, 381, 439, 444, 443, 485, 484, 477, 483, 481, 480, 478 of SEQ ID NO. 134, 476. 502, 499, 501, 500, 498, 497, 486, 479, 482, 475, and 442. In some embodiments, provided herein are CD98hc binding polypeptides that, when bound to human CD98hc, bind to residues at 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, the polypeptide (e.g., fc polypeptide) may comprise a sequence from table 2A, and the polypeptide (e.g., fc polypeptide) may be further modified to contain a CD98hc binding site in a modified CH3 domain as described herein.

TABLE 2A Fc sequences for other CD98hc binding site modifications or TfR binding site modifications

In other embodiments, a polypeptide described herein (e.g., an Fc polypeptide) can be further conjugated to another moiety, such as a Fab fragment, to produce an Fc-Fab fusion that binds to CD98 hc. In some embodiments, the Fc-Fab fusion that binds to CD98hc comprises a modified CH3 domain, a CH2 domain, a hinge region, and a Fab fragment. The Fab fragment can be directed against any target of interest, such as a therapeutic neurological target, wherein Fab is delivered to the target by endocytosis across the BBB mediated by binding of the modified CH3 domain polypeptide to CD98 hc.

CD98hc binding polypeptides (e.g., CD98hc binding Fc polypeptides) may also be fused to polypeptides of interest other than Fab. For example, in some embodiments, a CD98 hc-binding polypeptide (e.g., a CD98 hc-binding Fc polypeptide) can be fused to a polypeptide that desirably targets CD98 hc-expressing cells or is delivered across endothelial cells, such as the BBB, by endocytic transport. In some embodiments, a CD98hc binding polypeptide (e.g., a CD98hc binding Fc polypeptide) is fused to a soluble protein. In still other embodiments, a CD98 hc-binding polypeptide (e.g., a CD98 hc-binding Fc polypeptide) may be fused to a peptide or protein suitable for protein purification, such as polyhistidine, epitope tags (e.g., FLAG, c-Myc, lectin tags, and analogs thereof), glutathione S Transferase (GST), thioredoxin, protein a, protein G, and Maltose Binding Protein (MBP). In some cases, the peptide or protein fused to a CD98 hc-binding polypeptide (e.g., a CD98 hc-binding Fc polypeptide) may comprise a protease cleavage site, such as a cleavage site for factor Xa or thrombin.

CD98hc binding polypeptides

LLB2

In some embodiments, the polypeptide that binds to CD98hc is from the LLB2 family. In some embodiments, the polypeptide comprises at least eleven, twelve, thirteen, fourteen or fifteen substitutions in a 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 substitution is selected from 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 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. In some embodiments, the polypeptide comprises at least eleven, twelve, thirteen, fourteen or fifteen substitutions in a set of amino acid positions consisting of any of SEQ ID NOs 5-27: 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 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.

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 sequences 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 sequences SEQ ID NOs 28-43, wherein the modified constant domain comprises at least eleven, twelve, thirteen, fourteen, or fifteen substitutions in a set of amino acid positions 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, 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, 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 at least eleven, twelve, thirteen, fourteen or fifteen substitutions in a 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 at least eleven, twelve, thirteen, fourteen or fifteen substitutions in a set of amino acid positions consisting of any of SEQ ID NOs 5-27: 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 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 sequences SEQ ID NOs 28-43, wherein the modified constant domain comprises at least eleven, twelve, thirteen, fourteen, or fifteen substitutions in a 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 polypeptide that specifically binds to CD98hc (e.g., an Fc polypeptide) comprises a modified CH3 domain, wherein the modified CH3 domain comprises: (i) A first amino acid sequence LX ₁NX₂X₃X₄X₅ L (SEQ ID NO: 46), wherein X ₁ is any amino acid, wherein X ₂ is R, H or Q, wherein X ₃ is F or Y, wherein X ₄ is V, L, I, F, Y or E, wherein X ₅ is any amino acid; (ii) A second amino acid sequence X ₁X₂X₃AX₄X₅X₆X₇ (SEQ ID NO: 47), wherein X ₁ is E, N, Q or A, wherein X ₂ is I, V, T or P, wherein X ₃ and X ₄ are any amino acids, Wherein X ₅ is N or S, wherein X ₆ is any amino acid, wherein X ₇ is Y or W; and (iii) a third amino acid sequence X ₁X₂X₃X₄NX₅X₆ (SEQ ID NO: 48), wherein X ₁ is Y, R or W, wherein X ₂ is any amino acid, wherein X ₃ is F or W, Wherein X ₄ and X ₅ are any amino acid and wherein X ₆ is A, Q, K, R, H, M or S.

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., fc polypeptide) having 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. In one embodiment, the polypeptide (e.g., fc polypeptide) further comprises a T366W knob mutation.

LLB2-10-8

In certain embodiments, the polypeptide comprises SEQ ID NO. 29. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., fc polypeptide) having 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. In another embodiment, the bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., fc polypeptides) each having 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. In one embodiment, the polypeptide (e.g., fc polypeptide) further comprises a T366W knob mutation.

LLB2-10-8-d18

In certain embodiments, the polypeptide comprises SEQ ID NO. 30. In one embodiment, the bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., fc polypeptides) each having 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.

LLB2-10-8-d12

In certain embodiments, the polypeptide comprises SEQ ID NO. 31. In one embodiment, the bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., fc polypeptides) each having 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.

LLB2-10-8-d6

In certain embodiments, the polypeptide comprises SEQ ID NO 32. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., fc polypeptide) having 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. In another embodiment, the bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., fc polypeptides) each having 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. In one embodiment, the polypeptide (e.g., fc polypeptide) further comprises a T366W knob mutation.

LLB2-10-8-d3

In certain embodiments, the polypeptide comprises SEQ ID NO 33. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., fc polypeptide) having 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. In another embodiment, the bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., fc polypeptides) each having 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. In one embodiment, the polypeptide (e.g., fc polypeptide) further comprises a T366W knob mutation.

LLB2-10-8-d1

In certain embodiments, the polypeptide comprises SEQ ID NO 34. In one embodiment, the bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., fc polypeptides) each having 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.

LLB2.10.8.10.3

In certain embodiments, the polypeptide comprises SEQ ID NO. 35. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., fc polypeptide) having 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 one embodiment, the polypeptide (e.g., fc polypeptide) further comprises a T366W knob mutation.

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., fc polypeptide) having 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 one embodiment, the polypeptide (e.g., fc polypeptide) further comprises a T366W knob mutation.

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., fc polypeptide) having 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 one embodiment, the polypeptide (e.g., fc polypeptide) further comprises a T366W knob mutation.

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., fc polypeptide) having 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. In one embodiment, the polypeptide (e.g., fc polypeptide) further comprises a T366W knob mutation.

LLB2-37

In certain embodiments, the polypeptide comprises SEQ ID NO 39. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., fc polypeptide) having 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 another embodiment, the bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., fc polypeptides) each having 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 one embodiment, the polypeptide (e.g., fc polypeptide) further comprises a T366W knob mutation.

LLB2.10.8.9.11.N

In certain embodiments, the polypeptide comprises SEQ ID NO. 40. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., fc polypeptide) having 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.

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., fc polypeptide) having 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 one embodiment, the polypeptide (e.g., fc polypeptide) further comprises a T366W knob mutation.

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., fc polypeptide) having 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 one embodiment, the polypeptide (e.g., fc polypeptide) further comprises a T366W knob mutation.

LLB2.10.8.2.1

In certain embodiments, the polypeptide comprises SEQ ID NO. 43. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., fc polypeptide) having 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.

Additional polypeptides from the LLB2 family (e.g., fc polypeptides) that specifically bind CD98hc are shown in tables A2-A8 and a 12.

TABLE 2 illustrative CD98hc binding site modification

LLB1

In some embodiments, the polypeptide that binds to CD98hc (e.g., an Fc polypeptide) is from the LLB1 family. In some embodiments, a polypeptide (e.g., an Fc polypeptide) comprises at least eight, nine, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen substitutions in a 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, 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 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. 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 eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen substitutions in a set of amino acid positions 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, 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 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.

In some embodiments, the polypeptide (e.g., fc polypeptide) comprises a sequence having at least 85%, 90% or 95% sequence identity to amino acids 111-217 of sequence 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 sequence SEQ ID NOs 44-45, wherein the modified constant domain comprises at least eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen substitutions in a set of amino acid positions 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, 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 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.

In some embodiments, the polypeptide that binds to CD98hc (e.g., an Fc polypeptide) is from the LLB1 family. In some embodiments, a polypeptide (e.g., an Fc polypeptide) comprises at least eight, nine, ten, eleven, twelve, or thirteen substitutions in a 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 substitution is 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 the sequence of any of SEQ ID NOs 5-27 and at least eight, nine, ten, eleven, twelve or thirteen substitutions in a set of amino acid positions consisting of: 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 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 sequence SEQ ID NOs 44-45, wherein the modified constant domain comprises at least eight, nine, ten, eleven, twelve, or thirteen substitutions in a set of amino acid positions consisting of: 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 at least eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen substitutions in a 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 3839, 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, at least eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen of a 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 3839, 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 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 sequence SEQ ID NOs 44-45, wherein the modified constant domain comprises at least eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen of the amino acid positions of the group 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 3839, 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 at least eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen substitutions in a 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 substitution is 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 the sequence of any one of SEQ ID NOs 5-27 and at least eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen of the amino acid positions of the group consisting of: r at location 382, T at location 383, Y at location 384, K at location 385, P at location 386, Y at location 387, T at location 389, D at location 421, I at location 422, V at location 424, D at location 426, L at location 428, F at location 436, I at location 438, and K at location 440.

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 sequences SEQ ID NOs 28-43, wherein the modified constant domain comprises at least eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen of a set of amino acid positions consisting of: r at location 382, T at location 383, Y at location 384, K at location 385, P at location 386, Y at location 387, T at location 389, D at location 421, I at location 422, V at location 424, D at location 426, L at location 428, F at location 436, I at location 438, and K at location 440.

In some embodiments, the polypeptide that specifically binds to CD98hc (e.g., an Fc polypeptide) comprises a modified CH3 domain, wherein the modified CH3 domain comprises: (i) A first amino acid sequence X ₁X₂YKPYX₃ T (SEQ ID NO: 49), wherein X ₁ is E or R, wherein X ₂ is S or T, wherein X ₃ is any amino acid; (ii) A second amino acid sequence X ₁X₂X₃VX₄DX₅X₆ (SEQ ID NO: 50), wherein X ₁ is N or D, wherein X ₂ is V or I, wherein X ₃、X₄ and X ₅ are any amino acids, wherein X ₆ is M or L; and (iii) a third amino acid sequence X ₁X₂IX₃X₄ (SEQ ID NO: 51), wherein X ₁ is Y or F, wherein X ₂ and X ₃ are any amino acids, wherein X ₄ is S or K.

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., fc polypeptide) having 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. In one embodiment, the polypeptide (e.g., fc polypeptide) further comprises a T366W knob mutation.

LLB1-3-16

In certain embodiments, the polypeptide comprises SEQ ID NO. 45. In one embodiment, the monovalent dimer (e.g., monovalent Fc dimer) comprises a polypeptide (e.g., fc polypeptide) having 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 one embodiment, the polypeptide (e.g., fc polypeptide) further comprises a T366W knob mutation.

Additional polypeptides from the LLB1 family (e.g., fc polypeptides) that specifically bind CD98hc are shown in tables A9-a11 and a 13.

Illustrative Polypeptides binding to TFR

The CH3 domains of the disclosure may be joined to a CH2 domain, which may be a naturally occurring CH2 domain or a variant CH2 domain, typically located at the C-terminus of the CH2 domain to form a polypeptide (e.g., an Fc polypeptide) that binds to TfR. In some embodiments, the polypeptide (e.g., fc polypeptide) further comprises a portion or all of the hinge region of an antibody that is conjugated to the N-terminus of the CH2 domain. The hinge region may be from any immunoglobulin subclass or isotype. An illustrative immunoglobulin hinge is an IgG hinge region, such as an IgG1 hinge region, e.g., the human IgG1 hinge amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 4).

In some embodiments, the polypeptide (e.g., fc polypeptide) can comprise a sequence from table 2A, and the polypeptide (e.g., fc polypeptide) can be further modified to contain a TfR binding site in a modified CH3 domain as described herein.

In other embodiments, the polypeptide (e.g., fc polypeptide) may be further conjugated to another moiety, such as a Fab fragment, to produce an Fc-Fab fusion that binds to TfR. In some embodiments, the Fc-Fab fusion that binds TfR comprises a modified CH3 domain, a CH2 domain, a hinge region, and a Fab fragment. The Fab fragment can be directed against any target of interest, such as a therapeutic neurological target, wherein Fab is delivered to the target by modified CH3 domain polypeptide binding to TfR mediated endocytosis across the BBB.

TfR binding polypeptides (e.g., tfR binding Fc polypeptides) may also be fused to the polypeptide of interest, rather than Fab. For example, in some embodiments, a TfR-binding polypeptide (e.g., a TfR-binding Fc polypeptide) can be fused to a polypeptide that is desired to target TfR-expressing cells or to be delivered across endothelial cells, such as the BBB, by endocytic transport. In some embodiments, the TfR-binding polypeptide (e.g., tfR-binding Fc polypeptide) is fused to a soluble protein. In still other embodiments, tfR-binding polypeptides (e.g., tfR-binding Fc polypeptides) may be fused to peptides or proteins suitable for protein purification, such as polyhistidine, epitope tags (e.g., FLAG, c-Myc, lectin tags, and analogs thereof), glutathione S Transferase (GST), thioredoxin, protein a, protein G, and Maltose Binding Protein (MBP). In some cases, the peptide or protein fused to a TfR-binding polypeptide (e.g., a TfR-binding Fc polypeptide) may comprise a protease cleavage site, such as a cleavage site for factor Xa or thrombin.

TfR binding polypeptides

42.2.19

In certain embodiments, the polypeptide comprises the sequence SEQ ID NO. 72. Furthermore, the polypeptide may comprise the sequence of any of SEQ ID NOs 78, 84, 90, 96, 102, 108, 114 and 120. 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, 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. In one embodiment, the polypeptide (e.g., fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises a T366W knob mutation. In another embodiment, the polypeptide (e.g., fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises T366S, L a and Y407V mortar mutations. In another embodiment, the bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., fc polypeptides) each having 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.

42.2.3-1H

In certain embodiments, the polypeptide comprises the sequence SEQ ID NO 73. Furthermore, the polypeptide may 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, L at position 440, wherein the positions are determined according to EU numbering. In one embodiment, the polypeptide (e.g., fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises a T366W knob mutation. In another embodiment, the polypeptide (e.g., fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises T366S, L a and Y407V mortar mutations. In another embodiment, the bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., fc polypeptides) each 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, L at position 440, wherein positions are determined according to EU numbering.

42.8.196

In certain embodiments, the polypeptide comprises the sequence SEQ ID NO 74. Furthermore, the polypeptide may comprise the sequence of any 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, L at position 440, wherein positions are determined according to EU numbering. In one embodiment, the polypeptide (e.g., fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises a T366W knob mutation. In another embodiment, the polypeptide (e.g., fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises T366S, L a and Y407V mortar mutations. In another embodiment, the bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., fc polypeptides) each 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, L at position 440, wherein positions are determined according to EU numbering.

42.8.80

In certain embodiments, the polypeptide comprises the sequence SEQ ID NO 75. Furthermore, the polypeptide may 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, L at position 440, wherein positions are determined according to EU numbering. In one embodiment, the polypeptide (e.g., fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises a T366W knob mutation. In another embodiment, the polypeptide (e.g., fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises T366S, L a and Y407V mortar mutations. In another embodiment, the bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., fc polypeptides) each 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, L at position 440, wherein positions are determined according to EU numbering.

42.8.15

In certain embodiments, the polypeptide comprises the sequence SEQ ID NO 76. Furthermore, the polypeptide may comprise the sequence of any 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, L at position 440, wherein positions are determined according to EU numbering. In one embodiment, the polypeptide (e.g., fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises a T366W knob mutation. In another embodiment, the polypeptide (e.g., fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises T366S, L a and Y407V mortar mutations. In another embodiment, the bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., fc polypeptides) each 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, L at position 440, wherein positions are determined according to EU numbering.

42.8.17

In certain embodiments, the polypeptide comprises the sequence SEQ ID NO 77. Furthermore, the polypeptide may 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, L at position 440, wherein positions are determined according to EU numbering. In one embodiment, the polypeptide (e.g., fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises a T366W knob mutation. In another embodiment, the polypeptide (e.g., fc polypeptide) in a monovalent dimer (e.g., monovalent Fc dimer) further comprises T366S, L a and Y407V mortar mutations. In another embodiment, the bivalent dimer (e.g., bivalent Fc dimer) comprises two polypeptides (e.g., fc polypeptides) each 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, L at position 440, wherein positions are determined according to EU numbering.

IX. dimers for CD98HC binding site modification

In some embodiments, a polypeptide (e.g., an Fc polypeptide) that binds CD98hc can form a dimer (e.g., an Fc dimer) comprising both polypeptides (e.g., fc polypeptides). The dimer may be a heterodimer or a homodimer.

Dimer which binds divalent CD98hc

In some embodiments, the dimer is an Fc dimer comprising two Fc polypeptides, wherein each Fc polypeptide comprises a CD98hc binding site (i.e., bivalent binds CD98 hc). In the bivalent CD98 hc-binding Fc dimer, the first and second Fc polypeptides may comprise the same modified CH3 domain. In other embodiments, the second Fc polypeptide may comprise a modified CH3 domain that is different from the first Fc polypeptide to provide a second CD98hc binding site.

In some embodiments, the bivalent Fc dimer that specifically binds to CD98hc described herein comprises a first and second pair of Fc polypeptides from table 2C, and (i) each of the first and second Fc polypeptides is further modified to contain a CD98hc binding site in a modified CH3 domain as described herein, or (ii) wherein the first and second Fc polypeptides contain a CD98hc binding site in a modified CH3 domain as described herein. In other embodiments, the bivalent Fc dimer that specifically binds to CD98hc described herein comprises a first and second pair of Fc polypeptides 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 the sequence of the first Fc polypeptide sequence from table 2C, wherein 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, and wherein each of the first and second Fc polypeptides is further modified to contain a CD98hc binding site in a modified CH3 domain as described herein. In one embodiment, the bivalent Fc dimer that specifically binds to CD98hc described herein comprises a first and second pair of Fc polypeptides 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 the sequence of the first Fc polypeptide from table 2C, wherein the second polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89% identity to the second Fc polypeptide sequence from table 2C 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100%, and wherein each of the first and second Fc polypeptides is further modified to contain a CD98hc binding site in a modified CH3 domain comprising: (i) At least eleven, twelve, thirteen, fourteen or fifteen substitutions in a 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; (ii) At least eleven, twelve, thirteen, fourteen or fifteen substitutions in a set of amino acid positions 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, V, L, A, I, F, Y, S, T, H, R or E at position 386, L or I at position 387, a combination of two or more of the above, 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, A, V, M, Q, F, P, L at position 442, Y, K, R, H or M; or (iii) at least eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen or nineteen substitutions in a set of amino acid positions 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, T, P, E, K, A, V, D, T or F at position 386, N, L, Y at position 387, and, 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.

In one embodiment, the bivalent Fc dimer that specifically binds to CD98hc described herein comprises a first and second pair of Fc polypeptides 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 the sequence of the first Fc polypeptide sequence from table 2C, wherein 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 second Fc polypeptide sequence from table 2C, and wherein each of the first and second Fc polypeptides is further modified to contain a CD98hc binding site in a modified CH3 domain comprising a modification selected from the group of table 2B.

Dimer binding monovalent 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 CD98 hc. In addition, the Fc polypeptide may contain modifications for promoting heterodimerization of the Fc dimer (e.g., T366W; and T366S, L368A and Y407V). In some embodiments, the monovalent Fc dimer that specifically binds to CD98hc described herein comprises a first and second Fc polypeptide pair from table 2D, and the first Fc polypeptide is further modified to contain a CD98hc binding site in a modified CH3 domain as described herein. In other embodiments, a monovalent Fc dimer that specifically binds to CD98hc described herein comprises a first and second pair of Fc polypeptides 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 sequence of the first Fc polypeptide sequence from table 2D, wherein 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%, and wherein the first Fc polypeptide is further modified to contain a CD98hc binding site in 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 pair of Fc polypeptides 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 sequence of the first Fc polypeptide from table 2D, wherein the second polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89% identity to the second Fc polypeptide sequence from table 2D 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100%, and wherein the first Fc polypeptide is further modified to contain a CD98hc binding site in a modified CH3 domain comprising: (i) At least eleven, twelve, thirteen, fourteen or fifteen substitutions in a 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; (ii) At least eleven, twelve, thirteen, fourteen or fifteen substitutions in a set of amino acid positions 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, V, L, A, I, F, Y, S, T, H, R or E at position 386, L or I at position 387, a combination of two or more of the above, 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, A, V, M, Q, F, P, L at position 442, Y, K, R, H or M; or (iii) at least eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen or nineteen substitutions in a set of amino acid positions 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, T, P, E, K, A, V, D, T or F at position 386, N, L, Y at position 387, and, 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.

For example, the first Fc polypeptide of dimer pair K from Table 2D (i.e., SEQ ID NO: 11) is further modified to include 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.

In one embodiment, the monovalent Fc dimer that specifically binds to CD98hc described herein comprises a first and second pair of Fc polypeptides 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 sequence of the first Fc polypeptide from table 2D, wherein 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 second Fc polypeptide sequence from table 2B, and wherein the first Fc polypeptide is further modified to contain a CD98hc binding site in a modified CH3 domain comprising a modification selected from the group of table 2B.

Dimers for TFR binding site modification

In some embodiments, a polypeptide that binds to TfR (e.g., an Fc polypeptide) can form a dimer (e.g., an Fc dimer) comprising both polypeptides (e.g., fc polypeptides). The dimer may be a heterodimer or a homodimer.

Bivalent TfR-binding dimers

In some embodiments, the dimer is an Fc dimer comprising two polypeptides (e.g., fc polypeptides), wherein each Fc polypeptide contains a TfR binding site (i.e., divalent binding TfR). In a divalent TfR-binding Fc dimer, the first and second Fc polypeptides may comprise the same CH3 domain. In other embodiments, the second Fc polypeptide may comprise a CH3 domain that is different from the first Fc polypeptide to provide a second TfR binding site.

In some embodiments, the divalent Fc dimer that specifically binds to TfR described herein comprises a pair of first and second Fc polypeptides from table 2C, and (i) each of the first and second Fc polypeptides is further modified to contain a CD98hc binding site in a modified CH3 domain as described herein, or (ii) wherein the first and second Fc polypeptides contain a CD98hc binding site in a modified CH3 domain as described herein. In other embodiments, the divalent Fc dimer that specifically binds to TfR described herein comprises a first and second pair of 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, wherein 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 each of the first and second Fc polypeptides is further modified to contain a TfR binding site in a modified CH3 domain as described herein.

In one embodiment, the divalent Fc dimer that specifically binds to TfR described herein comprises a first and second pair of 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, wherein 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 each of the first and second Fc polypeptides is further modified to contain an r binding site in a modified CH3 domain comprising: three, four, five, six, seven or eight amino acid substitutions and/or one or two amino acid deletions in a set of amino acid positions comprising 380 and 382-389; and five, six or seven amino acid substitutions in a set of amino acid positions comprising 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 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.

In one embodiment, the divalent Fc dimer that specifically binds to TfR described herein comprises a first and second pair of 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, wherein 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 each of the first and second Fc polypeptides is further modified to contain an r binding site in a modified CH3 domain comprising: three, four, five, six, seven, or eight amino acid substitutions in a set of amino acid positions comprising 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 five, six, or seven amino acid substitutions in a set of amino acid positions comprising 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 positions are determined according to EU numbering.

Dimers of monovalent-binding 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 polypeptide may contain modifications for promoting heterodimerization of the Fc dimer (e.g., T366W; and T366S, L368A and Y407V). In some embodiments, the monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second pair of Fc polypeptides from table 2D, and the first Fc polypeptide is further modified to contain a TfR binding site in a modified CH3 domain as described herein. In some embodiments, the monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second pair of Fc polypeptides from table 2D, and the second Fc polypeptide is further modified to contain a TfR binding site in a modified CH3 domain as described herein.

In other embodiments, the monovalent Fc dimers that specifically bind to TfR described herein comprise a first and second pair of Fc polypeptides 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 sequence of the first Fc polypeptide sequence from table 2D, wherein 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 second Fc polypeptide sequence from table 2D, and wherein the first Fc polypeptide is further modified to contain a TfR binding site in the modified CH3 domain as described herein.

