Methods and compositions related to engineered transferrin receptor-binding molecules

Abstract

Provided herein are molecules that bind to a transferrin receptor (TfR), e.g., polypeptides (e.g., Fc polypeptides) including, for example, a modified CH3 domain as described herein, methods of generating such molecules, methods of using such molecules to transport agents across the blood brain barrier (BBB), and methods of using such molecules to treat a brain disease (e.g., a neurodegenerative disease).

Description

METHODS AND COMPOSITIONS RELATED TO ENGINEEREDTRANSFERRIN RECEPTOR-BINDING MOLECULESCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 387,895, filed on December 16, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes.BACKGROUND

[0002] Various techniques have been developed that engineer a protein to bind to a target that it does not normally bind. For example, libraries can be generated to screen for engineered proteins with desired binding or enzymatic activity.

[0003] Transferrin receptor (TfR) is a carrier protein for transferrin that, among other functions, is needed for the import of iron into the cell and is regulated in response to intracellular iron concentration. TfRs are expressed on endothelia, including the endothelium of the blood-brain barrier (BBB), and are expressed at increased levels on various cancer cells and inflammatory cells. It is one of the receptors that mediates transcytosis of cognate ligands across the BBB. TfRs can thus be desirable targets for introducing an agent into a cell for either endocytosis in the cell or transcytosis across the cell.

[0004] W02019033046 describes TfR-binding proteins with low binding affinities result in slower clearance. The present disclosure provides TfR-binding molecules with further lowered binding affinities and the surprising benefits of these molecules associated with transport across the BBB and therapeutic target engagement, which were unexpected from W02019033046.SUMMARY

[0005] The disclosure relates to methods and compositions related to transferrin receptor (TfR)-binding molecules. TfR-binding molecules described herein can be used in methods of transporting a therapeutic agent across the blood-brain barrier (BBB), as well as other methods such as methods of selecting a biological target or a therapeutic target. In other embodiments, the disclosure provides, in part, TfR-binding polypeptides with a binding affinty from about 2 pM to about 10 pM. In certain embodiments, such TfR-bindingpolypeptides have slower clearance, which leads to prolonged exposure, as compared to TfR- binding polypeptides with a higher binding affinity.

[0006] In one aspect, the disclosure provides a method of screening to identify a biological target for the treatment of a neurodegenerative disease, the method comprising: (a) selecting a biological target in the brain associated with a neurodegenerative disease; (b) providing a molecule comprising a biological target-binding moiety and a TfR-binding moiety; (c) testing whether the molecule comprising a biological target-binding moiety and a TfR-binding moiety has enhanced modulation of the biological target, as compared to a reference molecule comprising the biological target-binding moiety but lacking the TfR-binding moiety; and (d) selecting the biological target for treatment of the neurodegenerative disease based on whether the TfR-binding moiety enhances modulation of the target, wherein the biological target is not a TfR; and wherein the enhanced modulation is a greater modulating effect on the biological target as compared to the reference molecule.

[0007] In some embodiments, the molecule has a binding affinity to the TfR from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM).

[0008] In some embodiments of this aspect, the biological target is located on the cell surface of a brain cell selected from a microglial cell, an astrocyte, a neuron, and an oligodendrocyte. In particular embodiments, the cell surface receptor is located on a microglial cell. In certain embodiments, the cell surface receptor 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 Siglecl l.

[0009] In another aspect, the disclosure provides a method of screening to identify a biological target for the treatment of a cancer in the brain of a patient, the method comprising: (a) selecting a biological target selected from the group consisting of ALK, AXL, B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, CD79b, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL-3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, R0R1, TF (tissue factor), and TROP2; (b) providing a molecule comprising a biological target-binding moiety and a TfR-binding moiety; (c) testing whether the molecule comprising a biological target-binding moiety and a TfR-binding moiety has enhanced modulation of the biological target, as compared to a reference molecule comprising the biological target-binding moiety but lacking the TfR-binding moiety; and (d) selecting the biological target for treatment of cancer in the brain of the patient based on whether the TfR-binding moiety enhances modulation of the target; wherein the biological target is not a TfR; and wherein the enhanced modulation is a greater modulating effect on the biological target as compared to the reference molecule.