In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second pair of Fc polypeptides 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, wherein 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 wherein the first Fc polypeptide is further modified to contain a TfR binding site in a modified CH3 domain comprising: three, four, five, six, seven or eight amino acid substitutions and/or one or two amino acid deletions in a set of amino acid positions comprising 380 and 382-389; and five, six or seven amino acid substitutions in a set of amino acid positions comprising 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 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.

In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second pair of Fc polypeptides 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, wherein 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 wherein the first Fc polypeptide is further modified to contain a TfR binding site in a modified CH3 domain comprising: three, four, five, six, seven, or eight amino acid substitutions in a set of amino acid positions comprising 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 five, six, or seven amino acid substitutions in a set of amino acid positions comprising 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 positions are determined according to EU numbering.

In other embodiments, the monovalent Fc dimers that specifically bind to TfR described herein comprise a first and second pair of Fc polypeptides 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 sequence of the first Fc polypeptide sequence from table 2D, wherein 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 second Fc polypeptide sequence from table 2D, and wherein the second Fc polypeptide is further modified to contain a TfR binding site in the modified CH3 domain as described herein.

In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second pair of Fc polypeptides 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, wherein 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 wherein the second Fc polypeptide is further modified to contain a TfR binding site in a modified CH3 domain comprising: three, four, five, six, seven or eight amino acid substitutions and/or one or two amino acid deletions in a set of amino acid positions comprising 380 and 382-389; and five, six or seven amino acid substitutions in a set of amino acid positions comprising 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 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.

In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second pair of Fc polypeptides 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, wherein 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 wherein the second Fc polypeptide is further modified to contain a TfR binding site in a modified CH3 domain comprising: three, four, five, six, seven, or eight amino acid substitutions in a set of amino acid positions comprising 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 five, six, or seven amino acid substitutions in a set of amino acid positions comprising 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 positions are determined according to EU numbering.

For example, the first Fc polypeptide of dimer pair K from Table 2D (i.e., SEQ ID NO: 11) is further modified to include three, four, five, six, seven or eight amino acid substitutions in a set of amino acid positions containing 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 five, six, or seven amino acid substitutions in a set of amino acid positions comprising 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 positions are determined according to EU numbering.

In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second pair of Fc polypeptides 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, wherein 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 2D, and wherein the first Fc polypeptide is further modified to contain an r binding site in a modified CH3 domain comprising a modified group selected from the columns of table 29 (e.g., modified TfR binding site from clone 42.2.19, 42.2.3-1H, 42.8.196, 42.8.80, 42.8.15, or 42.8.17 in table 29). In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second pair of Fc polypeptides 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, wherein 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 2D, and wherein the second Fc polypeptide is further modified to contain an r binding site in a modified CH3 domain comprising a modified group selected from the columns of table 29 (e.g., modified TfR binding site from clone 42.2.19, 42.2.3-1H, 42.8.196, 42.8.80, 42.8.15, or 42.8.17 in table 29).

In another aspect, the present disclosure provides an Fc polypeptide dimer having the sequences of the first and second Fc polypeptides listed in tables 2C and 2D below:

table 2C dimer combinations for bivalent CD98hc binding site modification or TfR binding site modification

TABLE 2 pestle-mortar dimer combinations for monovalent CD98hc binding site modification or TfR binding site modification

XI conjugates

In some embodiments, a polypeptide described herein (e.g., an Fc polypeptide) is linked to an agent via a linker, e.g., an agent that internalizes into a cell and/or is for endocytic transport across an endothelial cell (such as the BBB). The linker may be any linker suitable for binding an agent to a polypeptide. In some embodiments, the linkage is enzymatically cleavable. In certain embodiments, the linkage may be cleaved by enzymes present in the central nervous system.

In some embodiments, the linker is a peptide linker. The peptide linker may allow the agent and the polypeptide to rotate relative to each other; and/or resistant to digestion by proteases. In some embodiments, the linker may be a flexible linker, e.g., containing amino acids such as Gly, asn, ser, thr, ala and the like. Such joints are designed using known parameters. For example, the linker may have a repeat sequence, such as a Gly-Ser repeat sequence.

In various embodiments, the conjugates can be produced using well-known chemical crosslinking reagents and protocols. For example, the cross-linking agent is a heterobifunctional cross-linking agent that can be used to link molecules in a stepwise manner. Heterobifunctional cross-linking agents provide the ability to design a more specific coupling method for conjugating proteins, thereby reducing the occurrence of undesired side reactions such as homologous protein polymers. A wide range of heterobifunctional cross-linking agents are known in the art, including N-hydroxysuccinimide (NHS) or its water soluble analogues N-hydroxysulfosuccinimide (sulfo-NHS), 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); n-succinimidyl aminobenzoate (4-iodoacetyl) ester (SIAB), succinimidyl 4- (p-maleimidophenyl) butyrate (SMPB), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-a-methyl-a- (2-pyridyldithio) -toluene (SMPT), N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), and succinimidyl 6- [3- (2-pyridyldithio) propionate ] hexanoate (LC-SPDP). Those crosslinkers having an N-hydroxysuccinimide moiety are available as N-hydroxysuccinimide analogs, which generally have greater water solubility. In addition, those cross-linking agents having disulfide bridges within the linking chain may be synthesized as alkyl derivatives instead to reduce the amount of in vivo linker cleavage. In addition to heterobifunctional crosslinkers, many other crosslinkers exist, including homobifunctional crosslinkers and photoreactive crosslinkers. Bis-succinimidyl suberate (DSS), bis-maleimidohexane (BMH) and dimethyl pimidate.2HCl (DMP) are examples of suitable homologous bifunctional cross-linkers, and bis- [ B- (4-azidosalicylamido) ethyl ] disulfide (BASED) and N-succinimidyl-6 (4 '-azido-2' -nitrophenylamino) hexanoate (SANPAH) are examples of suitable photoreactive cross-linkers.

The agent of interest may be a therapeutic agent, including cytotoxic agents, DNA or RNA molecules, antisense oligonucleotides, chemical moieties, and the like. In some embodiments, the agent may be a peptide or small molecule therapeutic or imaging agent. In some embodiments, the small molecule is less than 1000Da, less than 750Da, or less than 500Da.

The agent of interest may be linked to the N-or C-terminal region of the CD98hc binding polypeptide, or attached to any region of the polypeptide, provided that the agent does not interfere with the binding of the CD98hc binding polypeptide to CD98hc or CD98 heterodimer, i.e., does not interfere with the complexing of CD98hc to the CD98 light chain (LAT 1 (SLC 7 A5), LAT2 (SLC 7 A8), y ⁺LAT1(SLC7A7)、y⁺ LAT2 (SLC 7 A6), asc-1 (SLC 7a 10), or xCT (SLC 7a 11).

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

XI I methods for engineering polypeptides to bind CD98HC or TFR

In another aspect, methods of engineering a modified CH3 domain to bind CD98hc are provided. In some embodiments, the modification of the CH3 domain comprises substitution of various amino acids relative to amino acids 111-217 of SEQ ID NO.3 or relative to SEQ ID NO. 1. In some embodiments, the method comprises modifying a polynucleotide encoding a modified CH3 domain polypeptide to incorporate amino acid changes relative to sequence SEQ ID NO.3 or amino acids 111-217 relative to sequence SEQ ID NO. 1.

In some embodiments in which the polypeptide is engineered to bind CD98hc, the method comprises modifying a polynucleotide encoding a modified CH3 domain, the modified CH3 domain comprising a sequence having: a first sequence comprising at least one substitution or deletion relative to sequence EWESNGQP (SEQ ID NO:52; 380 to position 387, EU numbering of an Fc polypeptide (e.g., SEQ ID NO: 1)); (ii) A second sequence comprising at least one substitution relative to sequence NVFSCSVM (SEQ ID NO:53; fc polypeptide (e.g., SEQ ID NO: 1) 421 to position 428, EU numbering); and (iii) a third sequence comprising at least one substitution relative to sequence YTQKSLS (SEQ ID NO:53; fc polypeptide (e.g., SEQ ID NO: 1) 436 to position 442, EU numbering). In some embodiments, the method further comprises expressing and recovering a polypeptide comprising a modified CH3 domain; and determining whether the polypeptide binds to CD98 hc.

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

Amino acids introduced into desired positions can be generated by randomization or partial randomization to produce a library of CH3 domain polypeptides having amino acid substitutions at various positions described herein. In some embodiments, the modified CH3 domain polypeptide is mutated in the context of an Fc region, which may or may not contain a portion or all of the complete hinge region.

Polypeptides comprising modified CH3 domains may be expressed using any number of systems. For example, in some embodiments, the polypeptide is expressed in a display system. In other illustrative embodiments, the mutant polypeptide is expressed as a soluble polypeptide secreted from the host cell. In some embodiments, the expression system is a display system, e.g., a viral display system, a cell surface display system (such as a yeast display system), an mRNA display system, or a polysome display system. Libraries were screened using known methods to identify CD98hc binders, which may be further characterized to determine binding kinetics. Additional mutations can then be introduced into selected clones.

The CD98hc binding polypeptides of the disclosure can have a wide range of binding affinities, e.g., based on the form of the polypeptide. For example, in some embodiments, the affinity of the polypeptide comprising the modified CH3 domain for CD98hc binding is in the range of 1pM to 10 μm. In some embodiments, the polypeptide binds human CD98hc with an affinity of 15nM to 5 μm (e.g., 15nM、50nM、100nM、200nM、300nM、400nM、500nM、600nM、700nM、800nM、900nM、1μM、1.5μM、2μM、2.5μM、3μM、3.5μM、4μM、4.5μM or 5 μm). In another embodiment, the polypeptide binds to cynomolgus monkey CD98hc with an affinity of 80nM to 5 μm (e.g., 80nM, 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, 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 can be measured in a monovalent format. In other embodiments, affinity can be measured in bivalent form.

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

Methods for assaying 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 ^TM (GE HEAL THCARE, piscataway, NJ)), kinetic exclusion assays (e.g.) Flow cytometry, fluorescence Activated Cell Sorting (FACS), biolayer interferometry (e.g(Forte Bio, 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 example section below. In some embodiments, surface Plasmon Resonance (SPR) is used to determine binding affinity, binding kinetics, and/or cross-reactivity. In some embodiments, kinetic exclusion assays are used to determine binding affinity, binding kinetics, and/or cross-reactivity. In some embodiments, biological layer interferometry is used to determine binding affinity, binding kinetics, and/or cross-reactivity.

XIII nucleic acid, vector and host cell

CD98hc binding and TfR binding polypeptides as 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 one of the polypeptides as described herein; and a host cell into which the nucleic acid is introduced, the host cell being for replicating the nucleic acid encoding the polypeptide and/or expressing the polypeptide. In some embodiments, the host cell is a eukaryotic cell, such as a human cell.

In another aspect, polynucleotides comprising a nucleotide sequence encoding a polypeptide described herein are provided. The polynucleotide may be single-stranded or double-stranded. In some embodiments, the polynucleotide is DNA (e.g., cDNA). In some embodiments, the polynucleotide is RNA.

In some embodiments, the polynucleotide is included in a nucleic acid construct. In some embodiments, the construct is a replicable vector. In some embodiments, the vector is selected from the group consisting of a plasmid, a viral vector, a phagemid, a yeast chromosomal vector, and a non-episomal mammalian vector.

In some embodiments, the polynucleotide is operably linked to one or more regulatory nucleotide sequences in the expression construct. In a series of embodiments, the nucleic acid expression constructs are suitable for use as a surface expression library (e.g., yeast or phage). In another series of embodiments, the nucleic acid expression construct is suitable for expressing the polypeptide in a system that allows for isolation of the polypeptide in milligrams or picograms. In some embodiments, the system is a mammalian cell or yeast cell expression system.

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

The vector may be transformed into any suitable host cell. In some embodiments, host cells (e.g., bacterial or yeast cells) may be suitable for use as a surface expression library. In some cells, the vector is expressed in a host cell to express a relatively large amount 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, baby Hamster Kidney (BHK) cell, NS0 cell, Y0 cell, HEK293 cell, COS cell, vero cell, or HeLa cell.

The host cells transfected with an expression vector encoding a CD98 hc-binding or TfR-binding polypeptide can be cultured under appropriate conditions to allow expression of the polypeptide. The polypeptide may be secreted and isolated from a mixture of cells containing the polypeptide and the culture medium. Alternatively, the polypeptide may be retained in the cytoplasmic or membrane fraction, and the cells harvested, lysed and the polypeptide isolated using the desired method.

Xiv. delivery methods, targeting and therapeutic methods

The polypeptides described herein according to the present disclosure may be used to treat a variety of indications. In some embodiments, the polypeptide is used to deliver a therapeutic agent to a target cell type expressing CD98hc or TfR. In some embodiments, the polypeptides may be used to transport a therapeutic moiety across endothelial cells, such as the BBB, for uptake by the brain. Thus, the polypeptides of the present disclosure may be used, for example conjugated to a therapeutic agent, to deliver the therapeutic agent to treat a neurological disorder, such as a disease of the brain or Central Nervous System (CNS), to treat cancer, to treat an autoimmune or inflammatory disease, or to treat a cardiovascular disease.

In some embodiments, provided herein are methods of targeting an extracellular target in the brain using the polypeptides of the disclosure. In some embodiments, the polypeptide of the present disclosure is transported across the BBB and into the parenchyma, without being endocytosed into cells within the brain. In some embodiments, the method comprises delivering the therapeutic agent across the BBB to an extracellular target on or near an astrocyte, microglial cell, oligodendrocyte, or cancer cell. 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, targeted delivery is delivery to extracellular targets on microglial cells. In some embodiments, targeted delivery is delivery to an extracellular target on a cancer cell.

In some embodiments, provided herein are methods of treating a disease in a brain of a patient, the methods comprising using a polypeptide of the present disclosure to deliver a therapeutic agent to an extracellular target in the brain. In some embodiments, the method comprises delivering the therapeutic agent across the BBB and into the parenchyma without endocytosis into cells within the brain. In some embodiments, the method comprises delivering the therapeutic agent across the BBB to an extracellular target on or near an astrocyte, microglial cell, oligodendrocyte, or cancer cell. In other embodiments, the extracellular target is an antigen in the brain, such as a plaque, tangle, or other non-cellular target (e.g., abeta, tau, or alpha-synuclein). In some embodiments, the brain disease to be treated is selected from the group consisting of: frontotemporal dementia, lateral sclerosis, alzheimer's disease and Parkinson's disease. In some embodiments, the cancer is glioblastoma or metastatic cancer in the brain.

The polypeptides of the disclosure are administered to a subject in a therapeutically effective amount or dose. The dosage may vary depending on a variety of factors including the route of administration selected, the formulation of the composition, the patient's response, the severity of the condition, the weight of the subject, and the diagnosis of the prescribing physician. The dosage may be increased or decreased over time, depending on the needs of the individual patient. In some embodiments, the patient is initially given a low dose, which is then increased to an effective dose that the patient can tolerate. Determination of an effective amount is well within the ability of those skilled in the art.

In various embodiments, the polypeptides of the present disclosure are administered parenterally (e.g., intraperiostally, subcutaneously, intradermally, or intramuscularly). In some embodiments, the polypeptide is administered intravenously. Intravenous administration may be by infusion or by intravenous bolus injection. A combination of infusion and bolus administration may also be used. In other embodiments, the polypeptide may be administered orally, by pulmonary administration, intranasal administration, intraocular administration, or by topical administration. Pulmonary administration may also be employed, for example, by use of an inhaler or nebulizer, and formulation with a nebulizer.

XV. pharmaceutical compositions and kits

In another aspect, pharmaceutical compositions and kits comprising polypeptides according to the present disclosure are provided.

Pharmaceutical composition

Guidelines for preparing formulations for use in the present disclosure can be found in many manuals known to those of skill in the art regarding the preparation and formulation of drugs.

In some embodiments, the pharmaceutical composition comprises a polypeptide as described herein, and further comprises 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. Various pharmaceutically acceptable excipients are well known.

In some embodiments, the carrier is suitable for intravenous, intrathecal, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. The pharmaceutically acceptable carrier may contain one or more physiologically acceptable compounds that function, for example, to stabilize the composition or increase or decrease absorption of the polypeptide. Physiologically acceptable compounds may 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 clearance or hydrolysis of the active agent, or excipients or other stabilizers and/or buffers. Other pharmaceutically acceptable carriers and formulations thereof are also available in the art.

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

Generally, pharmaceutical compositions for in vivo administration are sterile. Sterilization may be accomplished according to methods known in the art, such as heat sterilization, steam sterilization, sterile filtration, or radiation.

The dosage and desired drug concentration of the pharmaceutical compositions of the present disclosure may vary depending upon the particular use envisaged. Determination of the appropriate dosage or route of administration can be determined by one skilled in the art.

Medicine box

In some embodiments, kits comprising the polypeptides described herein are provided. In some embodiments, the kit is for preventing or treating a neurological disorder, such as a brain or Central Nervous System (CNS) disease.

In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit comprises a polypeptide as described herein, and further comprises one or more additional therapeutic agents for 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 the composition across the BBB). Although instructional materials generally comprise written or printed materials, they are not limited thereto. The present disclosure contemplates any medium capable of storing and delivering such instructions to an end user. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, magnetic cassettes, chips), optical media (e.g., CD-ROMs), and the like. Such media may include addresses of internet sites that provide these instructional materials.

XVI. Examples

The present disclosure will be described in more detail by means of specific embodiments. 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 various non-critical parameters that may be changed or modified to produce substantially the same results. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the skill of the art. Such techniques are well explained in the literature. In addition, it will be apparent to those skilled in the art that engineering methods applied to certain libraries may also be applied to other libraries described herein.

Example 1 Generation of Limited reliability library and selection of CD98HC binding clones for engineering

We have developed a library of beta-sheets for use in the discovery of polypeptides capable of binding CD98 hc. The library includes randomization at human Fc residues 380, 382, 384-387, 422, 424, 426, 438, and 440 (EU numbering). We have observed that larger libraries (e.g., greater than 9 residue positions) can produce non-specific and/or poorly performing clones, as the libraries typically have a large number of unfavorable residues in adjacent positions. To reduce the frequency of residues that may produce such defects (particularly cysteines, arginines, tryptophan, and glycine as described below), libraries are engineered using codon bias to limit the frequency and position of these residues. Libraries embodying the art are referred to as "limited reliability" libraries. Such undesirable residues include arginine and tryptophan, which may enhance the affinity of interactions, but may also enhance non-specificity (entropy interactions); cysteine, which is one type of oxidative reliability; and glycine, which increases the flexibility of the protein structure and may disrupt the stability of the secondary structure. To avoid the occurrence of these four residues at defined positions, we introduced the NHK codon into our library. By alternating NNK (allowing all 20 amino acids) and NHK codons, this limits the placement of these four amino acids in adjacent positions without unduly limiting the diversity of the library. This method improves the quality of the polypeptides identified in the library.

Using the above method, libraries were designed to be limited to adjacent sites or to include reliability residues in general as shown in Table 2E below. Library was generated using the WT Fc gene in phage display vector using Kunkel mutation-inducing and mixture 6 oligonucleotides. Phage libraries called "LLB" for limited reliability B were screened for binding to human CD98 hc. Screening of these libraries resulted in five clones (shown in table A1) that weakly bound to CD98hc, designated LLB1, LLB2 and LLB3, LLB6, LLB7, which were selected for further engineering. LLB2 and LLB3 were found to be part of the same family (called LLB 2). Clones LLB1, LLB6 and LLB7 were found to be part of the same family (termed LLB 1).

Table 2E: mixing the oligonucleotides together to produce 9 different library variants which are pooled together

Example 2 production of LLB2 family CD98HC conjugates

LLB2 family affinity maturation (AM 1-AM 4)

Two affinity maturation libraries (LLB 2-AM1 and LLB2-AM2, table 3) were designed as LLB2 and LLB3 clones using Kunkel mutagenesis, as shown below. Codons were selected to include residues from LLB2 and LLB3 hits to increase library positions screened based on residues that contribute to affinity maturation of these libraries. Libraries were screened against human CD98hc using phage display, from which 13 unique clones were generated that had affinity for human and cyno CD98hc (measured by Surface Plasmon Resonance (SPR) using a Biacore ^TM 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 with anti-BACE 1 Fab with intact effector function (effector+) (i.e., no modification to modulate effector function, such as LALA or PG/PS) and in bivalent form (i.e., no pestle or mortar mutation). Cloned cell binding was also tested for huCD98hc binding in HeLa cells, for cyno binding in CHO cyCD98hc cells, and CHO cells served as negative controls.

TABLE 3 libraries LLB2-AM1 and LLB2-AM2

X = any amino acid

The affinity of human and cyno CD98hc was further engineered and improved against the LLB2 family using two additional libraries generated using LLB2-10 as background for additional mutations and positions to further explore the sequence space (table 4). 21 clones were selected from these libraries against human CD98hc using phage display. The sequences of selected clones are shown in table A3 and their affinities for human and cyno CD98hc (measured by Surface Plasmon Resonance (SPR) using a Biacore ^TM machine) are shown in table 9A. The affinity of the clones was measured by anti-BACE 1 Fab with complete effector function and in bivalent form (i.e. without pestle and socket mutation). Cloned cell binding was also tested for huCD98hc binding in HeLa cells, for cyno binding in CHO cyCD98hc cells, and CHO cells served as negative controls.

TABLE 4 libraries LLB2-AM3 and LLB2-AM4

Name of the name

Type(s)

378

380

382

383

384

385

386

387

389

391

LLB2-AM3

AA

X

L

N

X

R

F

VASL

L

X

Codons

NHK

CTG

AAT

NHK

CGC

TTT

KYR

CTG

NHK

LLB2-AM4

AA

·

L

X

F

X

L

·

Codons

·

CTG

NHK

TTT

NHK

CTG

·

Name of the name

Type(s)

421

422

424

426

428

436

438

440

441

442

LLB2-AM3

AA

X

I

A

N

L

X

F

N

·

X

Codons

NHK

ATC

GCC

AAT

CTG

NHK

TTT

AAT

·

NHK

LLB2-AM4

AA

·

I

A

N

L

·

F

N

LP

X

Codons

·

ATC

GCC

AAT

NHK

·

TTT

NHK

CYN

NHK

· = WT residue and codon

X = all amino acids except Arg, trp, gly, cys

While LLB2-AM3 and LLB2-AM4 were used for selection, clone LLB2-10 background was used to make rational design changes, including predicted residues, to further increase affinity for human and cyno CD98 hc. The cloning sequence is shown in table A4 and the affinity of the clones (measured by Surface Plasmon Resonance (SPR) using a Biacore ^TM machine) is shown in table 9A. The affinity of the clones was measured by anti-BACE 1 Fab with complete effector function and in bivalent form (i.e. without pestle or socket mutation). Clones LLB2-10-5, 2-10-6 and 2-10-8 were converted to monovalent forms and tested for affinity to CD98hc by SPR using the Biacore ^TM machine, with anti-BACE 1 Fab with intact effector function on both sides and with a CD98hc binding site on the pestle side (and no CD98hc binding site on the mortar side). The valency of the CD98hc binding molecule has no significant effect on the affinity of CD98hc, i.e., less than a 2-fold difference. Cloned cell binding was also tested for huCD98hc binding in HeLa cells, for cyno binding in CHO cyCD98hc cells, and CHO cells served as negative controls.

Yeast display LLB2 soft library

The ability to select good clones with higher surface expression levels using yeast display was used to improve the properties of the LLB2 family. To improve the properties of this family, soft mutation-inducing libraries (70:10:10:10 oligonucleotide bias) were made (Table 5). For this purpose, the desired codons were used, mixed with 70% of the original bases and 10% of each of the other three bases. This resulted in about 50% of the original amino acids, the remainder being a mixture of other amino acids. The original LLB2 clone backbone was used for soft mutation induction 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 first 20% of the expression clones that also tightly bound to human CD98hc were selected. The screen produced 9 clones, which were tested for binding to CD98hc by Surface Plasmon Resonance (SPR) using a Biacore ^TM machine, see table A5. The affinity of the clones was measured by Surface Plasmon Resonance (SPR) through a non-binding Fab with intact effector function using a Biacore ^TM machine and in bivalent form (i.e. without pestle and mortar mutations). Cloned cell binding was also tested for huCD98hc binding in HeLa cells, for cyno binding in CHO cyCD98hc cells, and CHO cells served as negative controls.

TABLE 5 LLB2 Soft mutation-inducing library

380

382

384

385

386

387

422

424

426

428

438

440

WT Fc residue

E

N

G

Q

P

V

S

M

Q

S

LLB2 Soft library

E

N

K

F

E

L

A

N

L

F

N

* Underlined residues were prepared with a 70:10:10:10 nucleotide mixture, 70% of which were the original bases and 10% were the other three bases.

Rational design to improve PK of LLB2 family

The M428L mutation in the various LLB2 backbones appears to contribute to poor HIC distribution and faster clearance in wild-type mice. This may be due to instability, poor specificity and/or differences in interactions with mouse FcRn compared to wild type IgG. Thus, the clones in table A6 were designed using previous clones further engineered with the M428Y mutation in the LLB2 background mutation. In addition, these clones also had the E380L mutation. These clones were made with non-binding Fab with intact effector function and in monovalent form (i.e. with CD98hc binding site on the knob side and no CD98hc binding site on the socket side).

Affinity matured LLB2-10-8 patch library for binding to CD98hc

The LLB2-10-8 pilot clone was affinity matured by 4-5 position mutation induction for each amino acid (NNK) of the surrounding patches. This is done to optimize each region separately and establish identity for the best sequence. 10 libraries are shown in Table 6 below, where NNK indicates that residues were mutated in the context of the LL2-10-8 clone. The library was assembled by two-step PCR and yeast homologous recombination. The first 24 clones were selected for affinity measurement by Surface Plasmon Resonance (SPR) using a Biacore ^TM machine. The affinities of the clones are shown in table 9A and the sequences of the clones are shown in table A7. Clones were made with non-binding Fab with intact effector function and in monovalent form (i.e., CD98hc binding site on the pestle side and no CD98hc binding site on the mortar side). Cloned cell binding was also tested for huCD98hc binding in HeLa cells, for cyno binding in CHO cyCD98hc cells, and CHO cells served as negative controls.

TABLE 6 LLB2 affinity de-maturation/rational design library

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 to residues previously observed to bind CD98hc with weaker affinity (Table 7; table A8). Variants were cloned, expressed, purified and tested for affinity to human and cyno CD98hc by Surface Plasmon Resonance (SPR) using a Biacore ^TM machine. The affinities of the clones are shown in table 9A. Combining these mutations allows the development of variants with a greater range of affinities. In summary, clones of LLB2 family had K _d values of 15nM to 5. Mu.M for human CD98hc and 80nM to 5. Mu.M for cyno CD98 hc. Cloned cell binding was also tested for huCD98hc binding in HeLa cells, for cyno binding in CHO cyCD98hc cells, and CHO cells served as negative controls.

TABLE 7 diversity for rational design of LLB2

To obtain other variants in the 600-5000nM affinity range, a second de-maturation was performed using LLB2-10-8-d6, LLB2-10-8-d12 and LLB2-10-8-d18 clones as templates, except that the following changes to human CD98hc affinity were predicted to be reduced based on previous data. Position 382 remains N or becomes S, position 385 alternates between Y or F, position 386 alternates between V, E, Q, and position 387 remains L or becomes P. Variants were made in previously untested combinations. Variants that bind with measurable affinities in this round of engineering are shown in table a12, and the corresponding human binding affinities obtained by Surface Plasmon Resonance (SPR) using the Biacore ^TM machine are shown in table 9B. Cloned cell binding was also tested for huCD98hc binding in HeLa cells, and CHO cells served as a negative control.

LLB2 family amino acid identity

Table 8 below is the residue that is allowed to bind CD98hc in the LLB2 family.

TABLE 8 acceptable amino acids in LLB2 family

To understand the nature of the interactions between the CD98hc conjugates described herein, the bivalent LLB2-10-6CD98hc conjugates and the bivalent LLB1-3-16CD98hc conjugates (neither of which is attached with any Fab) were each used toIs co-crystallized with the extracellular domain (ECD) of human CD98 hc. The structure indicated that the CD98hc conjugate bound to an epitope on CD98hc on the engineered surface that bound to a structured loop region located beside the alpha/beta barrel structure (fig. 28A and 28B). The CD98hc epitope of LLB2 is shown in fig. 42A and the CD98hc epitope of LLB1 is shown in fig. 42B. In addition, the use of the crystal structure generated a model of how FcRn bound to CD98hc in the presence and absence of CD98hc, and indicated that FcRn could bind in the absence of CD98hc but not in the presence of CD98hc (fig. 29A and 29B). A model of the interaction of the CD98hc conjugate with the complex of CD98hc with LAT1 on the membrane was also generated. Models of monovalent CD98hc conjugates that bind to the CD98hc/LAT1 complex indicate that CD98hc conjugates can readily bind to CD98hc on the surface (fig. 30C). Furthermore, the model shows that one bivalent TV can bind to both CD98hc/LAT1 complexes, albeit at extreme angles to the membrane (fig. 30A and 30B).

Table 9A: affinity measurement of LLB2 variants

Table 9B: affinity measurement of additional LLB2 variants

	Human CD98hc (uM)
		LLB2-10-8.d19	Not yet tested
LLB2-10-8.d20	2.3μM
		LLB2-10-8.d21	2.0μM
LLB2-10-8.d22	5.2μM
		LLB2-10-8.d23	1.8μM
LLB2-10-8.d24	2.6μM
		LLB2-10-8.d25	670nM
LLB2-10-8.d26	No combination
		LLB2-10-8.d27	No combination
LLB2-10-8.d28	4.2μM
		LLB2-10-8.d29	1.3μM
LLB2-10-8.d30	1.0μM
		LLB2-10-8.d31	460nM
LLB2-10-8.d32	3.1μM

Example 3 production of LLB1 family CD98HC conjugates

LLB1 family affinity maturation (LLB 1-AM1 and LLB1-AM 2)

Two phage display libraries (LLB 1-AM1 and LLB1-AM 2) were generated using residues found in clones of LLB1 family (i.e., LLB1, LLB6 and LLB 7) to increase affinity for CD98hc (Table 10). LLB1-AM1 did not amplify the number of positions in the library. LLB1-AM2 amplified to residues 428 and 434. These libraries were generated using Kunkel mutation induction and screened against human CD98hc using phage display. There are 16 clones selected for recombinant expression in bivalent form (i.e., without pestle and socket mutations) with anti-BACE 1 Fab with intact effector function. The cloned sequences are shown in Table A9. These clones showed binding to human CD98hc but not to cyno CD98hc. The leader clone was rearranged in a monovalent format called LLB1-3, wherein the anti-BACE 1 Fab had complete effector function, and wherein the CD98hc binding site was only on the pestle site, and the affinity of the leader clone to human CD98hc was measured by Surface Plasmon Resonance (SPR) using a Biacore ^TM machine. The valency of the CD98hc binding molecule has no significant effect on the affinity of CD98hc, i.e., less than a 2-fold difference. Cloned cell binding was also tested for huCD98hc binding in HeLa cells, for cyno binding in CHO cyCD98hc cells, and CHO cells served as negative controls.

TABLE 10 LLB1-AM1 and LLB2-AM2 affinity maturation libraries

X1=any amino acid

X2=any amino acid other than arginine, tryptophan, glycine and cysteine

LLB1 family affinity maturation (LLB 1-AM3 and LLB1-AM 4)

A second round of affinity maturation was performed using phage display to increase the affinity of LLB1 family for CD98hc (table 11). The backbone of the library was clone LLB1-3. Library LLB1-AM3 amplified library positions from original clones using NHK or ARY. Libraries LLB1-AM4 amplified the library and included a mixture of residues previously found at those positions of the LLB1 family. The Kunkel mutation-induced assembly library was used and screened against human CD98 hc. 20 clones were selected and affinity was measured by Surface Plasmon Resonance (SPR) using a Biacore ^TM machine. The sequence of the clone is shown in table a10 and the affinity of the clone is shown in table 12A. In the form of an anti-BACE 1 Fab with intact effector function and in the bivalent form (i.e. without pestle and socket mutations). Cloned cell binding was also tested for huCD98hc binding in HeLa cells, for cyno binding in CHO cyCD98hc cells, and CHO cells served as negative controls.

TABLE 11 LLB1-AM3 and LLB2-AM4 affinity maturation libraries

378

380

382

383

384

385

386

387

389

WT Fc

A

E

S

N

G

Q

P

N

LLB1-AM3

AA

X

D

R

X

Y

K

P

Y

X

Codons

NHK

GAC

CGG

NHK

TAC

AAG

CCC

TAC

NHK

LLB1-AM4

AA

ASTVILF

ED

KREG

X

YFNI

K

KQPT

YPFSLH

Codons

DYM

GAN

RRR

NHK

WWY

AAG

MMR

YHY

421

422

424

426

428

434

436

438

440

442

WT Fc

N

V

S

M

N

Y

Q

S

LLB1-AM3

AA

X

I

V

D

X

NS

X

I

K

X

Codons

NHK

ATC

GTG

GAC

NHK

ARY

NHK

ATC

AAG

NHK

LLB1-AM4

AA

IKVE

SVALF

DSAY

ML

NS

X

IVFL

KT

Codons

RWA

KYN

KMY

MTG

ARY

NHK

NTY

AMR

LLB1 rational design and PK adjustment

Clones 1-3-16 were prepared using all the beneficial binding mutations from the previous library, and E380 was reversed to wild type to improve PK. Additional clones were prepared in which E380 was reversed to wild type. The sequences and affinities of these clones are shown in table a11 and table 12A. Cloned cell binding was also tested for huCD98hc binding in HeLa cells, for cyno binding in CHO cyCD98hc cells, and CHO cells served as negative controls.

Cyno cross-reactive LLB1 engineering

To engineer the LLB1 family to have cyno cross-reactivity, a new library was designed using LLB1-3-16 clones as templates. In some libraries of the right register, position 382 remains R or NNK, position 383 remains mostly S or T and in some cases changes to NNK, position 384 mainly changes to NNK and in a few cases limits to Y, position 385 is fixed to NNK, position 386 remains at P and occasionally changes to NNK, position 387 randomizes to NNK, position 388 is unchanged and fixed to E, and position 389 switches between N and T. On the left register, position 424 is mostly fixed to V and in some libraries NNK, and so is position 440, which is fixed to K and only occasionally changes to NNK. Library size was kept at 1e6 and 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 a 13. The affinities of these clones were measured with non-binding Fab with intact effector function (effector+) (i.e., no modification to modulate effector function, such as LALA or PG/PS) and in bivalent form (i.e., no pestle or mortar mutation). Cloned cell binding was also tested for huCD98hc binding in HeLa cells, and CHO cells served as a negative control.

Table 12a. Llb1 variants affinity measurements

TABLE 12 affinity measurement of additional LLB2 variants

	Human CD98hc (M)
		LLB1-3-16.A1	1.55E-07
LLB1-3-16.A4	5.34E-07
		LLB1-3-16.B2	1.54E-07
LLB1-3-16.B8	2.07E-07
		LLB1-3-16.B10	1.84E-07
LLB1-3-16.C6	1.78E-07
		LLB1-3-16.D2	4.31E-08
LLB1-3-16.D4	1.80E-07
		LLB1-3-16.D5	4.03E-07
LLB1-3-16.E5	2.61E-07
		LLB1-3-16.E6	9.50E-07
LLB1-3-16.E7	5.33E-07
		LLB1-3-16.H2	2.91E-07
LLB1-3-16.H9	5.20E-07

EXAMPLE 4 plasma PK of LLB2 and LLB1 CD98HC conjugates

To begin characterization of CD98hc binding molecules, pharmacokinetics (PK) were evaluated in wild-type mice to demonstrate in vivo stability in models lacking CD98 hc-mediated clearance, as these CD98hc binding molecules bind only human CD98hc and do not bind murine CD98hc. The study design is shown in table 13 below. 6-8 week old C57B16 (WT) mice were dosed intravenously and lifelong (in-life) bleeding was collected via submandibular bleeding at time points as shown in Table 13 below. Blood was collected in EDTA plasma tubes, spun at 14,000rpm for 5 minutes and plasma was subsequently isolated for subsequent analysis.

TABLE 13 study design

The total huIgG concentration in plasma was quantified using a universal anti-human IgG sandwich format ELISA. Briefly, donkey anti-human IgG (JIR#709-006-098) was coated overnight at 1 μg/mL in sodium bicarbonate solution (Sigma#C3041-50 CAP) at 4deg.C. The plates were then washed 3 times with wash buffer (pbs+0.05% tween 20). Assay standards and samples were diluted in PBS+0.05% Tween 20 and 1% BSA (10 mg/mL). Standard curves were prepared in the range of 0.41 to 1,500ng/mL or 0.003 to 10nM (BLQ <0.03 nM). The standard and diluted samples were incubated with agitation for 2 hours at room temperature. After incubation, the plates were washed 3 times with wash buffer. The antibodies were detected, goat anti-human IgG (jir#109-036-098) was diluted in blocking buffer (PBS +0.05%Tween 20+5%BSA (50 mg/mL)) to a final concentration of 0.02 μg/mL, and the plates were incubated with agitation for 1 hour at room temperature. After the last 3 washes, the plates were developed by adding TMB substrate and incubating for 5-10 minutes. The reaction was quenched by addition of 4N H ₂SO₄ and read using absorbance at 450 nM.

The results are shown in fig. 1. All molecules exhibited clearance values similar to control IgG.

The Pharmacokinetics (PK) of the additional LLB1 and LLB2 clones were tested in WT mice according to the study design in table 14. Pharmacokinetic (PK) was assessed according to the sample collection and analysis protocol described above. The results are shown in fig. 2. The additional LLB1 and LLB2 clones tested exhibited clearance values similar to control IgG.

TABLE 14 study design

Pharmacokinetic (PK) was assessed according to the sample collection and analysis protocol described above. The results are shown in fig. 2. All molecules exhibited clearance values similar to control IgG.

According to the study design in table 15, the Pharmacokinetics (PK) of affinity matured LLB2 clones (i.e. strong binding to CD98 hc) were tested in WT mice. Pharmacokinetic (PK) was assessed according to the sample collection and analysis protocol described above. The results are shown in fig. 3. All affinity matured LLB2 clones tested exhibited clearance values similar to control IgG.

TABLE 15 study design

Pharmacokinetic (PK) was assessed according to the sample collection and analysis protocol described above. The results are shown in fig. 3. All molecules exhibited clearance values similar to control IgG.

The Pharmacokinetics (PK) of the affinity-depleted mature LLB2 clone (i.e. weak binding to CD98 hc) was tested in WT mice according to the study design in table 16. Pharmacokinetic (PK) was assessed according to the sample collection and analysis protocol described above. The results are shown in fig. 4. All affinity-depleted mature LLB2 clones tested exhibited clearance values similar to control IgG.

TABLE 16 study design

Pharmacokinetic (PK) was assessed according to the sample collection and analysis protocol described above. The results are shown in fig. 4. All molecules exhibited clearance values similar to control IgG.

EXAMPLE 5 brain absorption of LLB2 and LLB1 CD98HC conjugates

Next, to characterize CD98 hc-dependent brain absorption of LLB2 and LLB1 CD98hc binding molecules, homozygous CD98hc ^mu/hu KI mice at 6 months of age were intravenously dosed with 50mg/kg according to the study design in Table 17 below.

A full ECD knock-in CD98hc mouse model (i.e., CD98hc ^mu/hu KI or SLC3A2 ^huECD/huECD) was designed and generated for this study. The construct used to humanize the extracellular domain (ECD) of CD98hc contains 5 major elements. First, the 3 'and 5' arms are homologous to the endogenous mouse SLC3A2 locus to effect homologous recombination. Second, point mutations were made in murine exons 2, 3 and 4 to humanize only extracellular mouse residues that differ from the heterologous homologous human residues. This second element is capable of preserving mouse introns 1,2 and 3, which are predicted to have promoter regulatory regions, as well as endogenous splice sites at the junction of intron 1-exon 1/2, intron 2-exon 2/3, intron 3-exon 3/4 and intron 4-exon 4. The third element is the FRT-flanked Neo cassette (neomycin resistance gene) into murine intron 4, which serves to disrupt long regions of mouse homology that may lead to incomplete incorporation of the entire construct, and enables selection of partial incorporation based on neomycin antibiotic resistance. Since intron 4 also contains predicted promoter regulatory regions, we worry that Neo cassettes might disrupt Slc3a2 expression, so the FRT site provides an option to remove cassettes in ES after confirmation of incorporation. The fourth element is the cDNA for the human residues E335 to STOP codon, replacing the STOP codon in exon 10 from residue S268 in exon 5 of the murine genomic DNA, which allows for humanization of the remainder of the CD98hc ECD while providing sufficient differentiation from endogenous mouse sequences to allow for homologous recombination of the entire construct. The fifth element is the F3 'flanking hygro cassette (hygro-resistance gene) downstream of the murine 3' utr, which enables selection of the incorporation of the entire construct by addition of hygromycin and neomycin to the ES cell culture medium. Since hygromycin after the stop codon we did not expect it to disrupt the expression of Slc3a2 and the cassette would automatically excise in the male reproductive series of the resulting mice. This construct was electroporated into ES cells from C57Bl6 mice. ES cells with the appropriate homologous recombination are selected by growing the cells in the presence of neomycin and hygromycin. Incorporation was confirmed by PCR. Neo cassettes were removed in vitro by electroporation of constructs expressing Flp recombinase. This step is critical because the Neo cassette is suspected to disrupt the expression of the SLC3A2 gene and the CD98hc protein is essential for sperm function. This method resulted in surface expression of hCD98hc on ES cells. In contrast, ES cells that retain the Neo cassette do not express huCD98hc on ES cells. ES cells containing the appropriate humanized SLC3A2 gene but without Neo cassette are injected into goGermline embryonic cells (Ozgene) and the embryo is then transferred to pseudopregnant females. The originating male is selected from female offspring that receive the embryo and is mated with the wild female to produce an F1 heterozygous mouse. Homozygous mice were then generated from the breeding of F1 heterozygous mice.

TABLE 17 study design

48 Hours after dosing, blood was collected via cardiac puncture and mice were perfused with PBS. Brain tissue was homogenized using Qiagen TissueLyser in 10 times tissue weight lysis buffer containing 1% NP-40 in PBS with protease inhibitors. Blood was collected in EDTA tubes to prevent clotting and spun at 14000rpm for 7 minutes to separate plasma. Brain samples were homogenized in 1% np40 lysis buffer and lysates were diluted 1:2 and 1:20 for PK analysis. huIgG was measured using a universal anti-human IgG sandwich format ELISA, as described above. Dosing solutions were also analyzed on the same plate to confirm correct dosing.

The huIgG levels in plasma and brain 48 hours after the 50mg/kg IV dose of LLB1 and LLB2 variants in CD98hc ^mu/hu KI mice are shown in fig. 5A-5C. 48 hours after administration, the plasma levels of CD98hc binding molecules were lower than those of control 1, probably due to the clearance of the antibodies via binding to peripherally expressed huCD98 hc. The positive control anti-CD 98hc/BACE1 antibody and bivalent LLB2 and LLB1 clones were present in plasma at reduced concentrations, possibly due to target-mediated drug Treatment (TMDD) (fig. 5A). In the whole brain, an increase in the concentration of CD98hc binding molecules by a factor of 2-3 compared to control 1 was observed (FIG. 5B). The ratio of huIgG in brain to plasma is shown in fig. 5C. The brain to plasma ratio was higher for all CD98hc binding molecules compared to the control molecule, and the ratio was highest for bivalent LLB1-3 d380 e. The significant accumulation of CD98hc binding molecules in brain parenchyma is due to CD98hc mediated endocytic transport at the BBB.

After perfusion with PBS, the brain was dissected and the meninges and choroid plexus were removed. Fresh brain was homogenized in HBSS using a Dounce homogenizer. The homogenized sample was centrifuged (1,000 g for 10 min). After homogenization, an aliquot of the supernatant (non-cell associated fraction) was taken. The cell pellet was resuspended in 17% dextran. Additional aliquots of total centrifuged cells (representing all cells: cell association) were collected, washed and lysed in lysis buffer containing 1% NP-40 in PBS with protease inhibitors. The remaining resuspended cells were centrifuged at 4,122g for 15min. The resulting cell pellet contained vasculature and the supernatant contained parenchymal cells. The supernatant was added to a tube containing 10mL of HBSS and centrifuged at 4,122g for 15min. The cell pellet contains parenchymal cells. Vascular and parenchymal cell pellets were resuspended in lysis buffer containing 1% NP-40 in PBS with protease inhibitors. The BCA was used to measure the total protein concentration of the sample. huIgG concentrations were measured as described above using a human IgG assay (a universal anti-human IgG sandwich format ELISA) and then normalized to total protein concentration in the sample.

In the parenchymal fraction, a 5-8 fold increase in the concentration of all CD98hc binding molecules was observed compared to control 1 (negative control molecule with non-binding Fab (NBF), demonstrating that CD98hc binding molecules cross the BBB into the brain parenchyma. All huIgG values were normalized to the total protein concentration measured by BCA (fig. 6).

EXAMPLE 6 CNS biodistribution of LLB2 and LLB1 CD98HC conjugates

To characterize LLB2 and LLB1 CNS biodistribution, IHC against huIgG was performed to determine cell type specific localization of 3 clones selected from example 5 above: clone 4 (bivalent LLB1 clone), 9 (monovalent LLB2 clone) and 12 (monovalent LLB2 clone). After infusion with PBS, the hemispheres were instilled into 4% pfa for fixation overnight. Sagittal brain sections (40 μm) were cut using a microtome, blocked in 5%BSA+0.3%Triton X-100, followed by fluorescent staining with Alexa488 anti-huIgG (Jackson Immunoresearch-545-003, 1:500), rabbit anti-Iba 1 (Abcam AB178846, 1:500) or rabbit aquaporin 4 (Millipore AB2218, 1:500) +goat anti-rabbit 568 (Invitrogen A-11011, 1:500). Brain images were taken using a Leica SP8 lighting confocal microscope with a 40-fold objective. For CD98hc binding molecules, extensive cerebrovascular system and parenchymal staining was observed.

Immunohistochemistry for huIgG, huIgG and Iba1 (microglial markers), and huIgG and AQP4 (astrocyte process and tail markers) on brain sections from CD98hc ^mu/hu KI mice 48 hours after 50mpk dose of LLB2 and LLB1 molecules are shown in fig. 7-9. LLB2 molecules were significantly localized to microglia, while LLB1 molecules were more weakly localized to microglia and had significant diffuse punctate staining (FIG. 7). The anti-huIgG signal of 2 monovalent LLB2 molecules (clones 9 and 12) co-localized with Iba1, consistent with the monovalent LLB2 molecules localized to microglia. The anti-huIgG of the bivalent LLB1 molecule (clone 4) was also co-localized with Iba1 but weaker than LLB2 (FIG. 8). Diffuse punctate staining of bivalent LLB1 molecules strongly co-localized with AQP4, indicating that LLB1 molecules localized to astrocyte processes consistent with CD98hc expression (fig. 9). Thus, different CD98hc binding families have different biodistribution in the CNS.

EXAMPLE 7 brain absorption and peripheral tissue localization of LLB2 and LLB1 variants

Additional variants of LLB2 and valance matched LLB1 CD98hc binding molecules were tested for brain absorption in homozygous CD98hc ^mu/hu KI mice. The study design is shown in table 18 below. 50mg/kg was intravenously administered to 2-4 month old homozygous CD98hc ^mu/hu KI mice.

TABLE 18 study design

The huIgG concentrations in plasma and brain 48 hours after the 50mg/kg IV dose of the additional LLB1 and LLB2 variants in CD98hc ^mu/hu KI mice were assessed as described above and the results are shown in fig. 10A and 10B. The plasma concentration of CD98hc binding molecules was lower than control 1, while the higher affinity LLB1 clone was the lowest (fig. 10A). An increase in the concentration of all huCD98hc binding molecules was observed compared to control IgG. Higher affinity LLB1 also had the highest concentration in the brain (fig. 10B).

The concentration of huIgG in peripheral tissues (plasma, kidney, testis, bone marrow, lung, liver) was also measured (fig. 11A to 11F). Kidney and testis express high levels of CD98hc, bone marrow has moderate levels of expression, spleen has low levels of expression, and lung and liver do not express CD98hc. LLB2 and LLB1 variants were dosed at 50mpk to CD98hc ^mu/hu KI mice, and huIgG concentrations were assessed in 48 mice after dose. LLB2 and LLB1 variants were localized to peripheral tissues, consistent with CD98hc expression. The highest concentrations (relative to control IgG) of LLB2 and LLB1 molecules were observed in the kidneys and testes, and lower concentrations were measured in the bone marrow but still 2-3 times higher than the control. LLB2 and LLB1 concentrations were slightly higher than or equal to control IgG in spleen, liver and lung.

EXAMPLE 8 plasma and brain PK of monovalent and bivalent LLB2 CD98HC conjugates

To further characterize LLB2 molecules, molecular exposure in plasma and brain over time was assessed in plasma, brain, kidney, testis, pancreas, lung, liver, spleen, intestine and bone marrow. The study design is shown in table 19 below. According to the groups in Table 19, 6-8 month old homozygous CD98hc ^mu/hu KI mice were dosed intravenously at 50mg/kg and plasma, brain and peripheral tissues were collected 1,2, 4,7 and 10 days after dosing.