[0010] In some embodiments of this aspect, the cancer is glioblastoma or a metastatic cancer in the brain. In some embodiments, the molecule has a binding affinity to the TfR from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM).

[0011] In another aspect, the disclosure provides a method for optimizing the activity of a therapeutic molecule that binds to both a therapeutic target and a TfR, the method comprising: (a) selecting a therapeutic target in the brain associated with a neurodegenerative disease; (b) providing the therapeutic molecule comprising a therapeutic target-binding moiety and a TfR-binding moiety; (c) testing whether the therapeutic molecule enhances modulation and / or target engagement of the therapeutic target relative to a reference molecule comprising the the therapeutic target-binding moiety but lacking the TfR-binding moiety; (d) modifying the binding affinity of the therapeutic target-binding moiety and / or the TfR- binding moiety to achieve the desired acivity of the therapeutic molecule.

[0012] In some embodiments, the method comprises repeating steps (c) and (d) until the desired activity of the therapeutic molecule is achieved. In some embodiments, the reference molecule binds to the TfR with a higher binding affinity than that of the used molecule.

[0013] In another aspect, the disclosure provides a method of modulating a biological activity of a cell, the method comprising contacting the cell with a molecule that binds to: (i) a biological target that is expressed by the cell; and (ii) a transferrin receptor (TfR) with a binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM), in an amount sufficient to modulate at least one biological activity of the cell.

[0014] In another aspect, the disclosure provides a method of modulating a biological activity of a cell the method comprising: (a) providing a molecule that binds to: (i) a biological target that is expressed by the cell; and (ii) a transferrin receptor (TfR) with a binding affinity from about 2 pM to about 10 pM; and (b) contacting the cell with the molecule of step (a) in an amount sufficient to modulate at least one biological activity of the cell.

[0015] In some embodiments of this aspect, the cell is an in vitro assay. In other embodiments, the cell is in a subject, e.g., in the brain of the subject.

[0016] In further embodiments, the molecule has a greater modulating effect on the cell than a corresponding molecule that does not bind to the TfR or a corresponding molecule that binds to the TfR with a binding affinity stronger than 2 pM.

[0017] In some embodiments, the biological target is expressed on the cell surface of the cell. In certain embodiments, the cell also expresses a TfR. In certain embodiments, the biological target is not a TfR. In further embodiments, the cell is selected from the group consisting of a microglial cell, an astrocyte, a neuron, an oligodendrocyte, and a tumor cell.

[0018] In another aspect, the disclosure provides a method of modulating a biological target of a subject, the method comprising: (a) selecting a molecule that binds to a transferrin receptor (TfR) and to the biological target, wherein the molecule binds to the TfR with a binding affinity from about 2 pM to about 10 pM; and (b) peripherally administering the molecule to the subject in an effective amount to modulate the biological target, wherein the biological target is not a TfR. In certain embodiments, the biological target is in the brain of the subject.

[0019] In another aspect, the disclosure provides a method of screening to identify enhanced target engagement of a biological target in the brain of a model organism that expresses a TfR molecule, the method comprising: (a) administering to the model organism a molecule that binds a TfR and the biological target that is not a TfR; (b) measuring activity of the biological target; and (c) comparing the activity measured in step (b) with that of a reference molecule that binds to the biological target, wherein the reference molecule does not bind to the TfR, or binds to the TfR with a binding affinity that differs from the binding affinity of the molecule. In some embodiments, the target engagement of the biological target by the moleucle is enhanced relative to that of the reference molecule. In some embodiments, the molecule and the reference molecule have approximately equivalent brain exposure.

[0020] In another aspect, the disclosure provides a method of screening to identify enhanced target engagement of a biological target in the brain of a model organism that expresses a TfR molecule, the method comprising: (a) administering to the model organism a first molecule that bind a TfR and the biological target that is not a TfR; (b) measuring activity of the biological target; (c) repeating steps (a) and (b) using a second molecule that binds the TfR and the same biological target, wherein the first and second molecules bind to the TfR with different binding affinities; and (d) comparing the activities of the biological target measured using the first molecule and the second molecule. In some embodiments, the method further comprises repeating steps (a) and (b) using other molecules one at a time, wherein the other molecules bind to the TfR with different binding affinities and bind to the same biological target. In certain embodiments, the molecules that bind to the TfR have a TfR-binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM).