TABLE 19 study design

The concentration of huIgG was assessed as described above. The Pharmacokinetics (PK) of huIgG in plasma and brain after administration of monovalent and bivalent LLB2 variants at 50mg/kg IV in CD98hc ^mu/hu KI mice are shown in FIGS. 12A and 12B. CD98hc binding molecules cleared faster in plasma than control IgG (fig. 12A). The clearance value of monovalent LLB2 is 9-12mL/d/kg, while bivalent molecules clear faster: 16-21mL/d/kg. An increase in huIgG concentration in the brain was observed for all huCD98hc binding molecules compared to control IgG (fig. 12B). The brain concentrations of monovalent and divalent LLB2 generally increased up to 7 days post-dose, and the levels remained elevated 10 days post-dose. Thus, the kinetics of brain absorption of CD98hc binding molecules appears to be slower and longer than that observed in TfR binding molecules (i.e., T _max and concentration decrease in brain about 24-48 hours after dose). The concentration of huIgG in the brain was similar to that of the tested CD98hc binding molecule, except for the weaker monovalent clone (LLB 2-37), which was consistently lower.

Furthermore, capillary depletion indicated that monovalent and bivalent LLB2 variants cross the BBB into the brain parenchyma (fig. 13). The concentration of all CD98hc binding molecules in brain parenchyma was increased compared to the negative control molecule. The concentration of huIgG in the parenchyma generally also increases over time. All huIgG values were normalized to the total protein concentration measured by BCA.

Fig. 14A to 14H show huIgG Pharmacokinetics (PK) in peripheral tissues of CD98hc with different expression levels after a 50mg/kg IV dose of monovalent and bivalent LLB2 variants in CD98hc ^mu/hu K I mice. Kidney, testis and pancreas express high levels of CD98hc, bone marrow and intestine have moderate levels of expression, spleen have low levels of expression, and lung and liver do not express CD98hc. Monovalent and bivalent LLB2 variants localize to peripheral tissues, consistent with CD98hc expression. The highest concentration of LLB2 molecules (relative to control IgG) was observed in kidney, testis and pancreas, and lower concentrations were measured in bone marrow but still 2-3 fold higher than control. LLB2 concentrations in spleen, liver and lung were similar to or less than control IgG. There is a higher concentration of monovalent clones in tissues with high CD98hc expression, which may be driven by higher plasma concentrations of monovalent clones. Unlike the brain, the concentration of LLB2 clones in all peripheral tissues tested was reduced during the course of the study.

EXAMPLE 9 biodistribution time course of monovalent and divalent LLB2-10-8

To assess the biodistribution of monovalent and bivalent LLB2-10-8 molecules over time, brain sections of CD98hc ^mu/hu KI mice 1,2, 4, 7 and 10 days after the 50mpk dose of monovalent LLB2-9-10 (clone 9) and bivalent LLB2-10-8 (clone 26) were subjected to immunohistochemistry for huIgG. The fixed brains were collected and stained as described above. The results are shown in fig. 15. At early time points, there was an increase in the staining of parenchymal huIgG, consistent with huIgG ELISA quantification of whole brain lysates and the parenchymal capillary depleted fraction of all molecules. For CD98hc binding molecules, extensive cerebrovascular system and parenchymal staining was observed. Monovalent LLB2 (clone 9) was significantly localized to microglial cells, while divalent LLB2 (clone 26) had significant diffuse punctate staining, as shown in fig. 9, co-localized with astrocyte process (AQP 4). CD98hc localization to astrocytes was consistent with CD98hc expression. Thus, monovalent and bivalent LLB2 variants appear to have different CNS biodistribution.

Immunohistochemical results of huIgG and Iba1 (microglial cells) on brain sections from CD98hc ^mu/hu KI mice 1,2, 4, 7 and 10 days after the 50mpk dose of monovalent and bivalent LLB2-10-8 are shown in FIG. 16. Monovalent LLB2 (clone 9) was robustly co-localized with Iba1, while divalent LLB2 (clone 26) was minimally co-localized with Iba 1. Thus, monovalent CD98hc binding appears to be necessary for microglial localization of LLB2 variants.

Example 10 plasma and brain Exposure to repeated administration of monovalent and bivalent LLB2 variants and biodistribution

To investigate the effect of chronic dosing on safety and transport capacity over time, plasma and brain exposure was assessed after repeated dosing at 50 mg/kg. In addition, the difference between LLB2 molecules with effector function (i.e. effector positive) and without effector function (i.e. effector negative via LALAPG mutation) was analyzed. The study design is shown in tables 20A and 20B. Homozygous CD98hc ^mu/hu KI mice of 2-4 months of age were given 50mg/kg intravenously weekly for 4 weeks (5 doses). huIgG in plasma was measured 30 minutes and 6 days (C _max and C _trough) after the first 3 doses each. After dose 4, only C _max samples were collected. Animals were sacrificed 24 hours after the 5 th dose and final plasma was collected. Brain, blood and surrounding tissue were also collected 24 hours after the 5 th dose.

TABLE 20A monovalent LLB2 study design

Plasma and brain exposures after repeated dosing of monovalent LLB2 variants are shown in fig. 17A and 17B. The monovalent LLB2 variants had lower plasma concentrations than the control IgG (fig. 17A). huIgG in brain was evaluated 24 hours after the 5 th dose. In contrast to the single dose study (i.e., fig. 12A and 12B), there was accumulation of monovalent variants of LLB2 in the brain after repeated dosing (fig. 27B). In addition, no significant findings were observed in histopathology or hematology or clinical chemistry of the surrounding tissues. For all monovalent CD98hc TV, the reticulocyte, lymphocyte or monocyte numbers were unaffected, regardless of effector function.

Plasma and brain exposures after repeated dosing of bivalent LLB2 variants are shown in fig. 27A and 27B. The plasma concentration of the bivalent LLB2 variants was lower than the control IgG (fig. 27A). huIgG in the brain was assessed 24 hours after dose 5 and compared to single dose studies (i.e., fig. 12A and 12B, fig. 32A-32F, fig. 34A-34F), there was accumulation of LLB2 bivalent variants in the brain after repeated dosing. For all bivalent CD98hc TV, the reticulocyte, lymphocyte or monocyte numbers were unaffected, regardless of effector function. Such safety means that CD98hc binding molecules with monovalent or divalent binding can be used as targets for therapy targeting where wild-type effector function is ideally retained.

Furthermore, the fixed brains were collected and stained as described above. Immunohistochemistry of huIgG on brain sections of CD98hc ^mu/hu KI mice administered 50mpk weekly for 4 weeks is shown in fig. 18, wherein monovalent LLB2-10-8 variants have WT Fc (i.e., effector positive) and LALAPG mutations such that Fc does not bind fcγr (i.e., effector negative). For CD98hc binding molecules, extensive cerebrovascular system and parenchymal staining was observed. LLB2-10-8 (clone 9), which binds to FcgammaR, is robustly localized to microglial cells as shown by co-localization with TMEM119 (cell surface microglial marker) and astrocyte processes as shown by co-localization with AQP 4. LLB2-10-8 (clone 27) without effector function was minimally co-localized with TMEM119, but co-localized with AQP 4. These data indicate that localization of monovalent LLB2 variants to microglial cells requires effector functions, i.e. effector functions affect the biodistribution of these molecules in the CNS. These data indicate that binding to fcγrs on microglia can at least partially cover CD98 hc-driven localization to astrocytes for monovalent LLB2CD98 hc-binding molecules.

TABLE 20B bivalent LLB2 study design

Example 11 design and characterization of engineered TFR binding polypeptides based on beta-sheet libraries

We designed a library with residue diversity predominantly on the solvent exposed side of the beta-sheet surface of the CH3 domain (designated 6.5.11). This region has a number of advantages. For example, the β -sheet structure is stable and should allow for amino acid diversity without compromising the stability of the CH3 domain folding. Furthermore, 6.5.11-register-based libraries do not involve a large randomization of the flexible loop region that may introduce unwanted conformational flexibility. The beta-sheet region also forms a concave surface, which may be the ideal choice for protein-protein interactions. The concave surface is different from the FcRn and fcγr binding surfaces. The library was mapped onto the structure of the Fc polypeptides in fig. 19A and 19B.

6.5.11 Register-based initial engineering

The 6.5.11 register positions include amino acid positions 380, 382, 387, 422, 424, 426, 438, 440, which are predominantly β -sheet, especially according to EU numbering. A "NNK walk" library was generated, which involved NNK mutating residues one by one at these positions. The library was constructed and expressed on yeast surface. The library was sorted into full-length human TfR1, circularly arranged human TfR top domains, and circularly arranged cyno TfR top domains by one round of magnetic bead sorting. The library was then three rounds of FACS sorting using human TfR ECD or human TfR top domain, and the final round of negative selection for the secondary centered avidin-650 antibody. The resulting population was tested for binding to human TfR1 in the presence or absence of excess holoTf or competing clones. The results indicated that TfR binding of the library population did not compete with holo-Tf, but with clone 35.21. The resulting populations were sequenced and displayed by yeast display that the first 6 clones bound human TfR ECD and human TfR top domain. A single clone 6.5.11.1 (see table 21 for clone sequence) was selected for further processing because it had better affinity for TfR and no potential sequence defects.

Clone 6.5.11.1 was expressed recombinantly and binding of human TfR top domain was assessed by Biacore testing with an affinity of about 20-40 μm with very weak cross-reactivity with cyno TfR top domain (see table 22). "clone 6.5.11.1 bivalent" is a bivalent Fc-Fab fusion polypeptide comprising two Fc polypeptides each comprising the sequence of clone 6.5.11.1 fused to the anti-BACE 1 Fab domain (1A 11).

Affinity maturation Using clone 6.5.11.1-based NNK patch library

Other libraries based on clone 6.5.11.1 were generated to increase binding affinity to human TfR1 and cyno TfR. Six patch libraries (6.5.11.5, 6.5.11.6, 6.5.11.7, 6.5.11.8, 6.5.11.9, and 6.5.11.10) were generated with codons NNW randomly at 4 or 5 positions in different surface patches (table 21). In some libraries, the residues at position 384 (which are not part of the original register) are also randomized.

Six patch libraries were screened by one round of MACS sorting using the human TfR top domain. This round was sorted using human TfR top domain pre-mixed with streptavidin-650, followed by three rounds of FACS sorting using human or cyno TfR ECD. Libraries 6.5.11.5 and 6.5.11.7 returned clones with improved binding affinity for human and cyno TfR ECD. The first 12 clones (6.5.11.5.23、6.5.11.5.42、6.5.11.5.50、6.5.11.5.58、6.5.11.5.59、6.5.11.5.60、6.5.11.5.64、6.5.11.5.66、6.5.11.5.67、6.5.11.5.74、6.5.11.5.75 and 6.5.11.7.122) were sequenced and tested as single clones for human TfR ECD, cyno TfR ECD, circularly arranged human TfR top domain and circularly arranged cyno TfR top domain.

Analysis of the first 12 clones showed that the six β -sheet positions at positions 382, 422, 424, 426, 438 and 440 were unchanged in the clone, which showed an increase in binding affinity to TfR. In addition, the three positions at 380, 384 and 387 resulted in increased binding affinity compared to the original parent clone 6.5.11.1. Several clones with amino acid deletions between residues at positions 383 to 387 were found to have improved binding affinity for human TfR. The binding affinities of clones 6.5.11.5.42 and 6.5.11.5.50 were measured by Biacore, with lead clone 6.5.11.5.42 having a binding affinity of 2 μm for the circularly permuted human TfR top domain (table 22). In this embodiment, the term "bivalent" refers to an Fc dimer, wherein both Fc polypeptides contain a TfR binding site. The term "monovalent" refers to an Fc dimer in which one of the two Fc polypeptides contains a TfR binding site and the other Fc polypeptide does not contain a TfR binding site. Each of the clones shown in table 22 was fused to an anti-BACE 1 Fab domain.

PK/PD evaluation of clone 6.5.11.5.42 in chimeric HuTfR ^{Top end} knock-in mice

Transgenic mice expressing the human Tfrc top domain within the murine Tfrc gene (chimeric huTfR top knock-in mice) were generated using CRISPR/Cas9 technology. The resulting chimeric TfR is expressed in vivo under the control of an endogenous promoter. Chimeric huTfR ^{Top end} knock-in mice are described in international patent publication WO 2018/152285.

The Fc fragment containing clone 6.5.11.5.42 fused to anti-BACE 1 Fab (2H 8) was intravenously administered to chimeric huTfR ^{Top end} knock-in mice to measure amyloid beta 40 (Abeta 40) reduction in the mouse brain. "clone 6.5.11.5.42 bivalent: 2H8" is a bivalent Fc-Fab fusion polypeptide comprising two Fc polypeptides each comprising the sequence of clone 6.5.11.5.42 fused to the anti-BACE 1 Fab domain (2H 8). "clone 6.5.11.5.42 monovalent: 2H8" is a monovalent Fc-Fab fusion polypeptide comprising a first Fc polypeptide comprising the sequence of clone 6.5.11.5.42 and a T366W knob mutation, and a second Fc polypeptide comprising T366S, L368A and Y407V knob mutations and free of a TfR binding site fused to an anti-BACE 1 Fab domain (2H 8). The term "bivalent" refers to an Fc dimer, wherein both Fc polypeptides contain a TfR binding site. The term "monovalent" refers to an Fc dimer in which one of the two Fc polypeptides contains a TfR binding site and the other Fc polypeptide does not contain a TfR binding site. An "anti-BACE 1 control" is a negative control without any TfR binding sites.

Clone 6.5.11.5.42 bivalent: 2H8, clone 6.5.11.5.42 monovalent: 2H8 and anti-BACE 1 control were dosed at 50mg/kg at 24 hours in chimeric huTfR ^{Top end} knock-in mice. Clone 6.5.11.5.42 bivalent: 2H8 and clone 6.5.11.5.42 monovalent: 2H8 both had a decrease in brain aβ40 and significantly decreased compared to the negative control (fig. 20A-20C). Clone 6.5.11.5.42 was bivalent and clone 6.5.11.5.42 was monovalent. The brain concentration of 2H8 was also higher than negative control.

Peripheral wander library of clone 6.5.11.5.42

Clone 6.5.11.5.42 was additionally engineered by yeast display for further affinity maturation to human and cyno TfR, as well as to improve PK in wild-type mice. Additional mutations are added to backbone (i.e., non-temporal) positions that are expected to enhance binding via direct interactions, second shell interactions, or structural stabilization. By observing the structure of wild-type human Fc (PDB No.:4W 4O), 12 peripheral residues were chosen which hypothesized to increase affinity for TfR. The mutated peripheral residues are located at positions 378, 385, 386, 389, 390, 391, 421, 436, 437, 439, 441 and 442 (table 21). These surrounding residues and the original library residues were mutated to NNK in a separate library. NNK walk involves NNK mutation one by one of the residues near the original register. As described previously, libraries were generated separately using degenerate primers and assembly of two PCR products and expressed on yeast surfaces.

Independent analysis of each library population was performed by flow cytometry to determine that clones displayed binding to 50nM human TfR top domain and 50nM cyno TfR ECD through the yeast surface. If improvement over the parent is observed, the top 5% library is sorted and sequenced. In addition, to have improved TfR binding compared to the parent, the library is pooled and sorted twice to obtain a circular arrangement of human or cyno TfR top domains. Residues that appear in the sorting showing improved binding are shown in table 21. Interestingly, only positions 380, 384 and 387 in the original register were able to accept any residue change without complete loss of target binding, while the residues at positions 382, 422, 424, 426, 438 and 440 were identical to those in the parent clone 6.5.11.5.42.

Combination of NNK wander libraries

Residues from peripheral wandering at positions 378, 380, 386, 387, 389, 390 and 391 that have the greatest increase in TfR affinity (table 21) were incorporated into both libraries and screened for increased TfR binding using yeast surface display. The first library (hotspot library 1) included E or Y at position 380, and NNKs at positions 384, 385, 386, 387 and 389 (table 21). The second library (hotspot library 2) includes NNKs at positions 378, 386, 389, 390 and 391, E or Y at position 380, and P or R at position 387. Each library was sorted once from human or cyno TfR top domains by magnetic bead sorting, followed by three times from human TfR ECD, cyno TfR ECD, or circularly permuted human TfR top domains by FACS sorting. For the empty cells in table 21, the amino acids at that position are identical to those in the wild-type Fc.

Five best clones (clones 6.5.11.5.42.1, 6.5.11.5.42.2, 6.5.11.5.42.3, 6.5.11.5.42.4 and 6.5.11.5.42.8 in table 21) were expressed recombinantly and tested by Biacore and cell binding. These five clones bound to human TfR with a binding affinity between 380nM and 960nM, and cyno TfR with a binding affinity between 4.6 μm and less than 25 μm (table 22).

Table 22

PK/PD evaluation after Hot spot affinity maturation

Clones 6.5.11.5.42.1, 6.5.11.5.42.2, 6.5.11.5.42.3, 6.5.11.5.42.4 and 6.5.11.5.42.8 containing Fc polypeptides as listed in table 23 were tested in wild-type TfR mice for PK deficiency. In FIGS. 21A and 21B, all clones used were monovalent, except for "clone 6.5.11.5.42 bivalent: ab 153". All clones tested had normal clearance relative to the non-TfR binding control with clearance value of 10 mL/day/kg (fig. 21A and table 23).

Table 23

To test brain uptake of clones with improved PK, clone 6.5.11.5.42.2 was selected based on its affinity for human TfR (K _d =650 nM). In FIGS. 21B-21G, a "clone 6.5.11.5.42.2 bivalent: ab 153" is a bivalent Fc-Fab fusion polypeptide comprising two Fc polypeptides each comprising the sequence of clone 6.5.11.5.42.2 fused to a high affinity anti-BACE 1 Fab domain (Ab 153) and a LALA mutation; and "clone 6.5.11.5.42.2 monovalent: ab 153" is a monovalent Fc-Fab fusion polypeptide comprising a first Fc polypeptide comprising the sequence of clone 6.5.11.5.42.2, a T366W knob mutation, and a LALA mutation, and a second Fc polypeptide comprising a T366S, L a and Y407V knob mutation, a LALA mutation, and not comprising a TfR binding site fused to a high affinity anti-BACE 1 Fab domain (Ab 153).

Clones and controls were intravenously dosed to huTfR ^{Top end} knock-in mice at 50 mg/kg. Brain concentrations in the mouse brain were measured at 24, 96 and 198 hours (fig. 21B). Clone 6.5.11.5.42.2 monovalent Ab153 and clone 6.5.11.5.42.2 divalent Ab153 exhibited good brain absorption at 24 hours post-dose at 19.1nM and 19.9nM, respectively, compared to the negative control exhibiting brain concentrations of 3.3 nM. These two clones also showed a decrease in aβ40 in the brain 24 hours after dosing (fig. 21C). The brain concentration and brain aβ40 concentration of these two clones had sustained brain concentration and brain PD (aβ40 reduction) for up to 96 hours. As expected, plasma PK exhibited target-mediated clearance of all TfR binding clones compared to the control (fig. 21D).

It has been previously shown that molecules that bind to two TfR molecules simultaneously can result in a decrease in circulating reticulocytes and TfR ⁺ bone marrow cells. Whereas the crystal structure indicated that only one TfR molecule could be bound at a time, the levels of blood reticulocytes, ter119 ⁺ red blood cells (fig. 21E), and CD71 ⁺ bone marrow reticulocytes (fig. 21F) were measured. No change in these values compared to the anti-BACE 1 control was observed. The effect of clones on TfR levels was also measured (fig. 21G). These results indicated no difference between TfR levels, indicating that cloning did not cause TfR degradation.

De-maturation/reversion of clone 6.5.11.5.42.2

To attenuate the affinity of clone 6.5.11.5.42.2, several residues were reversed to wild-type residues or residues that had been previously identified to affect affinity for the human TfR top domain. These are performed as single point mutations or combinations. Clones were expressed and purified from HEK293 cells as described previously, and their affinity for the human TfR top domain was measured by Biacore.

Table 24 shows a library of 6.5.11.5.42.2 mutants. Each mutant contains 6.5.11.5.42.2 amino acid substitutions of 1, 2 or 3. For example, one mutant may contain D384N, and the amino acids at the remaining positions are identical to those in 6.5.11.5.42.2. The positions shown in table 24 are numbered according to the EU numbering scheme. Amino acids different from those in 6.5.11.5.42.2 are shown in bold in each mutant.

Table 24

* Clone 6.5.11.5.42.2.d5 was not expressed.

HIC evaluation of library clones

Clones 6.5.11.5.42.1, 6.5.11.5.42.2, 6.5.11.5.42.3, 6.5.11.5.42.4 and 6.5.11.5.42.8 containing the Fc polypeptides listed in table 23 were evaluated by Hydrophobic Interactions (HIC) during the developability evaluation. Each clone is a monovalent Fc-Fab fusion polypeptide comprising (i) a first Fc polypeptide having the sequence of the identified clone, a T366W knob mutation, and a LALA mutation; (ii) A second Fc polypeptide comprising a T366S, L a and Y407V acetabulum mutation and a LALA mutation; and (iii) a high affinity anti-BACE 1 Fab domain (Ab 153) linked to an Fc polypeptide. The control was a high affinity anti-BACE 1 Fab domain (Ab 153) linked to an Fc domain with LALA mutation and no TfR binding site.

Five (5) μg of each clone was injected into two Thermo ProPac HIC-10 columns (5 μm, 4.6X100 mm, cat. No. 63655) arranged in series. The mobile phases used in the column are as follows: mobile phase a:1 XPBS, pH 7.4; mobile phase B:1 XPBS, 0.9MNA ₂SO₄; flow rate 0.75mL/min, linear gradient: starting from 80% mobile phase B, for t=0 to 3 minutes, t=3 to 19 minutes gradually decreases to 0% mobile phase B, t=19 to 26 minutes remains at 0% mobile phase B, and t=27 to 32 minutes returns to 80% mobile phase B and remains, column temperature 25 ℃. Detection was performed using fluorescence (excitation 290 nm/emission 325 nm) and internal standard was 1mg/mL of NIST monoclonal antibody. The results are shown in Table 25.

Table 25

All clones exhibited higher recombinant protein titers and greater hydrophobicity than the wild-type control. Clone 6.5.11.5.42.2 exhibited minimal change in hydrophobicity relative to wild-type controls (as shown by retention time or RT) and was selected for further evaluation.

Low pH stability of clone 6.5.11.5.42

Clones 6.5.11.5.42.2 with and without LS mutations evaluated low pH stability during the developability evaluation. The protein tested was a bivalent Fc-Fab fusion polypeptide comprising an anti-HER 2 Fab domain linked to two Fc polypeptides, each having the sequence of the identified clone and LALA mutation, with or without LS mutation. An Fc control comprising an anti-HER 2 Fab domain linked to an Fc domain with LALA mutation and without TfR binding site was included for comparison.

Aliquots (100. Mu.L) of each clone were added to wells of 96-well plates containing 6. Mu.L of 5% (v/v) acetic acid and mixed well. The plates were sealed and incubated at room temperature for about three (3) hours. Subsequently, 35. Mu.L of 1M Tris HCl (pH 7.5) was added to the sample and mixed well. The plates were resealed and incubated at room temperature for 24 hours. The control conditions were stored in 1 XPBS without any change in pH during incubation. The binding affinity of the samples was then analyzed by Biacore and by size exclusion chromatography to detect the intact fusion proteins. Fig. 22 and table 26 provide the analysis results.

Table 26

As shown in fig. 22, low pH exposure (followed by neutralization) did not affect protein stability in clone 6.5.11.5.42.2. Minimal changes in clones were observed using Size Exclusion Chromatography (SEC) analysis, regardless of the presence or absence of LS mutations in the Fc polypeptide. In addition, low pH exposure and subsequent neutralization treatments did not affect cloned TfR binding either (table 26). These results demonstrate the stability of the clones, albeit affected by low pH stress conditions.

Additional clones from structural libraries

The structure of clone 6.5.11.5.42 (fig. 23A-23C) with the top domain of the human TfR circular arrangement was used to determine the residue position in contact with or close to the TfR top domain. These positions were made into four independent libraries using the clone 6.5.11.5.42.2 sequence, with some residue positions mutated to codon NNK or deleted. Libraries were made with 1 or 2 residue deletions so that transferrin side chains have more binding space.

(1) Library 1: locations 383, 385, 388, 389 and 391.

(2) Library 2: deletions at positions 383, 384, 385, 386, 387 and 388.

(3) Library 3 is an equal mix of three libraries: library 3a: deletions at positions 383, 384, 385, deletions at 386, 387 and 388; library 3b: deletions at positions 386, 387, 388, 390 and 391; library 3c: 383. 384, 385, 386.

(4) Library 4: positions 419-421 and 442-443.

Each mutant in table 27 contains several amino acid substitutions relative to wild-type human IgG1 Fc. For empty cells in table 27, the amino acid at this position is identical to that in wild-type Fc. Recombinant expression clones and testing for human TfR top domain binding, estimated affinities of about 43-1000nM, were very weak cross-reactivity with cyno TfR top domain (table 28).

Table 27

-It indicates that the amino acid at position 383 is absent.

Table 28

PK/PD evaluation of clone 42.2.1.2

Clones 42.2.1.2 monovalent and 42.2.1.2 bivalent PK were tested in wild-type TfR mice to ensure that they were free of PK deficiency. The clones were tested along with the previously tested clone 6.5.11.5.42.2 unit price and 6.5.11.5.42.2 unit price. Clone 42.2.1.2 as used herein monovalent contains a 42.2.1.2tfr binding site with mutations T366W knob, P329G and LALA as the first Fc polypeptide and an Fc sequence with mutations T366S, L368A and Y407V knob, P329G and LALA as the second Fc polypeptide. Both Fc polypeptides in the bivalent clone 42.2.1.2 used herein contained a 42.2.1.2tfr binding site with P329G and LALA mutations. Clone 6.5.11.5.42.2 as used herein monovalent contains as a first Fc polypeptide a 6.5.11.5.42.2tfr binding site with mutations T366W knob, P329G and LALA, and as a second Fc polypeptide an Fc sequence with mutations T366S, L368A and Y407V knob, P329G and LALA. Both Fc polypeptides in the bivalent clone 6.5.11.5.42.2 used herein contained a 6.5.11.5.42.2tfr binding site with P329G and LALA mutations.

Clones and controls were intravenously dosed to huTfR ^{Top end} knock-in mice at 50 mg/kg. Brain and plasma concentrations were measured at 24 hours (fig. 24A and 24B). Clone 42.2.1.2 monovalent and clone 42.2.1.2 divalent showed good brain uptake 24 hours after dosing compared to negative controls (figure 24A). As expected, plasma PK showed clearance of all TfR binding clones compared to the control (fig. 24B).

The effect of cloning on circulating reticulocytes was also studied. Fig. 25 shows the measurement of Ter119 ⁺ erythrocytes. No change in this value compared to the control was observed.

In addition, during the time course experiments, the concentration of clones in brain and plasma was also measured (see fig. 3B to 3D). In this experiment clone 42.2.1.2 monovalent Ab153 contains as a first Fc polypeptide a 42.2.1.2TfR binding site with T366W knob and LALA mutations, as a second Fc polypeptide an Fc sequence with T366S, L368A and Y407V knob and LALA mutations, and a high affinity anti-BACE 1 Fab domain (Ab 153) linked to the Fc polypeptide. Clone 42.2.1.2 bivalent Ab153 contains two Fc polypeptides, each having a 42.2.1.2TfR binding site with a LALA mutation and Ab153 linked to the Fc polypeptide. anti-BACE 1 control Ab153 contained Ab153 linked to an Fc domain with P329G and LALA mutations and did not have a TfR binding site. Clones showed a decrease in aβ40 in the brain 24 hours after dosing (see fig. 3C). Cloned brain concentration and brain aβ40 concentration had sustained brain concentration and brain PD (aβ40 reduction) for more than 144 hours (see fig. 3B and 3C). As expected, plasma PK exhibited target-mediated clearance of all TfR binding clones compared to the control (see figure 3D).

Additional clones from rational design

Based on previous sorting results and sequences, the optimal residues in the clone at key positions conducive to high affinity TfR binding were determined. To understand the relative impact of a few positions on binding and human/cyno cross-reactivity, a set of rational mutations were made at positions 382-389. Clones contained 1,2,3,4, 5,6,7 or 8 mutations at positions 382-389. 216 clones were prepared based on the sequence of clone 6.5.11.5.42.2 (table 29), with the following substitutions in each combination: at position 383: s or Y; at position 384: G. d or E; at location 385: D. a or G; at position 386: q or S; at position 388: e or L; and at position 389: n, T or S. Some additional clones are also listed in table 29. The positions listed in table 29 are numbered according to EU numbering. For positions not listed in table 29, the amino acids at these positions are identical to the amino acids of the wild-type Fc polypeptide except for clone 42.2.19, which has P at position 419, R at position 420, G at position 421, G at position 442, and E at position 443. To explore the affinity of each sequence combination at these positions for TfR, these clones were expressed in mammalian supernatants and their affinities for human and cyno top domains were measured using Biacore ^TM.

Clones were expressed in HEK293 cells. Supernatants were captured for SPR by GE HEAL THCARE anti-human Fab capture kit and affinity was measured for human and cyno TfR top domains (table 30). The clones in table 30 were bivalent and each TfR binding Fc polypeptide also contained P329G and LALA mutations. Clones showed estimated affinities of approximately 293-10432nM with very weak cross-reactivity with the cyno TfR top domain.

Table 30

EXAMPLE 12 PK evaluation

Clones 42.8.17, 42.8.15, 42.8.80, 42.8.196, 42.2.3-1H and 42.2.19 in monovalent and 42.2.1.2 bivalent forms were PK tested in huTfR ^{Top end} knock-in mice to ensure no PK defects. The monovalent clones used herein contained TfR binding sites with T366W knob, P329G and LALA mutations as the first Fc polypeptide and Fc sequences with T366S, L a and Y407V knob, P329G and LALA mutations as the second Fc polypeptide. Both Fc polypeptides in the bivalent clones used herein contained TfR binding sites with P329G and LALA mutations.

Clones and controls were intravenously dosed to huTfR ^{Top end} knock-in mice at 50 mg/kg. Brain and plasma concentrations were measured at 24 hours (fig. 26A to 26D). As expected, plasma PK showed clearance of all TfR binding clones compared to the control (fig. 26A and 26B). The cloned brain concentrations had sustained brain concentrations over 10 days (fig. 26C and 26D).

EXAMPLE 13 monovalent CD98HC affinity variants to 21 day plasma and brain PK

To further investigate the plasma and brain PK of CD98hc binding molecules, a single 50mg/kg dose was administered via IV to CD98hc ^mu/hu KI mice, with monovalent CD98hc LLB2-10-8 affinity variants in the range of 20nM to 550nM, according to the group in Table 31A below. Mice were sacrificed on days 1,2, 7, 14 or 21 post dose and blood and brain tissue were collected.

TABLE 31A study design

HuIgG PK in plasma and whole brain lysates was assessed 21 days post dose according to the sample collection and analysis protocol described above (i.e., example 4 for plasma PK and example 5 for brain PK). The results are shown in fig. 31A and 31B. There was a correlation between stronger affinity of CD98hc binding molecules and faster plasma clearance (fig. 31A). An increase in huIgG concentration was observed for all huCD98hc binding molecules compared to control non-binding IgG (fig. 31B). Up to 7 days post dose, brain concentration of LLB2 CD98hc binding molecules increased and levels remained higher than control IgG at 21 days post dose. In these experiments, the CD98hc molecule had a delayed T _max compared to the TfR binding molecule, and the concentration in the brain remained higher for longer than the control antibody. For the strongest and lowest affinity clones, a slightly lower AUC was observed.

Furthermore, capillary depletion (via the protocol in example 5 described above) indicated that all monovalent LLB2 affinity variants cross the BBB into the brain parenchyma (fig. 31C and 31D). Although the concentration of C _max was similar, the concentration of the weaker affinity LLB2 variant in the parenchymal fraction was lower. Higher concentrations of weaker affinity variants were detected in the non-cell associated fraction, indicating that the variants have a substantial contribution to whole brain huIgG (fig. 31E). There was a correlation between higher affinity and higher huIgG concentration in the cell associated fraction (fig. 31F). All huIgG values were normalized to the total protein concentration measured by BCA. Control IgG was lower than LLOQ in all fractions.

EXAMPLE 14 bivalent CD98HC affinity variants to 28 day plasma and brain PK

To further investigate the plasma and brain PK of the CD98hc binding molecules, a single 50mg/kg dose was administered via IV to CD98hc ^mu/hu KI mice, with bivalent CD98hc LLB2-10-8 affinity variants in the range of 275nM to 2100nM, according to the group in Table 31B below. Mice were sacrificed on days 1,2, 7, 14 or 28 post dose and blood and brain tissue were collected.

Table 31B: study design

HuIgG PK in plasma and whole brain lysates was assessed 28 days post dose according to the sample collection and analysis protocol described above (i.e., example 4 for plasma PK and example 5 for brain PK). Given the trend of higher brain exposure for the bivalent CD98hc binding molecules in fig. 12B, we hypothesize that bivalent CD98hc molecules may have even longer brain exposure times than monovalent molecules, so the last time point of the study was set to 28 days (instead of 21 days). The results are shown in fig. 32A and 32B. There was a correlation between stronger affinity of CD98hc binding molecules and faster plasma clearance (fig. 32A). An increase in huIgG concentration was observed for all huCD98hc binding molecules compared to control non-binding IgG (fig. 32B). Until 7 days post dose, the brain concentration of LLB2 TV increased and at 28 days post dose, the levels remained higher than control IgG for all clones except the weakest affinity. Like monovalent CD98hc TV, divalent CD98hc TV has a delayed T _max and the concentration in the brain remains elevated longer than TfR TV.

Furthermore, capillary depletion (via the protocol described in example 5 above) indicated that all bivalent LLB2 affinity variants cross the BBB into the brain parenchyma (fig. 32C and 32D). Higher concentrations of weaker affinity variants were detected in the non-cell associated fraction (fig. 32E). All bivalent LLB2 variants were detected in the cell-associated fraction except the weakest affinity clone (fig. 32F). All huIgG values were normalized to the total protein concentration measured by BCA. Control IgG was lower than LLOQ in all fractions except the cell associated fraction.

Example 15 plasma and brain PK of affinity-matched LLB1 and LLB2 variants

To elucidate any differences between affinity-matched monovalent LLB1 and LLB2 variants in terms of plasma and brain Pharmacokinetics (PK), a 50mg/kg IV dose was administered to CD98hc ^mu/hu KI mice according to the group in table 31C below. Mice were sacrificed on days 1,2, 4, 10 or 21 post dose and blood and brain tissue were collected.

Table 31C: study design

HuIgG PK in plasma and whole brain lysates was assessed 21 days post dose according to the sample collection and analysis protocol described above (i.e., example 4 for plasma PK and example 5 for brain PK). The results are shown in fig. 33A and 33B. LLB1 and LLB2 variants have similar plasma clearance (FIG. 33A). huIgG PK in whole brain lysates. LLB1 and LLB2 had similar brain exposures at all test time points (fig. 33B). No plasma or brain PK differences were observed in affinity matched monovalent LLB1 and LLB2 variants.

EXAMPLE 16 plasma and brain PK and CNS and cell type biodistribution of CD98HC LLB2-10-8 variants (monovalent and divalent; effector positive and negative)

To further elucidate the differences in PK and brain biodistribution of monovalent and bivalent CD98hc LLB2-10-8 with and without effector function modifying mutations, a single 50mg/kg dose was administered via IV to CD98hc ^mu/hu KI mice according to the group in table 31D below. Mice were sacrificed on days 1, 4, 7, 14 or 21 post-dose and blood and brain tissue were collected.

Table 31D: study design

HuIgG PK in plasma and whole brain lysates was assessed 21 days post dose according to the sample collection and analysis protocol described above (i.e., example 4 for plasma PK and example 5 for brain PK). The results are shown in fig. 34A and 34B. The bivalent variants cleared from plasma faster than the monovalent variants, and the effector function mutations did not affect plasma clearance (fig. 34A). An increase in huIgG concentration was observed for all huCD98hc binding molecules compared to control non-binding IgG (fig. 34B). Brain concentrations were similar in all tested variants until a later time point at which the concentration of bivalent variants retrained in the brain was higher than the monovalent variants. There was a slight increase in brain exposure of bivalent LLB2 TV with wild-type effector function (i.e. effector positive), but no effect on the effector function mutant of monovalent LLB2 (i.e. effector negative).

Furthermore, capillary depletion (via the protocol in example 5 described above) indicated that all LLB2 variants cross the BBB into the brain parenchyma (fig. 34C and 34D). Higher concentrations of monovalent LLB2 variants were found in the non-cell associated fraction, while higher concentrations of divalent LLB2 variants were found in the cell associated fraction (fig. 34E and 34F). All huIgG values were normalized to the total protein concentration measured by BCA. Consistent with data indicating that higher affinity for CD98hc results in association of the molecule with more cells, these data indicate that the higher valency of CD98hc binding also results in association of the molecule with more cells.

In addition, brain sections of CD98hc ^mu/hu KI mice were subjected to huIgG immunohistochemistry at 1,7, 14 and 21 days post-dose according to the protocol described above (i.e., in example 6). The results are shown in fig. 35. There was an increase in the staining of the parenchymal huIgG at 1 to 7 days, consistent with the huIgG ELISA quantification and the parenchymal capillary depletion fraction of the whole brain lysates. Monovalent LLB2 with WT effector function (i.e., effector positive) had significant glial localization, whereas divalent LLB2 (regardless of effector functional status) and monovalent LLB2 with LALAPG (i.e., effector negative mutant) had significant diffuse punctate staining. Thus, monovalent LLB2 with WT effector function has a different CNS biodistribution compared to the other versions, indicating that the combination of monovalent CD98hc and fcγr binding drives the localization of these molecules to microglia.

Immunohistochemistry for huIgG and CNS cell type markers (i.e., iba1 for microglial cells, AQP4 for astrocyte processes, and NeuN for neurons) were performed on brain sections from CD98hc ^mu/hu KI 7 days post dose according to the protocol described above (i.e., in example 6). The results are shown in fig. 36A to 36C. Monovalent LLB2 with WT effector function had significant microglial localization, whereas divalent LLB2 (regardless of effector function status) and monovalent LLB2 with LALAPG had significant co-localization with AQP4, consistent with astrocyte localization. No localization of LLB2 variants to neurons was observed.

One explanation for this data is that the delayed peak brain concentration (Cmax) observed in examples 16 and 17 above is due to the slower rate of internalization (compared to TfR binding molecules). Slow cellular internalization may also be helpful in finding CD98hc TV in the non-cellular fraction. This interpretation is consistent with the observation that increasing the likelihood of CD98hc binding molecule detachment via weaker affinity and/or monovalent state increases the proportion of CD98hc binding molecules found in the non-cell associated brain fraction.

EXAMPLE 17 PK, PD and CNS biodistribution of CD98HC TV with BACE1 FAB

To study the effect of binding Fab on PK, PD and CNS biodistribution of CD98hc molecules, monovalent and bivalent LLB2-10-8TV was constructed with anti-BACE 1 Fab. A single 50mg/kg dose was administered via IV to CD98hc ^mu/hu KI mice according to the group in Table 31E below. Mice were sacrificed on days 1, 4, 7, 14 or 21 post-dose and blood and brain tissue were collected.

TABLE 31E study design

HuIgG PK in plasma and whole brain lysates was assessed 21 days post dose according to the sample collection and analysis protocol described above (i.e., example 4 for plasma PK and example 5 for brain PK). The results are shown in fig. 37A and 37B. PK of huIgG in plasma 21 days post dose indicated that bivalent CD98hc binding resulted in faster plasma clearance (fig. 37A). This is consistent with studies with unbound Fab. In brain, consistent with the study of non-binding Fab, we observed that ATV.CD98hc: BACE molecule T _max was between 4-7 days, but that ATV.CD98hc: BACE1 maximum concentration was lower than that of CD98hc TV with non-binding Fab. In addition, ATV.CD98hc BACE1 cleared from the brain more rapidly and at a concentration similar to the negative control antibody 14 days after dose. This is in contrast to brain exposures of over 21 days we observed using CD98hc TV with non-binding Fab. These data indicate that BACE1 binding resulted in less retention in the brain than was observed with non-binding Fab (fig. 37B). This is probably due to BACE1 binding directing CD98hc to neurons, particularly lysosomes within neurons, leading to degradation of the LLB2: BACE1 molecule. These data also indicate that brain exposure to CD98hc TV paired with different fabs should be assessed on a target-by-target basis.

BACEl inhibition of amyloid precursor protein APP cleavage was used as a pharmacodynamic readout of antibody activity in the brain. Brain tissue was homogenized in 5M guanidine-HCl at 10 times the tissue weight and subsequently diluted 1:10 in 0.25% casein buffer in PBS. Mouse aβ40 levels in brain lysates were measured using sandwich ELISA. 384-well MaxiSorp plates were coated overnight with polyclonal capture antibodies specific for the C-terminus of the aβ40 peptide (Millipore # ABN 240). Casein-diluted guanamine lysate was further diluted 1:2 on ELISA plates and added simultaneously with detection antibody, biotinylated anti-mouse/rat β -amyloid M3.2. Samples were incubated overnight at 4 ℃ followed by the addition of streptavidin-HRP followed by the addition of TMB substrate. A standard curve of 0.78-50pg/mL msA beta 40 was fitted using four-parameter logistic regression.

Aβ40 measurements indicated that BACE1 inhibition occurred 1, 4 and 7 days post-dose, consistent with when LLB2: BACE1 molecule concentration was greater than control anti-BACE 1 antibody (FIG. 37C).

In addition, immunohistochemistry for huIgG, neuN (neurons), and LAMP2 (lysosomes) was performed on brain sections from CD98hc ^mu/hu KI according to the protocol described above (i.e., in example 6) 7 days after the 50mpk dose of monovalent and bivalent LLB2: BACE1 molecules. The results are shown in fig. 38A and 38B. Monovalent LLB2 BACE1 is mainly localized to neurons. Divalent LLB2 BACE1 also has some localization to neurons, and diffuse spots might be consistent with CD98hc driving localization to astrocyte processes. The anti-BACE 1 control also showed localization to neurons (fig. 38A). All 3 molecules tested showed co-localization with Lamp2 positive lysosomes in neurons, consistent with BACE1 localization in the endolysosomal pathway (fig. 38B). This data suggests that BACE1 drives degradation of CD98hc binding molecules in lysosomes of neurons. These data indicate that monovalent CD98hc TV can be kept away from CD98hc by binding to other proteins (such as therapeutic targets). For example, fcγr binding appears to drive the localization of monovalent LLB2 molecules to microglia (see example 10), whereas, as discussed above, neuronal anti-BACE 1 Fab drives monovalent LLB2 to neurons. Divalent CD98hc TV retained some of the CD98 hc-driven biodistribution.

EXAMPLE 18.15 plasma and brain PK of monovalent and bivalent LLB2 variants of MPK

To characterize plasma and brain PK of CD98hc binding molecules at lower dose levels, we dosed monovalent and bivalent LLB2 variants with affinities in the range of 20nM to 550nM at 15mg/kg IV in CD98hc ^mu/hu KI mice according to the groups in table 31F and table 31G below. Mice were sacrificed on days 1,4, 7, 14 or 21 post-dose and blood and brain tissue were collected.

TABLE 31F study design of monovalent LLB2 variants

TABLE 31G study design of bivalent LLB2 variants

HuIgG PK in plasma and whole brain lysates were evaluated according to the sample collection and analysis protocol described above (i.e., example 4 for plasma PK and example 5 for brain PK). The results are shown in fig. 39A to 39D. The stronger affinity and higher valency of the CD98hc binding molecules results in faster clearance from plasma (fig. 39A is monovalent and fig. 39C is divalent). An increase in huIgG concentration was observed for all huCD98hc binding molecules compared to control non-binding IgG (figure 39B is monovalent and figure 39D is bivalent). Until 7 days post dose, the brain concentration of LLB2 TV increased and at 21 days post dose, levels were still higher than control IgG, except for the highest affinity monovalent clones. Consistent with the study of higher doses of CD98hc TV, at lower doses CD98hc TV also delayed T _max and huIgG concentrations remained elevated longer than TfR TV.

EXAMPLE 19 plasma and brain uptake of LLB2 and TV42 in non-human primate (NHP) and cell type biodistribution

To evaluate the translatability of the above data regarding CD98hc binding molecules in mice, plasma and brain exposure studies of LLB2 (CD 98hc binding molecule) compared to TV42 (TfR binding molecule) were performed. A single 30mg/kg dose was administered via IV to a cynomolgus group according to Table 31H below. Monkeys were sacrificed 4 days after dose and blood and brain tissue were collected.

TABLE 31H study design

The total test concentration in monkey serum and brain lysate samples was quantified using a universal anti-human IgG sandwich format electrochemiluminescence immunoassay (ECLIA) on a Meso Scale Discovery (MSD) platform. Briefly, 1% casein-based PBS blocking buffer (Thermo Scientific, wal tham, MA) was added to MSD GOLD 96-well small spot streptavidin-coated microtiter plates (Meso Scale Discovery, rockville, MD) and incubated for about 1h. After the plate blocking and washing steps, biotinylated goat anti-human IgG antibody (SouthernBiotech, birmingham, AL) was added at a working concentration of 0.5 μg/mL to coat the assay plates and allowed to incubate for 1-2h. Subsequently, the test samples (1:100 MRD in 0.5% casein-based PBS assay buffer) were diluted and added to the assay plates. After incubation for 1-2h in the capture step, pre-adsorbed secondary ruthenium-based (SULFO-TAG) goat anti-human IgG antibody (Meso Scale Discovery, rockville, MD) was added to the assay plate at 0.5 μg/mL working solution and incubated for approximately 1h. An assay read buffer (1X MSD read buffer T) was then added to generate an Electrochemiluminescent (ECL) assay signal represented by ECL units (ECLU). All assay reaction steps were performed at ambient temperature and were shaken (as appropriate) on a plate shaker. In serum, the measured MRD was 100 in a pure matrix with 8 standard spots (serial dilutions at 1:2, including blank matrix samples) and dynamic calibration standard ranges from 19.5 to 2500ng/mL. Brain lysate required 50 MRD, with a dynamic range of 4.9-2500ng/mL, with 10 standard point curves (serial dilutions at 1:2 plus blank brain lysate samples). Serum and brain lysate sample concentrations were back calculated from a determination specific calibration standard curve fitted with a weighted four parameter nonlinear logistic regression. The back-calculated concentration of the sample in ng/mL is then converted to nanomole (nM) or micromolar (μM) as the final sample result.

The results are shown in fig. 40A and 40B. Due to the expression of TfR and CD98hc on peripheral tissues, bivalent TV42 and LLB2 cleared more rapidly from serum than non-binding control antibodies. Consistent with efficient lower affinity CD98hc binding due to lower valency and lack of avidity, monovalent LLB2 molecules have moderate serum clearance compared to bivalent TV and control non-binding antibodies. The concentration of TV42 and all versions of LLB2 in the brain was increased 5-13 fold compared to control non-binding antibodies. One animal dosed with the bivalent LLB2 molecule was excluded from the analysis due to signs of BBB destruction.

Capillary depletion was performed using the method described above, and huIgG concentrations were measured in each fraction using MSD described above. The results indicate that LLB2 and TV42 are absorbed into NHP brain parenchyma (fig. 40C and 40D). TV42 is highly cell associated, divalent LLB2 is present in both the cell associated and non-cell associated fractions, whereas monovalent LLB2 is highly non-cell associated (fig. 40E and 40F). The high cellular association of TV42 is consistent with rapidly internalizing TfR. The increase in cell association of bivalent LLB2 compared to monovalent was consistent with the mouse study. For huIgG in the parenchymal, cell associated and non-cell associated fractions, 2 control treated animals were below the lower limit of quantitation determined.

Furthermore, to study the biodistribution differences of any CNS cell type in NHPs, NHP brain sections were subjected to immunohistochemistry for huIgG and CNS cell type labeling. After infusion with PBS, the right hemisphere of the brain was collected and cut into 4mm thick coronal slices. A 4mm thick sheet was placed in an individual tissue cassette and immersed in a solution of 4% Paraformaldehyde (PFA) and refrigerated (4 ℃ to 9 ℃) for 48 hours. Immediately after fixation, the tissue was transferred to pbs+0.1% sodium azide. The slabs were further coronally cut into 40 μm sections on a microtome. Brain sections were blocked in 5%BSA+0.3%Triton X-100, followed by fluorescent staining with Alexa647 anti-huIgG (Southern Biotech 2049-31, 1:500) and rabbit anti-Iba 1 (Abcam ab178846, 1:500) +goat anti-rabbit 568 (Invitrogen A-11011, 1:500). In addition, sections were stained with Alexa647 anti-huIgG (Southern Biotech 2049-31, 1:500), rabbit aquaporin 4 (Millipore AB2218, 1:500) +goat anti-rabbit 568 (Invitrogen A-11011, 1:500), and mouse anti-NeuN (Millipore MAB377, 1:500) +anti-msIgG 1-488 (Invitrogen A21121, 1:500). Brain images were taken using a Leica SP8 lighting confocal microscope with a 40-fold objective.