[0021] In some embodiments of this aspect, the first molecule and the second molecule have approximately equivalent brain exposure.

[0022] In some embodiments, the model organism expresses a TfR molecule that has a partially humanized or fully human extracellular domain.

[0023] In some embodiments, the biological target is a cell surface receptor.

[0024] In another aspect, the disclosure provides a method of transporting a therapeutic agent across the blood-brain barrier (BBB) of a subject, the method comprising: (a) selecting a molecule that binds to a transferrin receptor (TfR) of the subject with a binding affinity from about 2 pM to about 10 pM (c.g, from about 2 pM to about 9 pM, from 2 pM to about8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM) to attach to the therapeutic agent; and (b) peripherally administering to the subject the molecule attached to the therapeutic agent in a therapeutically effective amount.

[0025] In another aspect, the disclosure provides a method of transporting a therapeutic agent across the blood-brain barrier (BBB) of a subject, the method comprising peripherally administering to the subject a molecule comprising the therapeutic agent linked to a molecule that binds a transferrin receptor (TfR), wherein the molecule has been selected as having a binding affinity to the TfR from about 2 pM to about 10 pM (c.g, from about 2 pM to about9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM).

[0026] In some embodiments, the therapeutic agent is a polypeptide that binds to a cell surface receptor, wherein the cell surface receptor is located on a microglial cell or a cancer cell. In certain embodiments, the cell surface receptor is located on a microglial cell. In particular embodiments, the cell surface receptor 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 Siglecl l. In some embodiments, the cell surface receptor is located on a cancer cell. In particular embodiments, the cancer cell is a solid tumor cancer cell. In particular embodiments, the cell surface receptor is selected from the group consisting of ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL-3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, ROR1, TF (tissue factor), andTR0P2. In some embodiments, the the cancer cell is a hematological cancer cell. In particular embodiments, 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.

[0027] In another aspect, the disclosure provides a method of producing a therapeutic molecule that has optimized activity towards a therapeutic target, the method comprising: (a) providing a molecule that binds (i) a transferrin receptor (TfR) and (ii) the therapeutic target; (b) generating one or more variants of the molecule that have different binding affinities to the TfR; and (c) measuring at least one biological activity of the molecule that is associated with the therapeutic target to determine whether the changes in the TfR binding affinity of the molecule alter the biological activity of the molecule, thereby producing a therapeutic molecule with optimized activity against the therapeutic target. In some embodiments, the therapeutic target is expressed on the cell surface of a cell that also expresses a TfR. In particular embodiments, the cell is a microglial cell. In some embodiments, the cell is a cancer cell in the brain.

[0028] In some embodiments, the biological activity is measured in a cell or model organism, e.g., a mouse. In some embodiments, the mouse expresses a human TfR. In particular embodiments, the mouse is a huTfRapicalknock-in mouse.

[0029] In some embodiments, the portion of the molecule that binds to the TfR comprises: (a) antibody variable regions that bind to the TfR; or (b) a polypeptide that has been engineered to bind the TfR; or (c) a polypeptide comprising an immunoglobulin or a portion thereof; or (d) a fibronectin type III domain.

[0030] In another aspect, the disclosure provides a method of producing a polypeptide for transport across the blood-brain barrier (BBB) of a subject, the method comprising: (a) providing a polypeptide that binds to a transferrin receptor (TfR) with a binding affinity stronger than 2 pM; (b) making one or more amino acid substitutions, insertions, or deletions in the polypeptide, thereby creating one or more modified polypeptides, each having an altered binding affinity to the TfR; and (c) selecting a polypeptide of step (b) having a binding affinity to the TfR from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM), thereby producing the polypeptide for transport across the BBB.

[0031] In some embodiments, the polypeptide transports a therapeutic agent across the BBB. In certain embodiments, the polypeptide is a part of a dimer that is monovalent for TfR binding. In some embodiments of the method, the method further comprises producing a therapeutic molecule that comprises the polypeptide selected from step (c) and a therapeutic agent, wherein the polypeptide and the therapeutic agent are linked.

[0032] In another aspect, the disclosure provides a method of treating a human subject having a brain disease, the method comprising: (a) selecting a therapeutic molecule for the treatment of the brain disease by (i) its modulation of a biological target in the brain, wherein the biological target is not a transferrin receptor (TfR), and (ii) its binding to a TfR at an affinity of about 2 to about 10 pM; and (b) peripherally administering the therapeutic molecule to the human subject in a therapeutically effective amount to treat the brain disease. In some embodiments, the biological target is a cell surface receptor.