The results are shown in fig. 41A to 41C. TV42 was robustly co-localized with NeuN positive neurons (fig. 41C). Monovalent LLB2 with WT effector function had significant microglial (Iba 1) localization (fig. 41B), whereas divalent LLB2 (regardless of effector function status) and monovalent LLB2 with LALAPG (i.e. effector negative) were significantly co-localized with AQP4, consistent with astrocyte localization (fig. 41A). No localization of LLB2 variants to neurons was observed (fig. 41C). CNS cell type biodistribution of LLB2 and TV42 in non-human primates was similar to that in mice.

Example 20 design and characterization of engineered TFR binding polypeptides from Natural phage libraries

Library generation for phage display

Display phagemids were generated using human Fc sequences fused to the 6XHis tag, the c-Myc tag and the truncated P3 protein from M13 phage. Mutagenic oligonucleotides containing degenerate codons at library positions were purchased from INTEGRATED DNA Technologies. The use of kunkel mutation induced, uridine containing ssDNA templates were generated and annealed to phosphorylating mutagenic oligonucleotides. T7 DNA polymerase and T4 DNA ligase are used to form a covalently closed circular dsDNA (CCC-dsDNA) library. The CCC-dsDNA library was electroporated into TG1 E.coli cells (Lucigen, 60502-2). Transformed cells were grown in SB medium and infected with M13K07 helper phage. Carbenicillin and kanamycin antibiotics were used to maintain display and M13K07 helper phagemid. Cultures were grown overnight at 37℃with shaking. Phage particles were precipitated from the medium with PEG/NaCl solution and resuspended in PBS.

The registers are designed to have a residue diversity that is predominantly on the solvent exposed side of the beta-sheet surface of the CH3 domain with additional residues adjacent to the beta-sheet. Libraries were generated by randomizing positions (according to EU numbering) 380, 382, 384, 385, 386, 387, 422, 424, 426, 438, and 440 on the Fc domain of hIgG1 with "NNK" degenerate codons. Library positions map to the structure of the Fc domain of human IgG 1. The library was cloned onto phagemids and displayed on phages using the methods described above.

Selection of TfR binding clones by phage display

To select human TfR-binding clones, the recombinant biotinylated human TfR top domain was captured by streptavidin magnetic beads (Invitrogen, 11206D). Human TfR coated beads were washed and incubated with phage library in PBS with 1% bsa for at least 1 hour at room temperature. The beads were washed 3 times, each in PBS with 0.05% Tween-20 for 1 min. Bound phage were eluted with 100mM glycine pH 2.7 for 15 min and neutralized with Tris-HCl buffer. The eluted phage were used to infect TG1 cells for additional rounds of selection. Four-wheel selection is performed. In each subsequent round, the concentration of soluble biotinylated human TfR top domain was reduced and the washing time was increased to provide selective binding pressure.

For libraries intended for maturation binding affinity, phage libraries were incubated with 20nM of soluble biotinylated human TfR top domain for 30 min. Streptavidin magnetic beads were added for 5min to capture the biotinylated human TfR top domain. The beads were washed 3 times, each in PBS with 0.05% Tween-20 for 15 minutes. The eluted phage were used to infect TG1 cells for additional rounds of selection. Four-wheel selection is performed. In each subsequent round, the wash time is increased to provide selective binding pressure.

Recombinant expression of clones

TfR binding clones identified by phage display were in a truncated P3 rearrangement format by removal of the 6XHis tag, the c-Myc tag. The Fab was fused to the N-terminus of the clone to allow Surface Plasmon Resonance (SPR) capture (GE HEALTHCARE, anti-human Fab capture kit, 28958325). The fusion was expressed recombinantly in Expi293 cells and purified by protein a.

Binding was measured by Biacore

Binding affinity of the Fab-clone fusion of TfR top domain was determined by surface plasmon resonance in 1X HBS-ep+ running buffer (GE HEALTHCARE, BR 100669) using a Biacore ^TM K instrument. The Biacore ^TM series S CM5 sensor chip was immobilized with anti-human Fab (GE HEALTHCARE, 28958325). Molecules were captured on each flow cell and serial 3-fold dilutions of human TfR apical domain (2, 0.67, 0.22, 0.074, 0.025 and 0 μm) and cynomolgus monkey TfR apical domain (4, 1.3, 0.44, 0.15, 0.05 and 0 μm) were injected at a flow rate of 30 μl/min using single cycle kinetics. Each sample was subjected to 60 second association and 3 minute dissociation analysis. After each cycle, the chip was regenerated with 10mM glycine-HCl (pH 2.1) at 50. Mu.L/min for 30 seconds. Binding reactions were corrected by subtracting RU from the reference flow cells. The 1:1Langmuir model fitted with K _{Association with} and K _{dissociation of} simultaneously using Biacore ^TM K evaluation software was used for kinetic analysis. Table 32A below summarizes the binding affinities of selected clones for human and cynomolgus monkey TfR top domains.

Table 32A

Cell uptake was measured for clone 1 with LALA and TfR positive cells with clone 3 with LALA, indicating uptake by human and cyno TfR positive cells, but not negative control cells that did not express TfR.

Preliminary identification and characterization of clone 1 and clone 3

Phage libraries were panned against biotinylated human TfR top domain immobilized on magnetic streptavidin beads. Four rounds of phage panning were performed and the enriched clones were sequenced. Unique hits were cloned onto anti-BACE 1 hIgG1 monoclonal antibodies containing the "LALA" mutation, expressed in HEK293 cells and purified by protein a chromatography. Affinity was measured by SPR for human and cyno TfR top domains. Clones were evaluated for TfR-specific cell binding (fig. 43A and 43B). Clone 1 and clone 3, each with LALA substitution, had related sequences and were found to bind to human and cyno TfR by SPR and on cells. Clone 1 with LALA bound to the human TfR top domain at 480nM K _D, the cyno TfR top domain at 1800nM K _D and clearance values in WT mice of 7.6 mL/day/kg. Clone 3 with LALA bound to human TfR top domain at K _D at 1500nM, cyno TfR top domain at K _D at 6000nM and clearance values in WT mice were 20.6 mL/day/kg.

Affinity maturation of Soft randomized clone 1

Clone 1 was selected for affinity maturation. Affinity maturation library AM1 was generated by soft randomizing positions 380, 382, 384, 385, 386, 387, 422, 424, 426, 438 and 440 of clone 1. Affinity maturation library AM2 was generated by keeping positions 382, 385 and 387 of clone 1 constant while hard randomizing positions 380, 386, 436 and 440 and soft randomizing positions 384, 422, 424, 426, 428 and 438. The library was cloned onto phagemids and displayed on phages using the methods described above. AM1 and AM2 phage library for immobilized on magnetic streptavidin beads on biotinylated human and cyno TfR top domain panning. Three rounds of phage panning were performed and the enriched clones were sequenced. Periplasmic extracts enriched for clones were prepared and screened for binding by SPR. The front hits were cloned onto anti-BACE 1 hIgG1 monoclonal antibodies containing the "LALA" mutation, expressed in HEK293 cells and purified by protein a chromatography. Affinity for human and cyno TfR top domains was measured by SPR (table 32B). Several clones were selected and shown to bind huTfR and cyTfR expressing cells. A multi-dose study was performed in which chimeric huTfR ^{Top end} knock-in mice were administered 350 mg/kg IP doses of clones 1-112 with LALA and M428L on days 0, 3, and 5. On day 6, clones 1-112 with LALA and M428L showed significant brain absorption and pharmacodynamic response via BACE1 inhibition.

Table 32B

The sequences of the clones shown in tables 32A and 32B are shown in Table 32B-1 below. For the unlisted positions, the amino acid at that position is identical to the amino acid in the wild-type Fc.

TABLE 32B-1

Mutation analysis of clones 1-112

Clones 1-112 were selected for mutation analysis. To understand the relative effect of each position of the register on binding and human/cyno cross-reactivity, a set of single, double or triple rational mutations were made to clones 1-112. These mutations include alanine scanning of the register, reversal of WT Fc scanning, conservative mutations, and mutations observed in self-sequencing affinity maturation libraries. Mutations were cloned onto 1-112 and expressed in HEK293 cells. Supernatants were captured for SPR by GE HEAL THCARE anti-human Fab capture kit and affinity was measured for human and cyno TfR top domains (tables 32C and 32D). The analysis discloses mutations critical for binding, affinity, and cross-reactivity of human and cyno TfR.

Tables 32C and 32D below show libraries of clone 1-112 mutants and their binding affinities. Each mutant contains one or more amino acid substitutions of clones 1-112. For example, one mutant may contain an E380A mutation, and the amino acids at the remaining positions are identical to those in clones 1-112. In addition, to generate K _D data in Table 32C, each of the clones in Table 32C (except for clones 1-112-102 through 1-112-104 and clones 1-112-106 through 1-112-114) also contained LALA and M428L. Clones 1-112-102 to 1-112-104 and clones 1-112-106 to 1-112-114 also contained LALA. The positions are numbered according to the EU numbering scheme.

Table 32C

Table 32D

Clone 1 family second round affinity maturation

For the second round of affinity maturation, three additional phage libraries were generated. Affinity maturation library AM3 was generated with degenerate or non-degenerate codons at the specific positions: "VWR" at 380, "GGG" at 382, "SHR" at 384, "GTG" at 385, "KCN" or "CAG" at 386, "ADA" at 422, "BCN" at 424, "RBY" at 426, "CTG" at 428, "ARY" at 434, "VTH" at 438, and "RBB" at 440. Affinity maturation library AM4 was generated with degenerate or non-degenerate codons at the specific positions: "NNK" at 378, "VWR" at 380, "GGG" at 382, "GAG" at 384, "GTG" at 385, "GCC" or "CAG" at 386, "NHK" at 391, "NHK" at 421, "ADA" at 422, "BCN" at 424, "RBY" at 426, "CTG" at 428, "ARY" at 434, "VTH" at 438, "RBB" at 440, and "NNK" at 442. Affinity maturation library AM5 was generated with degenerate or non-degenerate codons at the specific positions: "VWR" at 380, "NNK" at 382, "NNK" at 383, "NNK" at 384, "NNK" at 385, "NNK" at 386, "ADA" at 422, "BCN" at 424, "RBY" at 426, "CTG" at 428, "ARY" at 434, "VTH" at 438, and "RBB" at 440. The library was cloned onto phagemids and displayed on phages using the methods described above. Three rounds of solution phage panning were performed, with increasing stringency for each round, and enriched clones sequenced. Periplasmic extracts enriched for clones were prepared and screened for binding by SPR. The front hits were cloned into anti-BACE 1 hIgG1 monoclonal antibodies containing the "LALA" mutation.

Additional engineering of clone 1-131

Additional clones were designed by incorporating enrichment mutations from the second round of affinity maturation of clone 1 onto clones 1-131. These clones were expressed in HEK293 cells and purified by protein a chromatography. Affinity was measured by SPR for human and cyno TfR top domains.

Table 32E below shows the library of affinity matured clones and their binding affinities. Each mutant contains several amino acid substitutions relative to the wild-type Fc. For empty cells in table 32E, the amino acid at that position is identical to the amino acid in wild-type Fc. Furthermore, to generate the K _D data in table 32E, each of the clones in table 32E also contained LALA and M428L. The positions are numbered according to the EU numbering scheme.

Table 32E

Additional engineering of clones 1-112

The mutation N421L, Q438Y and S442R was found by SPR to increase affinity to human and cyno TfR top domains. These mutations were incorporated into clones 1-112 in order to expand the affinity range. These mutations were also combined with WT Fc reverse mutations in order to attenuate affinity and reduce the total number of mutations. Variants were cloned as monovalent dimers onto the heavy chain of anti-BACE 1 hIgG1 monoclonal antibodies containing "LALA" and knob mutations. Clones were expressed in HEK293 cells and purified by protein a chromatography. Affinity for human and cyno TfR top domains was measured by SPR (table 32F). Molecules containing the N421L, Q438Y or S442R mutations showed weaker cell binding than expected. Affinity for full-length human TfR is measured by SPR for a selected set of molecules. Clones 1-112 have similar binding affinities for full-length human TfR and the top domain of human TfR. Molecules containing the N421L, Q438Y or S442R mutations bind with less affinity to full-length human TfR than to the top domain of human TfR. It was concluded that the affinity improvements observed from these mutations were specific to the top domain construct.

TABLE 32F

To expand the affinity of clones 1-112, an additional set of variants was generated by combining mutations in the mutation analysis study. The engineered molecules in the panel contain the N434S mutation. Variants were cloned as monovalent dimers onto the heavy chain of anti-BACE 1 hIgG1 monoclonal antibodies containing "LALA" and knob mutations. The second anti-BACE 1 heavy chain contains "LALA", mortar and "LS" mutations. Clones were expressed in HEK293 cells and purified by protein a chromatography. Affinity for human and cyno TfR top domains was measured by SPR (table 32G).

TABLE 32G

Clone 1 family third round affinity maturation

For the third round of affinity maturation, affinity maturation library AM6 was generated with degenerate or non-degenerate codons at the specific positions: "NHW" at 378, "NHW" at 380, "GGC" at 382, "VHW" at 384, "GTG" at 385, "NHW" at 386, "NTY" at 422, "BCN" at 424, "ABY" at 426, "CTG" at 428, "TCC" at 434, "NHW" at 438, and "NNW" at 440. Three rounds of solution phage panning were performed, with increasing stringency for each round, and enriched clones sequenced. Periplasmic extracts enriched for clones were prepared and screened for binding by SPR. The front hit was cloned as a monovalent dimer onto the heavy chain of an anti-BACE 1hIgG1 monoclonal antibody containing a "LALA" and pestle mutation. The second anti-BACE 1 heavy chain contains "LALA" and a mortar mutation. The "LS" mutation is included on the "mortar" heavy chain of clone 1-244 through clone 1-334. These clones were expressed in HEK293 cells and purified by protein a chromatography. Affinity for human and cyno TfR top domains was measured by SPR (table 32H). All residues and mutations observed in the validated TfR conjugates are summarized in table 32I.

Table 32H

The sequences of the clones shown in Table 32H are shown in Table 32H-1 below. For the unlisted positions, the amino acid at that position is identical to the amino acid in the wild-type Fc.

TABLE 32H-1

PK/PD evaluation of clone 1 and clone 3

The PK containing Fc fragments of clone 1 and clone 3, each fused to anti-BACE 1Fab (2H 8), were tested in wild-type TfR mice to ensure that they were free of PK defects. "clone 1 bivalent: ab153" is a bivalent Fc-Fab fusion polypeptide comprising two Fc polypeptides each comprising the sequence of clone 1 with LALA and M428L fused to a high affinity anti-BACE 1Fab domain (Ab 153). "clone 3 bivalent: ab153" is a bivalent Fc-Fab fusion polypeptide comprising two Fc polypeptides each comprising the sequence of clone 3 with LALA and M428L fused to a high affinity anti-BACE 1Fab domain (Ab 153). An "anti-BACE 1 control" is a negative control without any TfR binding sites. Clone 1 had normal clearance relative to the negative control (figure 44). The clearance values for clones 1 and 3 were 7.6 mL/day/kg and 20.6 mL/day/kg, respectively, and the clearance value for the negative control was 6.3 mL/day/kg.

Table 32I further summarizes the possible amino acids at each of the positions leading to TfR binders according to the analysis of TfR binding clones. The positions are numbered according to the EU numbering scheme.

TABLE 32I

Additional clones from rational design and affinity maturation

To further expand the affinity of clones 1-112, an additional set of variants was generated by combining mutations in the mutation analysis study. Each mutant in table 32J contains several amino acid substitutions relative to the wild-type Fc. For empty cells in table 32J, the amino acid at that position is identical to the amino acid in wild-type Fc. Recombinant expression clones and human TfR top domain binding was tested by Biacore ^TM, estimated to have an affinity of about 57-2300nM, with very weak cross-reactivity with the cyno TfR top domain (table 32K).

Table 32J

Table 32K

PK/PD evaluation of clones 1-112_L, 1-112_LS, 1-292 and 1-321

Clones 1-112_l monovalent, 1-112_ls monovalent, 1-292 monovalent, and 1-321 monovalent PK were tested in wild-type TfR mice to ensure that they were free of PK defects. Clone 1-112_l as used herein monovalent contains 1-112TfR binding sites with T366W knob, M428L and LALA mutations as the first Fc polypeptide and Fc sequences with T366S, L a and Y407V knob, M428L and LALA mutations as the second Fc polypeptide. Clone 1-112_LS monovalent as used herein contains the 1-112TfR binding site with the T366W knob, M428L, N S and LALA mutations as the first Fc polypeptide and the Fc sequence with the T366S, L A and Y407V knob, M428L, N S and LALA mutations as the second Fc polypeptide. Clone 1-292 as used herein monovalent contains a 1-292TfR binding site with mutations T366W knob, M428L and LALA as the first Fc polypeptide and an Fc sequence with mutations T366S, L a and Y407V knob, M428L and LALA as the second Fc polypeptide. Clone 1-321 as used herein monovalent contains a 1-321TfR binding site with mutations T366W knob, M428L and LALA as the first Fc polypeptide and an Fc sequence with mutations T366S, L a and Y407V knob, M428L and LALA as the second Fc polypeptide. The anti-BACE 1 control is a negative control without any TfR binding sites.

Clones and controls were intravenously dosed to huTfR ^{Top end} knock-in mice at 50 mg/kg. Brain and plasma concentrations were measured at 24 hours (fig. 45A and 45B). All clones showed good brain uptake 24 hours after dose compared to the negative control (fig. 45A). As expected, plasma PK showed clearance of all TfR binding clones compared to the control (fig. 45B).

EXAMPLE 21 PK/PD evaluation of clone 6.5.11.5.42 and clone 1-112

To determine if clones were safe after multiple doses, a multi-dose safety study was performed using clone 6.5.11.5.42.2 and clones 1-112. "clone 6.5.11.5.42.2 bivalent: ab153" is a bivalent Fc-Fab fusion polypeptide comprising two Fc polypeptides each comprising the sequence of clone 6.5.11.5.42.2 fused to a high affinity anti-BACE 1 Fab domain (Ab 153); "clone 1-112 bivalent: ab153" is a bivalent Fc-Fab fusion polypeptide comprising two Fc polypeptides each comprising the sequence of clone 1-112 having LALA and M428L fused to a high affinity anti-BACE 1 Fab domain (Ab 153); and an "anti-BACE 1 control" is a negative control that does not have any TfR binding sites. Mice knocked in to chimeric huTfR ^{Top end} on day 0, day 3, and day 5 (n=5 mice/group) were dosed intravenously at 50 mg/kg. All mice were perfused with PBS 24 hours after dosing. Prior to perfusion, blood was collected via cardiac puncture in EDTA plasma tubes and spun at 14000rpm for 5 minutes. The plasma was then isolated for subsequent PK and PD analysis. Brains were extracted after perfusion and half brains were isolated to homogenize 10-fold in PBS (for PK) or 5M GuHCl (for PD) at tissue weight of 1% np-40.

BACE1 inhibition of amyloid precursor protein APP cleavage was used as a pharmacodynamic readout of antibody activity in the brain. Brain tissue was homogenized in 5M guanidine-HCl at 10 times the tissue weight and subsequently diluted 1:10 in 0.25% casein buffer in PBS. Mouse aβ40 levels in brain lysates were measured using sandwich ELISA. 384-well MaxiSorp plates were coated overnight with polyclonal capture antibodies specific for the C-terminus of the aβ40 peptide (Millipore # ABN 240). Casein-diluted guanamine lysate was further diluted 1:2 on ELISA plates and added simultaneously with detection antibody, biotinylated M3.2. Plasma was analyzed at 1:5 dilution. Samples were incubated overnight at 4 ℃ followed by the addition of streptavidin-HRP followed by the addition of TMB substrate. A standard curve of 0.78-50pg/mL msA beta 40 was fitted using four-parameter logistic regression.

FIG. 46 shows brain A.beta.40 reduction in mice treated with clone 6.5.11.5.42.2 and clones 1-112 with LALA and M428L.

EXAMPLE 22 Crystal Structure Generation method of clone 1-112 and a circularly arranged human TFR Top Domain Co-Complex

The His-tagged circularly permuted human TfR top domain (huTfR ^{Top end}) and clone 1-112Fc with LALA and M428L were expressed in HEK cells at an initial cell density of 2.5X10 ⁶ cells/mL. Conditioned medium was collected 3-4 days post-transfection and proteins were purified using Ni-NTA (Sigma) or protein a (Genescript) affinity chromatography, as appropriate. Proteins were further purified by size exclusion chromatography on Superdex 75/60 (clone 1-112 with LALA and M428L) and Superdex20026/60 (huTfR ^{Top end}) as well as gel filtration columns and eluted in 30mM Hepes pH 7.4, 150mM NaCl, 50mM KCl, 3% glycerol and 1mM TCEP.

To obtain huTfR ^{Top end}/clone 1-112 complexes, the protein was mixed with a 1.3 molar excess of huTfR ^{Top end} and incubated for 1 hour at room temperature. The complexes were purified from excess unbound protein by size exclusion chromatography on a Superdex200 column and buffer exchanged to 20mM HEPES pH 7.5, 200mM NaCl with 5% glycerol and 1mM TCEP to a final concentration of 12mg/mL.

Crystals were grown by sitting-drop vapor diffusion at 4 ℃ with a complex solution and a 2:1 mixture of pore solutions containing 0.10m 2ph 7.50, 10% amino acid mix (L-Na-glutamine; alanine (racemic); glycine; lysine HCl (racemic); serine (racemic)) and 30% w/v PEG550 MME and PEG20 k.

Crystals were flash frozen in liquid nitrogen using a crystallization mother liquor supplemented with 25% (v/v) ethylene glycol. The diffraction dataset of the complex was collected in PX1/XO6SA SWISS LIGHT SOURCE (SLS) at 100K using EIGERX M detector. Diffraction to crystalsAnd belongs to the space group P2 ₁2₁2₁, having two complexes in the same asymmetric unit. The data was indexed, integrated, and scaled using the CCP4 suite of programs (Xia 2-XDS and XSCALE).

Structure determination and refinement

The initial phase of the structure was obtained by molecular substitution using PHASER, and the coordinates of the crystal structure of the non-glycosylated human Fc fragment (PDB ID:3S 7G) were used as a search model. The model is refined by rigid body refinement, followed by constraint refinement using REFMAC. The data collection and refinement statistics are shown in table 32L. All crystallographic calculations were performed using the CCP4 suite of programs. The composite model was constructed as electron density using the graphic program COOT.

Table 32L

* Statistics of the highest resolution shell are shown in brackets.

EXAMPLE 23 Crystal Structure of clone 1-112 binding to the circulating human TFR Top Domain

The crystal structure of clones 1-112 with LALA and M428L co-complexed with the top domain of human TfR circulation was resolved (fig. 47C). Clones 1-112 with LALA and M428L bound to the human TfR top domain in a manner slightly offset from clone 6.5.11.5.42 with the epitope. Clones 1-112 were contacted with the β -sheet surface and surrounding loop region. When either the bivalent clones 1-112 or clone 6.5.11.5.42 were mimicked to bind to two full-length TfR ECDs, the slightly offset effect of the epitope could be seen as clone 6.5.11.5.42 (fig. 47A-47C) which would result in a spatial conflict between the two TfR ECDs (whereas 1-112 would not). The crystal structures of clones 1-112 and 6.5.11.5.42, as well as TfR ECD modeling, showed that bivalent clone 6.5.11.5.42 would bind full-length TfR in a monovalent manner, while bivalent clone 1-112 would bind full-length TfR in a bivalent manner under conditions where transferrin does not bind to TfR.

Example 24 production of tfr targets

Circularly permuted apical domains

The top domain of the human and cyno circular arrangement was cloned into the pET28-His ₁₀ -Smt3-Avi-PreScission-TfR vector, where TfR residues 326-379 are the N-terminus of TfR residues 194-296, to create a circular arrangement with new N-and C-termini and deletion of TfR loop residues 297-325. The sequences of human TfR1 and cyno TfR are shown in SEQ ID NOs 127 and 128, respectively. The apical domain construct was co-expressed with BirA in BL21 (DE 3) e.coli cells (Novagen) at 37 ℃ and in LB medium with antibiotics until OD was about 0.7, refrigerated on ice for 30 min, followed by treatment with 1mM IPTG at 18 ℃ for 16 hours. Cells were collected by centrifugation, resuspended in 50mM Tris-HCl (pH 7.5), 500mM NaCl, 10% glycerol and benzoate, incubated at 37℃for 1 hour, lysed using a microfluidizer, and insoluble material removed by centrifugation. The cyclically aligned TfR top domains were purified using 5mL His Trap (GEHEALTHCARE), washed with 25mM and 50mM imidazole, followed by elution with a 100-500mM imidazole gradient. Fractions were pooled and lysed with Ulp1 at a 100:1 molar ratio, incubated overnight and dialyzed into 50mM HEPES (pH 7.5), 150mM NaCl, 1mM DTT at 4 ℃. The lysed sample again flowed through equilibrated 5mL HisTrap and the effluent was collected. The samples were further purified by size exclusion chromatography on Superdex75 16/60 (GE HEALTHCARE).