[0033] In another aspect, the disclosure provides a method of prolonging brain exposure to a therapeutic agent in a subject, the method comprising administering to the subject a molecule that binds to a transferrin receptor (TfR) with a binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM), wherein the molecule is linked to the therapeutic agent.

[0034] In some embodiments, the molecule prolongs brain exposure to the therapeutic agent as compared to a corresponding molecule that does not bind to the TfR or a corresponding molecule that binds to the TfR with a binding affinity stronger than 2 pM.

[0035] In another aspect, the disclosure provides a method of sustaining a modulating effect of a therapeutic agent in the brain of a subject, the method comprising administering to the subject a molecule that binds to a transferrin receptor (TfR) with a binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM toabout 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM), wherein the molecule is linked to the therapetic agent. In some embodiments, the molecule increases the duration of the modulating effect of the therapeutic agent as compared to a corresponding molecule that does not bind to the TfR or a corresponding molecule that binds to the TfR with a binding affinity stronger than 2 pM. In some embodiments, the therapeutic agent binds to a biological target and the modulating effect is a reduction in the level of the biological target.

[0036] In certain embodiments, the therapeutic agent is a known therapeutic agent. In particular embodiments, the known therapeutic agent binds to a therapeutic target selected from the group consisting of ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL-3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, ROR1, TF (tissue factor), and TROP2. In particular embodiments, the known therapeutic agent binds to a therapeutic target selected from the group consisting of B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b. In certain embodiments, the known therapeutic agent is selected from the group consisting of lorlatinib, crizotinib, cabozantinib, basiliximab, daclizumab, bivatuzumab, promiximab, lorvotuzumab, polatuzumab, tusamitamab, sunitinib, cetuximab, panitumumab, nimotuzumab, necitumumab, rindopepimut (CDX-110), amivantamab, pemigatinib, erdafitinib, STRO-002, bevacizumab, naxitamab, ipilimumab, tebentafusp, glembatumumab, margetuximab-cmkb, enhertu, trastuzumab, pertuzumab, patritumab, seribantumab, lumretuzumab, elgemtumab, U3-1402, AV-203, KTN3379, AVE1642, MK- 0646, cixutumumab, ladiratuzumab, gemtuzumab, pembrolizumab, sacituzumab, samrotamab, amivantamab-vmjw, TEPMETKO, lifastuzumab,177lutetium-PSMA-617, cofetuzumab, Zt / g4-MMAE, VLS-101, brexucabtagene, CS5001, tisotumab, sacituzumab, teclistamab, atezolizumab, avelumab, cosibelimab, durvalumab, belantamab, benralizumab, tafasitamab, loncastuximab, obinutuzumab, ofatumumab, rituximab, MEN1309 / OBT076, inotuzumab, and brentuximab.

[0037] 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 one of the following: (a) F at position 384 and S at position 413; (b) E atposition 380 and the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389; (c) E at position 380, N at position 386, and E at position 387; (d) E at position 380, E at position 387, and Y at position 421; (e) E at position 380, V at position 389, and T at position 413; or (f) Y at position 384, V at position 389, and S at position 413, wherein the positions are determined according to EU numbering.

[0038] In some embodiments of this aspect, the modified CH3 domain comprises F at position 384 and S at position 413. In some embodiments, the modified CH3 domain further comprises one or more (e.g., one, two, three, four, five, six, or seven) amino acid substitutions in a set of amino acid positions comprising 386-390, 416, and 421. In some embodiments, in addition to F at position 384 and S at position 413, the modified CH3 domain comprises (i) N or T at position 386; (ii) E at position 387; (iii) W at position 388; (iv) a polar amino acid (e.g., S or T) or V at position 389; (v) S or N at position 390; (vi) E at position 416; and / or (vii) an aromatic amino acid (e.g., F or Y) at position 421, according to EU numbering.