TfR extracellular domain (ECD)

DNA encoding residues 121-760 of TfR extracellular domain (ECD) (human TfR1 (SEQ ID NO: 127) or cyno TfR (SEQ ID NO: 128)) was cloned into a mammalian expression vector with a C-terminal cleavable His tag and Avi tag. Plasmids were transfected and expressed in HEK293 cells. The extracellular domain was purified from the harvested supernatant using Ni-NTA chromatography followed by removal of any aggregated protein using size exclusion chromatography. The yield was about 5mg per liter of culture. Proteins were stored in 10mM K ₃PO₄ (pH 6.7), 100mM KCl, 100mM NaCl, 20% glycerol and frozen at-20 ℃.

Purified TfR ECD was biotinylated using EZ-link sulfo-NHS-LC-biotin kit (obtained from Thermo Scientific) with five-fold molar excess of biotin. Excess biotin was removed by extensive dialysis against PBS.

The Avi-tagged human TfR ECD and the top domain were biotinylated using BirA-500 (BirA biotin protein ligase standard reaction kit from Avidity, LLC). After the reaction, the labeled protein was further purified by size exclusion chromatography to remove excess BirA enzyme. The final material was stored in 10mM K ₃PO₄ (pH 6.7), 100mM KCl, 100mM NaCl, 20% glycerol and frozen at-20 ℃.

Full-length TfR

Full-length human and cyno TfR without Avi tag were prepared as previously described in international patent publication No. WO 2018/152326.

Example 25 Yeast display library Generation and selection methods

Library generation

A DNA template encoding a wild-type human Fc sequence was synthesized and incorporated into a yeast display vector. The Fc polypeptide is displayed on the Aga2p cell wall protein. The vector contains a leader peptide with Kex2 cleavage sequence and a c-Myc epitope tag fused to the Fc terminus.

Freshly prepared inductance-accepting yeasts (i.e., strain EBY 100) were electroporated with the linearized vector and assembled library inserts. After recovery in selective SD-CAA medium, the yeast was grown to confluence and split twice, followed by induction of protein expression by transfer to SG-CAA medium. Typical library sizes range from about 10 ⁷ to about 10 ⁹ transformants. Fc dimers are formed by pairing adjacently displayed Fc monomers.

Library selection

The selection of Magnetic Assisted Cell Sorting (MACS) and Fluorescence Activated Cell Sorting (FACS) was performed similarly to that described in Ackerman et al 2009Biotechnol. Prog.25 (3), 774. Streptavidin magnetic beads (Promega) were labeled with biotinylated targets and incubated with yeast (typically 5-10 fold library diversity). Unbound yeast is removed, the beads are washed, and the bound yeast is grown in selective medium and induced for selection for subsequent rounds.

For FACS selection, yeasts are labeled with anti-c-Myc antibodies to monitor expression and biotinylated targets (concentration varies depending on the sorting round). In some experiments, the target was combined with streptavidin-Alexa647 To enhance the affinity of the interaction. In other experiments, biotinylated targets were bound and treated with streptavidin-Alexa647 Washing and then detecting. The single-peak yeast with binding was sorted using FACS ARIA III cell sorter. The sorted yeasts are grown in selective medium and then induced for subsequent selection rounds.

After obtaining the enriched yeast population, the yeast was plated on SD-CAA agar plates and a single colony was grown and expression was induced, followed by labeling as described above to determine its propensity to bind to the target. Positive single clones were then sequenced for binding targets, after which some clones were expressed as soluble Fc fragments or fused to Fab fragments.

EXAMPLE 26 cell uptake method

HEK293T, CHO: cyTfR and CHO cells were plated in standard growth medium (DMEM (Gibco ^TM 11995073) +10% fbs (VWR 89510-188) +1xPen/Strep (Gibco 15140122)) at 40,000 cells/well in 96-well plates. After about 24 hours, the molecules were diluted in standard growth medium heated to 37 ℃. Old medium was removed from the cells and diluted molecules were added to the cells. Cells were incubated at 37℃for 45 min. Cells were then washed with PBS and then fixed in 4% PFA (Electron Microscopy Sciences 15714-S) for 10 min. Cells were washed with PBS and then blocked for 30min with 5% bsa, 0.3% tritonx100 in PBS. Cells were stained with 1% BSA, 0.3% Triton X100 in anti-human IgG-488 (1:1000;Jackson Immuno Research 109-545-003), cell mask (1:10,000;Thermo H32721) and DAPI (1:2000;Thermo D1306) diluted in PBS for at least 30min. Cells were washed with PBS, imaged on Opera phenoix, and images were analyzed with Harmony software.

It is understood that the examples and implementations described herein are for illustrative purposes only and that modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. The sequences of the sequence accession numbers cited herein are hereby incorporated by reference.

Informal sequence listing

Claims

1. A polypeptide comprising a modified constant domain that specifically binds to a CD98hc protein.

2. The polypeptide of claim 1, wherein the modified constant domain comprises a modified CH3 domain that specifically binds to the CD98hc protein.

3. The polypeptide of claim 2, wherein the modified CH3 domain is part of an Fc polypeptide.

4. A polypeptide according to any one of claims 1 to 3 wherein the CD98hc protein is a human CD98hc protein.

5. The polypeptide of any one of claims 1 to 4, wherein the CD98hc protein forms a complex with LAT1 (SLC 7 A5), LAT2 (SLC 7 A8), y ⁺LAT1(SLC7A7)、y⁺ LAT2 (SLC 7 A6), asc-1 (SLC 7a 10) or xCT (SLC 7a 11).

6. The polypeptide of claim 5, wherein the CD98hc protein forms a complex with LAT1 (SLC 7 A5).

7. The polypeptide of any one of claims 1 to 6, wherein the modified constant domain comprises a sequence having at least 85%, 90% or 95% sequence identity to amino acids 111-217 of the sequence of any one of SEQ ID NOs 28-45.

8. A polypeptide comprising a modified CH3 domain that specifically binds to a CD98hc protein, wherein the modified CH3 domain comprises at least five, six, seven, eight, or nine substitutions in a set of amino acid positions consisting of 382, 384, 385, 387, 422, 424, 426, 438, 440 according to EU numbering.

9. A polypeptide comprising a modified CH3 domain that specifically binds to a CD98hc protein, wherein the modified CH3 domain comprises at least eleven, twelve, thirteen, fourteen or fifteen substitutions in a 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.

10. The polypeptide of claim 9, wherein the modified CH3 domain comprises a sequence having at least 85%, 90% or 95% sequence identity to amino acids 111-217 of the sequence of any one of SEQ ID NOs 28-43, wherein the modified CH3 domain comprises at least eleven, twelve, thirteen, fourteen or fifteen substitutions in a 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.

11. The polypeptide of claim 9 or 10, wherein the 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 a at position 442.

12. The polypeptide of any one of claims 9 to 11, wherein the modified CH3 domain comprises SEQ ID No. 28.

13. The polypeptide of claim 9 or 10, wherein the 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, N at position 440, and a at position 442.

14. The polypeptide of any one of claims 9, 10 and 13, wherein the modified CH3 domain comprises SEQ ID No. 29.

15. The polypeptide of claim 9 or 10, wherein the modified CH3 domain comprises 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.

16. The polypeptide of any one of claims 9, 10 and 15, wherein the modified CH3 domain comprises SEQ ID No. 30.

17. The polypeptide of claim 9 or 10, wherein the modified CH3 domain comprises 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.

18. The polypeptide of any one of claims 9, 10 and 17, wherein the modified CH3 domain comprises SEQ ID No. 31.

19. The polypeptide of claim 9 or 10, wherein the 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, 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.

20. The polypeptide of any one of claims 9, 10 and 19, wherein the modified CH3 domain comprises SEQ ID No. 32.

21. The polypeptide of claim 9 or 10, wherein the 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, 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.

22. The polypeptide of any one of claims 9, 10 and 21, wherein the modified CH3 domain comprises SEQ ID No. 22.

23. The polypeptide of claim 9 or 10, wherein the 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.

24. The polypeptide of any one of claims 9, 10 and 23, wherein the modified CH3 domain comprises SEQ ID NO 34.

25. The polypeptide of claim 9 or 10, wherein the 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.

26. The polypeptide of any one of claims 9, 10 and 25, wherein the modified CH3 domain comprises SEQ ID No. 35.

27. The polypeptide of claim 9 or 10, wherein the 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.

28. The polypeptide of any one of claims 9, 10 and 27, wherein the modified CH3 domain comprises SEQ ID No. 36.

29. The polypeptide of claim 9 or 10, wherein the 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.

30. The polypeptide of any one of claims 9, 10 and 29, wherein the modified CH3 domain comprises SEQ ID No. 37.

31. The polypeptide of claim 9 or 10, wherein the modified CH3 domain comprises 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.

32. The polypeptide of any one of claims 9, 10 and 31, wherein the modified CH3 domain comprises SEQ ID No. 38.

33. The polypeptide of claim 9 or 10, wherein the 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.

34. The polypeptide of any one of claims 9, 10 and 33, wherein the modified CH3 domain comprises SEQ ID NO 39.

35. The polypeptide of claim 9 or 10, wherein the 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, 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.

36. The polypeptide of any one of claims 9, 10 and 35, wherein the modified CH3 domain comprises SEQ ID No. 40.

37. The polypeptide of claim 9 or 10, wherein the 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.

38. The polypeptide of any one of claims 9, 10 and 37, wherein the modified CH3 domain comprises SEQ ID No. 41.

39. The polypeptide of claim 9 or 10, wherein the 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.

40. The polypeptide of any one of claims 9, 10 and 39, wherein the modified CH3 domain comprises SEQ ID No. 42.

41. The polypeptide of claim 9 or 10, wherein the modified CH3 domain comprises 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.

42. The polypeptide of any one of claims 9, 10 and 41, wherein the modified CH3 domain comprises SEQ ID No. 43.

43. A polypeptide comprising a modified CH3 domain that specifically binds to a CD98hc protein, wherein the modified CH3 domain comprises:

(i) First amino acid sequence LX ₁NX₂X₃X₄X₅ L (SEQ ID NO: 46),

Wherein X ₁ is any amino acid,

Wherein X ₂ is R, H or Q,

Wherein X ₃ is F or Y,

Wherein X ₄ is V, L, I, F, Y or E,

Wherein X ₅ is any amino acid;

(ii) A second amino acid sequence X ₁X₂X₃AX₄X₅X₆X₇ (SEQ ID NO: 47),

Wherein X ₁ is E, N, Q or A,

Wherein X ₂ is I, V, T or P,

Wherein X ₃ and X ₄ are any amino acid,

Wherein X ₅ is N or S,

Wherein X ₆ is any amino acid,

Wherein X ₇ is Y or W; and

(Iii) Third amino acid sequence X ₁X₂X₃X₄NX₅X₆ (SEQ ID NO: 48),

Wherein X ₁ is Y, R or W,

Wherein X ₂ is any amino acid,

Wherein X ₃ is F or W,

Wherein X ₄ and X ₅ are any amino acid, and

Wherein X ₆ is A, Q, K, R, H, M or S.

44. The polypeptide of claim 43, wherein the polypeptide binds human CD98hc with an affinity of 15nM to 5. Mu.M.

45. The polypeptide of claim 43 or 44, wherein the polypeptide has cyno cross-reactivity.

46. The polypeptide of any one of claims 43 to 45, wherein the polypeptide binds cyno CD98hc with an affinity of 80nM to 5 μm.

47. A polypeptide comprising a modified CH3 domain that specifically binds to a CD98hc protein, wherein the modified CH3 domain comprises at least eight, nine, ten, eleven, twelve or thirteen substitutions in a 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.

48. The polypeptide of claim 47, wherein the 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.

49. A polypeptide comprising a modified CH3 domain that specifically binds to a CD98hc protein, wherein the modified CH3 domain comprises at least eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen substitutions in a 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.

50. The polypeptide of claim 49, wherein the 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.

51. A polypeptide comprising a modified CH3 domain that specifically binds to a CD98hc protein, wherein the modified CH3 domain comprises at least eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen substitutions in a 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.

52. The polypeptide of claim 51, wherein the modified CH3 domain comprises a sequence having at least 85%, 90% or 95% sequence identity to amino acids 111-217 of the sequence of any one of SEQ ID NOs 44-45, wherein the modified CH3 domain comprises at least eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen substitutions in a set of amino acid positions consisting of: r at location 382, T at location 383, Y at location 384, K at location 385, P at location 386, Y at location 387, T at location 389, D at location 421, I at location 422, V at location 424, D at location 426, L at location 428, F at location 436, I at location 438, and K at location 440.

53. The polypeptide of claim 51 or 52, wherein the modified CH3 domain comprises 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.

54. The polypeptide of any one of claims 51 to 53, wherein the modified CH3 domain comprises SEQ ID No. 44.

55. The polypeptide of claim 51 or 52, wherein the modified CH3 domain comprises 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.

56. The polypeptide of any one of claims 51, 52 and 55, wherein the modified CH3 domain comprises SEQ ID No. 45.

57. A polypeptide comprising a modified CH3 domain that specifically binds to a CD98hc protein, wherein the modified CH3 domain comprises:

(i) The first amino acid sequence X ₁X₂YKPYX₃ T (SEQ ID NO: 49),

Wherein X ₁ is E or R, wherein X ₂ is S or T,

Wherein X ₃ is any amino acid;

(ii) A second amino acid sequence X ₁X₂X₃VX₄DX₅X₆ (SEQ ID NO: 50),

Wherein X ₁ is N or D, wherein X ₂ is V or I,

Wherein X ₃、X₄ and X ₅ are any amino acid,

Wherein X ₆ is M or L; and

(Iii) Third amino acid sequence X ₁X₂IX₃X₄ (SEQ ID NO: 51),

Wherein X ₁ is Y or F,

Wherein X ₂ and X ₃ are any amino acid,

Wherein X ₄ is S or K.

58. The polypeptide of any one of claims 8 to 57, wherein the position is substituted relative to SEQ ID No. 1.

59. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises five, six, or seven amino acid substitutions in a set of amino acid positions comprising 422, 424, 426, 433, 434, 438, and 440,

Wherein the modified CH3 domain does not have a combination of G at position 437, F at position 438, and D at position 440, an

Wherein the position is determined according to EU numbering.

60. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises three, four, five, six, seven, or eight amino acid substitutions and/or one or two amino acid deletions in a set of amino acid positions comprising 380 and 382-389; and

Five, six or seven amino acid substitutions in a set of amino acid positions containing 422, 424, 426, 433, 434, 438 and 440, wherein the positions are determined according to EU numbering.

61. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises a sequence comprising at least one amino acid substitution in sequence VFSCSVMHEALHNHYTQKS (SEQ ID NO: 57),

Wherein the sequence SEQ ID NO:57 is from position 422 to position 440 of the Fc polypeptide, the sequence does not have a combination of G at position 437, F at position 438 and D at position 440, and the positions are determined according to EU numbering.

62. The polypeptide of claim 61, wherein the sequence comprises five, six, or seven amino acid substitutions in a set of amino acid positions comprising 422, 424, 426, 433, 434, 438, and 440.

63. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises a first sequence comprising at least one amino acid substitution and/or deletion in sequence AVEWESNGQPENN (SEQ ID NO: 56), and

A second sequence comprising at least one amino acid substitution in sequence VFSCSVMHEALHNHYTQKS (SEQ ID NO: 57),

Wherein sequence SEQ ID NO:56 is from position 378 to position 390 of the Fc polypeptide and sequence SEQ ID NO:57 is from position 422 to position 440 of the Fc polypeptide, and said positions are determined according to EU numbering.

64. The polypeptide of claim 63, wherein the modified CH3 domain comprises three, four, five, six, seven, or eight amino acid substitutions in a set of amino acid positions comprising 380 and 382-389.

65. The polypeptide of claim 63 or 64, wherein the modified CH3 domain comprises five, six, or seven amino acid substitutions in a set of amino acid positions comprising 422, 424, 426, 433, 434, 438, and 440.

66. The polypeptide of any one of claims 63 to 65, wherein the modified CH3 domain comprises one or two amino acid deletions in sequence SEQ ID No. 56.

67. The polypeptide of any one of claims 59 to 66, wherein the modified CH3 domain is part of an Fc polypeptide.

68. The polypeptide of any one of claims 59 to 67, wherein the modified CH3 domain comprises F at position 382.

69. The polypeptide of any one of claims 59 to 68, wherein the modified CH3 domain comprises an a or a polar amino acid at position 383.

70. The polypeptide of claim 69, wherein the polar amino acid is Y or S.

71. The polypeptide of any one of claims 59 to 70, wherein the modified CH3 domain comprises G, N or an acidic amino acid at position 384.

72. The polypeptide of claim 71, wherein the acidic amino acid is D or E.

73. The polypeptide of any one of claims 59 to 72, wherein the modified CH3 domain comprises N, R or a polar amino acid at position 389.

74. The polypeptide of claim 73, wherein the polar amino acid is S or T.

75. The polypeptide of claims 59 to 74, wherein said modified CH3 domain comprises at least one amino acid substitution at a β -sheet position relative to sequence SEQ ID No. 56.

76. The polypeptide of claim 75, wherein said modified CH3 domain comprises one, two or three amino acid substitutions at β -sheet positions relative to sequence SEQ ID No. 56.

77. The polypeptide of claim 75 or 76, wherein the β -sheet position is selected from the group consisting of: positions 380, 382 and 383, wherein the positions are determined according to EU numbering.

78. The polypeptide of claims 75 to 77, wherein said modified CH3 domain comprises an amino acid substitution at β -sheet position 380 relative to sequence SEQ ID No. 56.

79. The polypeptide of claim 78, wherein the modified CH3 domain comprises E, N, F or Y at position 380.

80. The polypeptide of claim 79, wherein the modified CH3 domain comprises E at position 380.

81. The polypeptide of any one of claims 75 to 80, wherein the modified CH3 domain comprises an amino acid substitution at β -sheet position 382 relative to sequence SEQ ID No. 56.

82. The polypeptide of claim 81, wherein the modified CH3 domain comprises F at position 382.

83. The polypeptide of any one of claims 75 to 82, wherein the modified CH3 domain comprises an amino acid substitution or amino acid deletion at β -sheet position 383 relative to sequence SEQ ID No. 56.

84. The polypeptide of claim 83, wherein the modified CH3 domain comprises Y or a at position 383.

85. The polypeptide of claim 83 or 84, wherein the modified CH3 domain comprises Y at position 383.

86. The polypeptide of any one of claims 59 to 85, wherein said modified CH3 domain comprises at least one amino acid substitution at a β -sheet position relative to sequence SEQ ID No. 57.

87. The polypeptide of claim 86, wherein the modified CH3 domain comprises one, two, three or four amino acid substitutions at β -sheet positions relative to sequence SEQ ID No. 57.

88. The polypeptide of claim 86 or 87, wherein the β -sheet position is selected from the group consisting of: positions 424, 426, 438, and 440 according to EU numbering.

89. The polypeptide of any one of claims 86 to 88, wherein the modified CH3 domain comprises an amino acid substitution at β -sheet position 424 relative to sequence SEQ ID No. 57.

90. The polypeptide of claim 89, wherein the modified CH3 domain comprises a at position 424.

91. The polypeptide of any one of claims 86 to 90, wherein the modified CH3 domain comprises an amino acid substitution at β -sheet position 426 relative to sequence SEQ ID No. 57.

92. The polypeptide of claim 91, wherein the modified CH3 domain comprises E at position 426.

93. The polypeptide of any one of claims 86 to 92, wherein the modified CH3 domain comprises an amino acid substitution at β -sheet position 438 relative to sequence SEQ ID No. 57.

94. The polypeptide of claim 93, wherein the modified CH3 domain comprises Y at position 438.

95. The polypeptide of any one of claims 86 to 94, wherein the modified CH3 domain comprises an amino acid substitution at β -sheet position 440 relative to sequence SEQ ID No. 57.

96. The polypeptide of claim 95, wherein the modified CH3 domain comprises L at position 440.

97. The polypeptide of any one of claims 59 to 96, wherein the modified CH3 domain comprises H or E at position 433.

98. The polypeptide of claim 97, wherein the modified CH3 domain comprises an H at position 433.

99. The polypeptide of any one of claims 59 to 98, wherein the modified CH3 domain comprises N or G at position 434.

100. The polypeptide of claim 99, wherein the modified CH3 domain comprises N at position 434.

101. The polypeptide of any one of claims 59 to 100, wherein the modified CH3 domain comprises at least one position selected from the group consisting of: e, N, F or Y at position 380, F at position 382, Y, S, A or 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.

102. The polypeptide of any one of claims 59 to 101, wherein the modified CH3 domain comprises five, six, seven or eight positions selected from the group consisting of: 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, N, T or S at position 389.

103. The polypeptide of any one of claims 59 to 102, wherein the modified CH3 domain comprises at least one position selected from the group consisting of: 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.

104. The polypeptide of any one of claims 59 to 103, wherein the modified CH3 domain comprises five positions selected from the group consisting of: l at position 422, a at position 424, E at position 426, Y at position 438, and L at position 440.

105. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises:

(i) Sequence AVX₁WFX₂X₃X₄X₅X₆X₇X₈N(SEQ ID NO:65), wherein X ₁ is E, N, F or Y; x ₂ is Y, S, A or absent; x ₃ is G, D, E or N; x ₄ is D, G, N or A; x ₅ is Q, S, G, A or N; x ₆ is K, I, R or G; x ₇ is E, L, D or Q; and X ₈ is N, T, S or R; and

(Ii) Sequence LFACEVMHEALX ₁X₂ HYTYKL (SEQ ID NO: 67), wherein X ₁ is H or E; and X ₂ is N or G.

106. The polypeptide of any one of claims 59 to 105, wherein the modified CH3 domain comprises 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).

107. The polypeptide of any one of claims 59 to 106, wherein the modified CH3 domain comprises sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64).

108. The polypeptide of any one of claims 59 to 107, wherein the modified CH3 domain comprises sequence AVEWFYDDSKLTN (SEQ ID NO: 58) and sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64).

109. The polypeptide of any one of claims 59 to 107, wherein the modified CH3 domain comprises sequence AVEWFYGNAKETN (SEQ ID NO: 59) and sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64).

110. The polypeptide of any one of claims 59 to 107, wherein the modified CH3 domain comprises sequence AVEWFYEAQKLNN (SEQ ID NO: 60) and sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64).

111. The polypeptide of any one of claims 59 to 107, wherein the modified CH3 domain comprises sequence AVEWFSEGSKETN (SEQ ID NO: 61) and sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64).

112. The polypeptide of any one of claims 59 to 107, wherein the modified CH3 domain comprises sequence AVEWFSGAQKESN (SEQ ID NO: 62) and sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64).

113. The polypeptide of any one of claims 59 to 107, wherein the modified CH3 domain comprises sequence AVEWFSGAQKLTN (SEQ ID NO: 63) and sequence LFACEVMHEALHNHYTYKL (SEQ ID NO: 64).

114. The polypeptide of any one of claims 59 to 113, wherein the modified CH3 domain further comprises one, two, three, four, or five amino acid substitutions at positions comprising 419-421, 442, and 443, wherein the positions are determined according to EU numbering.

115. The polypeptide of claim 114, wherein the modified CH3 domain comprises Q or P at position 419, G or R at position 420, N or G at position 421, S or G at position 442, and/or L or E at position 443.

116. The polypeptide of any one of claims 59 to 115, wherein 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.

117. The polypeptide of claim 116, wherein the modified CH3 domain comprises amino acids 111-217 of any one of SEQ ID NOs 72-77.

118. The polypeptide of any one of claims 59 to 117, wherein 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.

119. The polypeptide of claim 118, wherein the polypeptide comprises the sequence of any one of SEQ ID NOs 72-77.

120. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: f at location 382, Y at location 383, D at location 384, D at location 385, S at location 386, K at location 387, L at location 388, T at location 389, P at location 419, R at location 420, G at location 421, L at location 422, a at location 424, E at location 426, Y at location 438, L at location 440, G at location 442, and E at location 443,

Wherein the position is determined according to EU numbering.

121. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: f at location 382, Y at location 383, G at location 384, N at location 385, a at location 386, K at location 387, T at location 389, L at location 422, a at location 424, E at location 426, Y at location 438, L at location 440,

Wherein the position is determined according to EU numbering.

122. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: f at location 382, Y at location 383, E at location 384, a at location 385, K at location 387, L at location 388, L at location 422, a at location 424, E at location 426, Y at location 438, L at location 440,

Wherein the position is determined according to EU numbering.

123. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: f at location 382, E at location 384, S at location 386, K at location 387, T at location 389, L at location 422, a at location 424, E at location 426, Y at location 438, L at location 440,

Wherein the position is determined according to EU numbering.

124. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: f at location 382, G at location 384, a at location 385, K at location 387, S at location 389, L at location 422, a at location 424, E at location 426, Y at location 438, L at location 440,

Wherein the position is determined according to EU numbering.

125. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: f at location 382, G at location 384, a at location 385, K at location 387, L at location 388, T at location 389, L at location 422, a at location 424, E at location 426, Y at location 438, L at location 440,

Wherein the position is determined according to EU numbering.

126. A polypeptide comprising the sequence of any one of SEQ ID NOs 72, 78, 84, 90, 96, 102, 108, 114 and 120.

127. A polypeptide comprising the sequence of any one of SEQ ID NOs 73, 79, 85, 91, 97, 103, 109, 115 and 121.

128. A polypeptide comprising the sequence of any one of SEQ ID NOs 74, 80, 86, 92, 98, 104, 110, 116 and 122.

129. A polypeptide comprising the sequence of any one of SEQ ID NOs 75, 81, 87, 93, 99, 105, 111, 117 and 123.

130. A polypeptide comprising the sequence of any one of SEQ ID NOs 76, 82, 88, 94, 100, 106, 112, 118 and 124.

131. A polypeptide comprising the sequence of any one of SEQ ID NOs 77, 83, 89, 95, 101, 107, 113, 119 and 125.

132. An Fc polypeptide that specifically binds to TfR, comprising a modified CH3 domain, wherein the modified CH3 domain comprises a sequence that is at least 85% (e.g., at least 90%, 91%, 93%, 95%, 97%, 98%, or 99%) identical to amino acids 111-217 of sequence SEQ ID No. 137, wherein the modified CH3 domain comprises Ala, asp, his, tyr or Phe at position 378 according to EU numbering; ala, asp, phe, leu, gln, glu or Lys at position 380; gly at position 382; leu, ala, or Glu at position 384; val at location 385; gln or Ala at position 386; val, ile, phe or Leu at position 422; ser, ala or Pro at position 424; thr or Ile at position 426; ile or Tyr at position 438; and Gly, ser, thr or Val at location 440.

133. The Fc polypeptide of claim 132, wherein said modified CH3 domain has a Met or Leu at position 428.

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

135. The Fc polypeptide of any one of claims 132 to 134, wherein said modified CH3 domain comprises Ala or His at position 378 according to EU numbering; asp or Glu at position 380; gly at position 382; leu at position 384; val at location 385; gln or Ala at position 386; ile or Val at location 422; ala or Pro at position 424; thr or Ile at position 426; ile at position 438; and Gly or Thr at position 440.

136. The Fc polypeptide of claim 135, wherein said modified CH3 domain comprises His at position 378 according to EU numbering; glu at position 380; gly at position 382; leu at position 384; val at location 385; gln at position 386; ile at location 422; pro at position 424; ile at location 426; ile at position 438; and Thr at location 440.

137. The Fc polypeptide of claim 135, wherein said modified CH3 domain comprises His at position 378 according to EU numbering; glu at position 380; gly at position 382; leu at position 384; val at location 385; gln at position 386; ile at location 422; pro at position 424; ile at location 426; leu at location 428; ile at position 438; and Thr at location 440.

138. An Fc polypeptide that specifically binds to TfR, comprising a modified CH3 domain, wherein the modified CH3 domain comprises a sequence that is at least 85% (e.g., at least 90%, 91%, 93%, 95%, 97%, 98%, or 99%) identical to amino acids 111-217 of sequence SEQ ID No. 137, wherein the modified CH3 domain comprises His at position 378 according to EU numbering; glu at position 380; gly at position 382; leu at position 384; val at location 385; gln at position 386; ile at location 422; pro at position 424; ile at location 426; ile at position 438; and Thr at location 440.

139. The Fc polypeptide of claim 138, wherein said modified CH3 domain comprises Met or Leu at position 428.

140. An Fc polypeptide that specifically binds to TfR, comprising a modified CH3 domain, wherein the modified CH3 domain comprises a sequence that is at least 85% (e.g., at least 90%, 91%, 93%, 95%, 97%, 98%, or 99%) identical to amino acids 111-217 of sequence SEQ ID No. 138, wherein the modified CH3 domain comprises His at position 378 according to EU numbering; glu at position 380; gly at position 382; leu at position 384; val at location 385; gln at position 386; ile at location 422; pro at position 424; ile at location 426; leu at location 428; ile at position 438; and Thr at location 440.

141. The polypeptide of any one of claims 2 to 140, wherein the modified CH3 domain further comprises at least one modification that promotes heterodimerization.

142. The polypeptide of claim 141, wherein the at least one modification that promotes heterodimerization comprises a T366W substitution according to EU numbering.

143. The polypeptide of claim 141, wherein the at least one modification that promotes heterodimerization comprises T366S, L a and Y407V substitutions according to EU numbering.

144. The polypeptide of any one of claims 1 to 143, wherein the polypeptide further comprises L at position 428 and S at position 434.

145. The polypeptide of any one of claims 1 to 144, wherein the polypeptide further comprises a modified CH2 domain.

146. The polypeptide of claim 145, wherein the modified CH2 and CH3 domains form an Fc polypeptide.

147. The polypeptide of claim 145 or 146, wherein the modified CH2 domain comprises a modification that reduces effector function.

148. The polypeptide of claim 147, wherein the modification that reduces effector function comprises Ala at position 234 and Ala at position 235 according to EU numbering.

149. The polypeptide of claim 147 or 148, wherein said modified CH2 domain comprises Gly or Ser at position 329 according to EU numbering.

150. The polypeptide of claim 145 or 146, wherein the modified CH2 domain does not comprise a modification that reduces effector function.

151. The polypeptide of any one of claims 145 to 150, wherein the modified CH2 domain is a human IgG1, igG2, igG3, or IgG4 CH2 domain.

152. The polypeptide of any one of claims 1 to 151, wherein the polypeptide is part of a dimer.

153. The polypeptide of claim 152, wherein the dimer is an Fc dimer.

154. The polypeptide of any one of claims 1 to 153, wherein said polypeptide is further conjugated to a Fab.

155. The polypeptide of any one of claims 152 to 154, wherein the polypeptide is a first polypeptide of a dimer such that the dimer is monovalent for CD98hc binding.

156. The polypeptide of any one of claims 152 to 154, wherein the polypeptide is a first polypeptide of a dimer such that the dimer is bivalent for CD98hc binding.

157. The polypeptide of any one of claims 152-154, wherein said polypeptide is a first polypeptide of a dimer such that said dimer is monovalent for TfR binding.

158. The polypeptide of any one of claims 152-154, wherein said polypeptide is a first polypeptide of a dimer such that said dimer is bivalent for TfR binding.

159. The polypeptide of any one of claims 1-158, wherein the C-terminal lysine of the polypeptide is absent.

160. A polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of claims 1 to 159.

161. A vector comprising the polynucleotide of claim 160.

162. A host cell comprising the polynucleotide of claim 160.

163. A method for producing a polypeptide comprising a modified constant domain or a modified CH3 domain, the method comprising culturing a host cell under conditions that express a polypeptide encoded by the polynucleotide of claim 160.

164. A pharmaceutical composition comprising the polypeptide of any one of claims 1-159 and a pharmaceutically acceptable carrier.

165. A method of transcytosis of an endothelial cell, comprising contacting the endothelial cell with a composition comprising the polypeptide dimer of any of claims 152-158 fused to a therapeutic agent.

166. The method of claim 165, wherein the polypeptide dimer is capable of binding to CD98 hc.

167. The method of claim 165, wherein the polypeptide dimer is capable of binding to TfR.

168. The method of any one of claims 165-167, wherein the endothelial cell is the BBB.

169. A method for engineering a polypeptide comprising a modified CH3 domain to specifically bind to a CD98hc protein, the method comprising:

(a) Modifying a polynucleotide encoding the modified CH3 domain to comprise: (i) A first sequence comprising at least one substitution relative to sequence EWESNGQP (SEQ ID NO: 52); (ii) A second sequence comprising at least one substitution relative to sequence NVFSCSVM (SEQ ID NO: 53); and (iii) a third sequence comprising at least one substitution relative to sequence YTQKSLS (SEQ ID NO: 54);

(b) Expressing and recovering a polypeptide comprising the modified CH3 domain; and

(C) Determining whether said polypeptide binds to the CD98hc protein,

Wherein sequence SEQ ID NO:52 is from position 380 to position 387 of the Fc polypeptide, sequence SEQ ID NO:53 is from position 421 to position 428 of the Fc polypeptide, sequence SEQ ID NO:54 is from position 436 to position 442 of the Fc polypeptide, and said positions are determined according to EU numbering.

170. The method of claim 169, wherein the steps of expressing the polypeptide comprising the modified CH3 domain and determining whether the modified CH3 domain binds to CD98hc are performed using a display system.

171. A method for engineering a polypeptide comprising a modified CH3 domain to specifically bind to a TfR protein, the method comprising:

(a) Modifying a polynucleotide encoding the modified CH3 domain to comprise: (i) A first sequence comprising at least one amino acid substitution and/or deletion relative to sequence AVEWESNGQPENN (SEQ ID NO: 56); and (ii) a second sequence comprising at least one amino acid substitution in sequence VFSCSVMHEALHNHYTQK S (SEQ ID NO: 57);

(b) Expressing and recovering the polypeptide comprising the modified CH3 domain; and

(C) Determining whether said polypeptide binds to said TfR protein,

172. The method of claim 171, wherein the steps of expressing the polypeptide comprising the modified CH3 domain and determining whether the modified CH3 domain binds to the TfR protein are performed using a display system.

173. The method of claim 170 or 172, wherein 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.

174. A method of delivering a therapeutic agent across the BBB to the brain parenchyma, the method comprising contacting the BBB with a composition comprising the polypeptide dimer of any one of claims 152-158 fused to a therapeutic agent.

175. A method of delivering a therapeutic agent across a BBB to target an extracellular target, the method comprising contacting the BBB with a composition comprising the polypeptide dimer of any one of claims 152-158 fused to a therapeutic agent.

176. The method of claim 174 or 175, wherein one polypeptide of the polypeptide dimers comprises at least eleven, twelve, thirteen, fourteen or fifteen substitutions in a group 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.

177. The method of claim 174 or 175, wherein one polypeptide of the polypeptide dimers comprises at least eight, nine, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen substitutions in a 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.

178. The method of claim 176 or 193, wherein neither polypeptide of the polypeptide dimers has the substitution L234A, L a or P329G.

179. A method of delivering across the BBB to a biological target in the brain, the method comprising: (a) A CD98hc binding polypeptide according to any one of claims 1 to 58; and (b) means for binding the biological target in the brain.

180. The method of claim 179, wherein the biological target is a cell surface target in the brain.

181. The method of claim 180, wherein 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.

182. The method of claim 179, wherein the biological target is a cell surface target on a hematological cancer cell.

183. The method of claim 182, wherein 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.

184. The method of claim 179, wherein the biological target is located on a tumor cell.

185. The method of claim 184, wherein the biological target is selected from the group consisting of: ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD 66E), EGFR viii, ETBR, FGFR (1-4), folate receptor alpha, GAL-3BP (galectin binding protein), GD2, GD3, globohexacyl ceramide, gp100, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (15 containing leucine rich repeats), MET, naPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK 4; colon cancer kinase), RON, ROR1, TF (tissue factor) and TROP2.

186. The method of claim 179, wherein the biological target is an α -synuclein or derivative or fragment thereof, a β -amyloid peptide or derivative or fragment thereof, tau or derivative or fragment thereof, pTau, huntingtin, transthyretin, or TAR DNA binding protein 43 (TDP-43) or derivative or fragment thereof.

187. A method of targeting an extracellular target in the brain by a CD98hc binding polypeptide, the method comprising administering the CD98hc binding polypeptide to a patient, wherein the polypeptide is transported across the BBB and into the parenchyma, without endocytosis into cells within the brain.

188. The method of claim 187 wherein the extracellular target is on an astrocyte, microglial cell, oligodendrocyte or cancer cell.

189. The method of claim 187 or 188 wherein the extracellular target is an antigen in the brain.

190. The method of claim 189, wherein the antigen is a lysoplaque, tangle, or other non-cellular target.

191. The method of claim 187 wherein the extracellular target is a non-neuronal target.

192. The method of any one of claims 187-191 wherein the method comprises delivering a therapeutic agent to the extracellular target.

193. A method of delivering a therapeutic agent across the BBB to astrocytes, the method comprising contacting the BBB with a composition comprising the polypeptide dimer of any one of claims 152-158 fused to a therapeutic agent.

194. The method of claim 193, wherein both polypeptides in the polypeptide dimer comprise at least eleven, twelve, thirteen, fourteen or fifteen substitutions in a 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.

195. A method of delivering a therapeutic agent to a peripheral CD98 hc-expressing organ, the method comprising administering to a subject a composition comprising the polypeptide dimer of any one of claims 152-158 fused to a therapeutic agent.

196. The method of claim 195, wherein the peripheral CD98hc expression organ is a kidney, testis, bone marrow, spleen, or pancreas.

197. 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 the residues at positions selected from the group consisting of: 477, 478, 479, 480, 481, 482, 483, 486, 499, 497, 498, 500, 501 and 502 of SEQ ID NO. 144.

198. The CD98hc binding polypeptide of claim 197, wherein 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. 144.

199. The CD98hc binding polypeptide of claim 198, wherein when bound to human CD98hc, the polypeptide additionally binds to at least 1 additional residue at a position selected from the group consisting of: 229, 231, 232, 236, 235, 488, 495 and 496 of SEQ ID NO. 144.

200. The CD98hc binding polypeptide of claim 198, wherein when bound to human CD98hc, the polypeptide additionally binds to at least 1 additional residue at a position selected from the group consisting of: 312, 315, 348, 381, 439, 444, 443, 485, 484, 476, 475 and 442 of SEQ ID NO. 144.

201. 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 of residues at positions selected from the group consisting of: 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. 144.

202. 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 of residues at positions selected from the group consisting of: 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. 144.

203. The CD98hc binding polypeptide of any one of claims 197 to 202, wherein the polypeptide is an antibody or fragment thereof, a VHH domain or a polypeptide comprising a modified constant domain that specifically binds to CD98hc protein.

204. A method of increasing brain exposure of a subject to a therapeutic agent 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 20nM to about 550nM, wherein the molecule is linked to the therapeutic agent, and wherein the reference molecule comprises the therapeutic agent but does not comprise a CD98hc binding moiety.

205. A method of increasing brain exposure of a subject to a therapeutic agent 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 275nM to about 2100nM, wherein the molecule is linked to the therapeutic agent, and wherein the reference molecule comprises the therapeutic agent but no CD98hc binding moiety.

206. A composition for delivery across the BBB to a biological target in the brain, the composition comprising: (a) A CD98hc binding polypeptide according to any one of claims 1 to 58, and (b) a means for binding the biological target in the brain.

207. The composition of claim 206, wherein the biological target is a cell surface target in the brain.

208. The composition of claim 207, wherein 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.

209. The composition of claim 206, wherein the biological target is a cell surface target on a hematological cancer cell.

210. The composition of claim 209, wherein 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.

211. The composition of claim 206, wherein the biological target is located on a tumor cell.

212. The composition of claim 211, wherein the biological target is selected from the group consisting of: ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD 66E), EGFR viii, ETBR, FGFR (1-4), folate receptor alpha, GAL-3BP (galectin binding protein), GD2, GD3, globohexacyl ceramide, gp100, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (15 containing leucine rich repeats), MET, naPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK 4; colon cancer kinase), RON, ROR1, TF (tissue factor) and TROP2.

213. The composition of claim 206, wherein the biological target is α -synuclein or a derivative or fragment thereof, β -amyloid peptide or a derivative or 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.

214. A method for delivery across the BBB to a biological target in the brain of a subject, the method comprising:

(a) Providing a composition comprising: (i) A CD98hc binding polypeptide according to any one of claims 1 to 58, and (ii) means for binding to the biological target; and

(B) Peripherally administering the composition of step (a) to the subject.

215. 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 according to any one of claims 1 to 58, and (ii) means for binding to the biological target;

(b) Peripherally administering the composition of step (a) to the subject;

Wherein the composition binds the biological target in the brain of the subject.

216. A method of engineering a non-native binding site of a transferrin receptor (TfR) or CD98hc protein into a polypeptide, the method comprising:

(b) Contacting the library with a target protein;

(c) Selecting library members that bind to the target protein; and

217. The method of claim 216, wherein the method comprises repeating steps (b) - (d) using the library members isolated from first step (d).

218. The method of claim 216 or 217, wherein the library comprises at least 10 randomized positions.

219. The method of any one of claims 216 to 218, wherein the primary amino acid sequence of each polypeptide comprises positions of limited diversity separated by positions of no limited diversity.

220. The method of any one of claims 216 to 219, wherein each polypeptide comprises a β -sheet and at least three of the randomized positions are present in a single β -sheet.

221. The method of claim 220, wherein at least three of the randomized positions are present within at least two β -strands forming the β -sheet.

222. The method of claim 220 or 221, wherein at least three of said randomized positions are present within at least one β -strand forming said β -sheet.

223. The method of any one of claims 220 to 222 wherein at least three of the randomized positions form a surface on one side of the β -sheet.

224. The method of claim 223, wherein at least three of the randomized positions are surface exposed.

225. The method of any one of claims 220 to 224, wherein the β -sheet comprises at least one position having limited diversity.

226. The method of claim 225 wherein the β -sheet includes at least two positions having limited diversity.

227. The method of claim 226, wherein the at least two locations with limited diversity are separated by a location that does not have limited diversity.

228. The method of any one of claims 220 to 227, wherein the β -sheet comprises at least two positions that do not have limited diversity.

229. The method of claim 228, wherein the at least two locations that do not have limited diversity are separated by a location that has limited diversity.

230. The method of any one of claims 227 to 229, wherein said isolating is relative to the primary amino acid sequence of said polypeptide or relative to spatial three-dimensional localization of amino acids in the protein structure.

231. The method of any one of claims 216 to 230, wherein the positions with limited diversity are encoded by degenerate codons.

232. The method of claim 231, wherein at least one of the degenerate codons is NHK.

233. The method of claim 231 or 232, wherein the positions that do not have limited diversity are encoded by degenerate codon NNK.

234. The method of any one of claims 216 to 233, wherein the polypeptide comprises an immunoglobulin-like fold.

235. The method of claim 234, wherein the polypeptide comprises an immunoglobulin (IgG) domain.

236. The method of claim 235, wherein the IgG domain is from IgG, igA, igE, igM or IgD family.

237. The method of claim 236, wherein the IgG domain is selected from an IgG1, igG2, igG3 or IgG4 molecule.

238. The method of claim 237, wherein the IgG domain comprises a VH, CH1, CH2, CH3, VL, or CL domain.

239. The method of any one of claims 235-238, wherein the randomized positions are surface accessible.

240. The method of claim 238 or 239, wherein the randomized positions are selected from any of those listed in table 1B.

241. The method of claim 234, wherein the polypeptide comprises fibronectin.

242. A polypeptide having at least three modified positions in the β -sheet portion, wherein:

(Iii) The β -sheet does not bind to an antigen that does not have the modified position.

243. The polypeptide of claim 242, which has at least 4 or 5 modified positions in the β -sheet.

244. The polypeptide of claim 242 or 243, which has at least seven modified positions that form at least a portion of a binding site capable of binding CD98 hc.

245. The polypeptide of any one of claims 242 to 244, wherein the polypeptide comprises an immunoglobulin-like fold.

246. The polypeptide of claim 245, wherein the polypeptide comprises an immunoglobulin (IgG) domain.

247. The polypeptide of claim 246, wherein the IgG domain is from IgG, igA, igE, igM or IgD family.

248. The polypeptide of claim 247, wherein the IgG domain is selected from an IgG1, igG2, igG3, or IgG4 molecule.

249. The polypeptide of claim 248, wherein the IgG domain comprises a VH, CH1, CH2, CH3, VL, or CL domain.

250. The polypeptide of any one of claims 246 to 249, wherein the modified position is surface accessible.

251. The polypeptide of claim 249 or 250, wherein the modified positions are selected from any of those listed in table 1B.

252. The polypeptide of claim 245, wherein the polypeptide comprises fibronectin.

253. A polypeptide comprising a constant domain or a non-CDR portion of a variable domain of an immunoglobulin having at least three modified positions in a β -sheet, wherein:

254. The polypeptide of claim 253, wherein the constant domain comprises an Fc polypeptide.

255. The polypeptide of claim 253 or 254, wherein the at least two β -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, wherein the positions are determined according to EU numbering.

256. The polypeptide of claim 255, wherein the location is surface accessible.

257. The polypeptide of claim 256, wherein the positions are selected from those listed in table 1B.

258. The polypeptide of any one of claims 253 to 257, wherein the modified positions form a continuous surface on the β -sheet.

259. The polypeptide of any one of claims 253-258, wherein the modified positions are surface accessible residues.

260. The polypeptide of claim 259, wherein 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, wherein the positions are determined according to EU numbering.

261. The polypeptide of claim 259 or 260, wherein 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, wherein the positions are determined according to EU numbering.

262. The polypeptide of any one of claims 253-259, wherein the modified positions comprise three, four, five, six or seven amino acid substitutions in a set of amino acid positions comprising 380, 382, 383, 424, 426, 438 and 440, wherein the positions are determined according to EU numbering.

263. The polypeptide of any one of claims 253-259, wherein the modified positions comprise three, four, five, six, seven, eight, nine, ten, or eleven amino acid substitutions in a set of amino acid positions comprising 378, 380, 382, 383, 422, 424, 426, 428, 438, 440, and 442, wherein the positions are determined according to EU numbering.

264. The polypeptide of any one of claims 253 to 262, wherein the binding site includes one or more modified positions in at least one loop region.

265. The polypeptide of claim 264, wherein said one or more modified positions in at least one loop region are selected from the group consisting of: amino acid positions 387 and 422, wherein said positions are determined according to EU numbering.

266. The polypeptide of claim 264 or 265, wherein the loop region connects two β -strands.

267. A method of introducing a non-natural binding site of a TfR or CD98hc protein into a non-CDR region of a constant domain or variable domain of an immunoglobulin, the method comprising:

(b) Expressing the library to produce 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 the constant domain or non-CDR regions of the variable domain of the immunoglobulin.

268. The polypeptide of claim 267, wherein the immunoglobulin sequence comprises an Fc polypeptide.

269. The method of claim 267 or 268, wherein said at least two β -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.

270. The method of any one of claims 267-269, wherein the modified positions form a continuous surface on the β -sheet.

271. The method of any one of claims 267-270, wherein the modified position is a surface accessible residue.

272. The method of claim 271, wherein 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, wherein the positions are determined according to EU numbering.

273. The method of claim 271 or 272, wherein 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, wherein the positions are determined according to EU numbering.

274. The method of any one of claims 267-271, wherein the modified position comprises three, four, five, six, or seven amino acid substitutions in a set of amino acid positions comprising 380, 382, 383, 424, 426, 438, and 440, wherein the positions are determined according to EU numbering.

275. The method of any one of claims 267-271, wherein the modified position comprises three, four, five, six, seven, eight, nine, ten, or eleven amino acid substitutions in a set of amino acid positions comprising 378, 380, 382, 383, 422, 424, 426, 428, 438, 440, and 442, wherein the positions are determined according to EU numbering.

276. The method of any one of claims 267-275, wherein said binding site comprises one or more modified positions in at least one loop region.

277. The method of claim 276, wherein the one or more modified positions in at least one loop region are selected from the group consisting of: amino acid positions 387 and 422, wherein said positions are determined according to EU numbering.

278. A method of introducing a CD98hc binding site into a polypeptide comprising a β -sheet, the method comprising:

(b) Expressing the library to produce a library of sequence variants;

(c) Contacting said sequence variant with at least a portion of said CD98hc protein; and

(D) Isolating the sequence variant that binds to the Cd98hc protein.

279. The method of claim 278, wherein the polypeptide has at least seven modified positions in the β -sheet.

280. The method of claim 278 or 279, wherein the polypeptide comprises an immunoglobulin-like fold.

281. The method of claim 280, wherein the polypeptide comprises an immunoglobulin (IgG) domain.

282. The method of claim 281, wherein the IgG domain is from IgG, igA, igE, igM or IgD family.

283. The method of claim 282, wherein the IgG domain is selected from an IgG1, igG2, igG3, or IgG4 molecule.

284. The method of claim 283, wherein the IgG domain comprises a VH, CH1, CH2, CH3, VL, or CL domain.

285. The method of any one of claims 278-284, wherein the modified position in the β -sheet is surface accessible.

286. The method of claim 284 or 285, wherein the modified position is selected from any one of those listed in table 1B.

287. The method of claim 280, wherein the polypeptide comprises fibronectin.