[0039] In some embodiments of this aspect, the modified CH3 domain comprises E at position 380 and the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389. In some embodiments, the modified CH3 domain further comprises one or more (e.g., one, two, three, four, five, or six) amino acid substitutions in a set of amino acid positions comprising 384, 390, 413, 415, 416, and 421. In certain embodiments, in addition to E at position 380 and the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389, the modified CH3 domain comprises: (i) Y at position 384; (ii) S or N at position 390; (iii) a polar amino acid (e.g., S or T) at position 413; (iv) S or E at position 415; (v) E at position 416; and / or (vi) F at position 421, according to EU numbering.

[0040] In some embodiments of this aspect, the modified CH3 domain comprises E at position 380, N at position 386, and E at position 387. In some embodiments, the modified CH3 domain further comprises one or more (e.g., one, two, three, four, five, six, seven, or eight) amino acid substitutions in a set of amino acid positions comprising 384, 388-390, 413, 415, 416, and 421. In certain embodiments, in addition to E at position 380, N at position 386, and E at position 387, the modified CH3 domain comprises: (i) Y at position 384; (ii) W at position 388; (iii) S at position 389; (iv) S or N at position 390; (v) a polar amino acid at position 413; (vi) S or E at position 415; (vii) E at position 416; and / or (viii) F at position 421, according to EU numbering.

[0041] In some embodiments, the modified CH3 domain comprises E at position 380, E at position 387, and Y at position 421. In some embodiments, the modified CH3 domain further comprises one or more (e.g., one, two, three, four, five, six, seven, or eight) amino acid substitutions in a set of amino acid positions comprising 384, 386, 388-390, 413, 415, and 416. In some embodiments, in additon to E at position 380, E at position 387, and Y at position 421, the modified CH3 domain comprises: (i) Y at position 384; (ii) T at position 386; (iii) W at position 388; (iv) S or V at position 389; (v) S or N at position 390; (vi) a polar amino acid at position 413; (vii) S or E at position 415; and / or (viii) E at position 416, according to EU numbering.

[0042] In some embodiments, the modified CH3 domain comprises E at position 380, V at position 389, and T at position 413. In some embodiments, the modified CH3 domain further comprises one or more (e.g., one, two, three, four, five, six, or seven) amino acid substitutions in a set of amino acid positions comprising 384, 386-388, 390, 416, and 421. In some embodiments, in addition to E at position 380, V at position 389, and T at position 413, the modified CH3 domain comprises: (i) Y at position 384; (ii) T at position 386; (iii) E at position 387; (iv) W at position 388; (v) N at position 390; (vi) E at position 416; and / or (vii) F at position 421, according to EU numbering.

[0043] In some embodiments, the modified CH3 domain comprises Y at position 384, V at position 389, and S at position 413. In some embodiments, the modified CH3 domain further comprises one or more (e.g., one, two, three, four, five, or six) amino acid substitutions in a set of amino acid positions comprising 386-388, 390, 416, and 421. In some embodiments, in addition to Y at position 384, V at position 389, and S at position 413, the modified CH3 domain comprises: (i) T at position 386; (ii) E at position 387; (iii) W at position 388; (vi) N at position 390; (v) E at position 416; and / or (vi) F at position 421, according to EU numbering.

[0044] 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:28-51 (e.g., SEQ ID NO:36 or 37) and one of the following: (a) F at position 384 and S at position 413; (b) E at position 380 and the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389; (c) E at position 380, N at position 386, and E at position 387; (d) E at position 380, E at position 387, and Y at position 421; (e) E at position 380, V at position 389, and T at position 413; or (f) Y at position 384, V at position389, and S at position 413, wherein the positions are determined according to EU numbering. In particular embodiments, the modified CH3 domain comprises amino acids 111-217 of any one of SEQ ID NOS:28-51 (e.g., SEQ ID NO:36 or 37).

[0045] In some embodiments of this aspect, the polypeptide comprises a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to a sequence of any one of SEQ ID NOS:28-51 (e.g., SEQ ID NO:36 or 37) and one of the following: (a) F at position 384 and S at position 413; (b) E at position 380 and the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389; (c) E at position 380, N at position 386, and E at position 387; (d) E at position 380, E at position 387, and Y at position 421; (e) E at position 380, V at position 389, and T at position 413; or (f) Y at position 384, V at position 389, and S at position 413, wherein the positions are determined according to EU numbering. In particular embodiments, the polypeptide comprises a sequence of any one of SEQ ID NOS:28-51 (e.g., SEQ ID NO:36 or 37).

[0046] 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: F at position 384, N or T at position 386, E at position 387, W at position388, a polar amino acid (e.g., S or T) or V at position 389, S or N at position 390, S at position 413, E at position 416, and an aromatic amino acid (e.g., F or Y) at position 421, wherein the positions are determined according to EU numbering. 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 SEQ ID NO: 68 and F at position 384, N or T at position 386, E at position 387, W at position 388, a polar amino acid (e.g., S or T) or V at position389, S or N at position 390, S at position 413, E at position 416, and an aromatic amino acid (e.g., F or Y) at position 421, wherein the positions are determined according to EU numbering. In particular embodiments, the polypeptide binds to the TfR with a binding affinity between about 1 pM and about 36 pM.

[0047] 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: E at position 380, Y at position 384, the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389, S or N at position 390, a polar amino acid (e.g., S or T) at position 413, S or E at position 415, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering. In some embodiments, thepolypeptide comprises a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 68 and E at position 380, Y at position 384, the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389, S or N at position 390, a polar amino acid (e.g., S or T) at position 413, S or E at position 415, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering. In particular embodiments, the polypeptide binds to the TfR with a binding affinity between about 3.5 pM and about 12 pM.

[0048] 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: E at position 380, Y at position 384, N at position 386, E at position 387, W at position 388, S at position 389, S or N at position 390, a polar amino acid at position 413, S or E at position 415, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering. 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 SEQ ID NO: 68 and E at position 380, Y at position 384, N at position 386, E at position 387, W at position 388, S at position 389, S or N at position 390, a polar amino acid at position 413, S or E at position 415, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering. In particular embodiments, the polypeptide binds to the TfR with a binding affinity between about 5 pM and about 19 pM.

[0049] 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: E at position 380, Y at position 384, T at position 386, E at position 387, W at position 388, S or V at position 389, S or N at position 390, a polar amino acid at position 413, S or E at position 415, E at position 416, and Y at position 421, wherein the positions are determined according to EU numbering. 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 SEQ ID NO: 68 and E at position 380, Y at position 384, T at position 386, E at position 387, W at position 388, S or V at position 389, S or N at position 390, a polar amino acid (e.g., S or T) at position 413, S or E at position 415, E at position 416, and Y at position 421, wherein the positions are determined according to EU numbering. In particular embodiments, the polypeptide binds to the TfR with a binding affinity between about 1 pM and about 15 pM.

[0050] 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: E at position 380, Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, N at position 390, and T at position 413, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering. 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 SEQ ID NO: 68 and E at position 380, Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, N at position 390, and T at position 413, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering. In particular embodiments, the polypeptide binds to the TfR with a binding affinity about 2.5 pM.

[0051] 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: Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, N at position 390, and S at position 413, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering. 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 SEQ ID NO: 68 and Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, N at position 390, and S at position 413, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering. In particular embodiments, the polypeptide binds to the TfR with a binding affinity about 3.6 pM.

[0052] In some embodiments of the aspects described herein, the modified CH3 domain further comprises at least one modification that promotes heterodimerization. In particular embodiments, the at least one modification that promotes heterodimerization comprises a T366W substitution, according to EU numbering. In certain embodiments, the at least one modification that promotes heterodimerization comprises T366S, L368A, and Y407V substitutions, according to EU numbering.

[0053] In some embodiments, the polypeptide further comprising L at position 428 and S at position 434.

[0054] In some embodiments, the polypeptide further comprises a CH2 domain.

[0055] In certain embodiments, the CH2 and CH3 domains form an Fc polypeptide.

[0056] In some embodiments, the CH2 domain comprises modifications that reduce effector function. In certain embodiments, the modifications that reduce effector function comprise Ala at position 234 and Ala at position 235, according to EU numbering. In certain embodiments, the CH2 domain comprises Gly or Ser at position 329, according to EU numbering.

[0057] In other embodiments, the CH2 domain does not comprise modifications that reduce effector function.

[0058] In some embodiments, the CH2 domain is derived from a human IgGl, IgG2, IgG3, or IgG4 CH2 domain.

[0059] In some embodiments of the aspects described herein, the polypeptide is part of a dimer. In certain embodiments, the dimer is an Fc dimer. In some embodiments, the polypeptide is a first polypeptide of a dimer such that the dimer is monovalent for TfR binding. In some embodiments, the polypeptide is a first polypeptide of a dimer such that the dimer is bivalent for TfR binding.

[0060] In certain embodiments, the polypeptide is further joined to a Fab.

[0061] In some embodiments, the C-terminal lysine of the polypeptide is absent.

[0062] In another aspect, the disclosure provides a polynucleotide comprising a nucleic acid sequence encoding the polypeptide described herein.

[0063] In another aspect, the disclosure provides a vector comprising the polynucleotide comprising a nucleic acid sequence encoding the polypeptide described herein.

[0064] In another aspect, the disclosure provides a host cell comprising the polynucleotide comprising a nucleic acid sequence encoding the polypeptide described herein.

[0065] In another aspect, the disclosure provides a method for producing a polypeptide comprising a modified CH3 domain, comprising culturing a host cell under conditions in which the polypeptide encoded by the polynucleotide described herein is expressed.

[0066] In another aspect, the disclosure provides a pharmaceutical composition comprising the polypeptide described herein and a pharmaceutically acceptable carrier.

[0067] In another aspect, the disclosure provides a method of transcytosis of a therapeutic agent across an endothelium, the method comprising contacting the endothelium with acomposition comprising a polypeptide described herein linked to the therapeutic agent. In particular embodiments, the endothelium is the blood-brain barrier (BBB).

[0068] In another aspect, the disclosure provides an improvement to a method of modulating a biological activity of a cell by providing a molecule that binds to the biological target that is expressed by the cell, wherein the biological target is not a TfR, the improvement comprising: (a) providing a molecule that binds to a TfR with a binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM); and (b) contacting the cell with the molecule of step (a) in an amount sufficient to modulate at least one biological activity of the cell.

[0069] In some embodiments, the cell is a microglial cell, an astrocyte, an oligodendrocyte, a neuron, and a tumor cell. In particular embodiments, the cell is a microglial cell. In particular embodiments, the cell surface receptor 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 Siglecl l. In some embodiments, the cell is a cancer cell, e.g., a solid tumor cancer cell. In particular embodiments, the cell surface receptor is selected from the group consisting of ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL-3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, ROR1, TF (tissue factor), and TROP2. In particular embodiments, the cancer cell is a hematological cancer cell. In certain embodiments, 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.

[0070] In another aspect, the disclosure provides an improvement to a method of selecting a biological target for treatment of a disease, wherein the biological target in the brain is located on a cell surface that also contains a TfR, the improvement comprising: (a) testingwhether a molecule comprising a biological target-binding moiety and a TfR-binding moiety has enhanced modulation of the biological target, as compared to a molecule comprising the biological target-binding moiety but lacking the TfR-binding moiety; and (b) selecting the biological target for treatment based on the enhanced modulation observed in step (a), wherein the disease is selected from the group consisting of Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and brain cancer; and wherein said treatment of disease is by the administration of a molecule comprising a TfR-binding moiety and target-binding moiety to said biological target.

[0071] In another aspect, the disclosure provides an improvement to a method of producing a therapeutic molecule that has optimized activity towards a known therapeutic target, the improvement comprising: (a) providing a molecule that binds (i) a transferrin receptor (TfR) and (ii) the known therapeutic target; (b) generating one or more variants of the molecule that have different binding affinities to the TfR; and (c) measuring at least one biological activity of the molecule that is associated with the known therapeutic target to determine whether the changes in the TfR binding affinity of the molecule alter the biological activity of the molecule, thereby producing a therapeutic molecule with optimized activity against the therapeutic target. In some embodiments, the known therapeutic target is selected from the group consisting of ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL- 3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, ROR1, TF (tissue factor), and TROP2. In certain embodiments, the known therapeutic target is selected from the group consisting of B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b.

[0072] In certain embodiments, the portion of the therapeutic molecule that binds the known therapeutic target is selected from the group consisting of lorlatinib, crizotinib, cabozantinib, basiliximab, daclizumab, bivatuzumab, promiximab, lorvotuzumab, polatuzumab, tusamitamab, sunitinib, cetuximab, panitumumab, nimotuzumab, necitumumab, rindopepimut (CDX-110), amivantamab, pemigatinib, erdafitinib, STRO-002, bevacizumab, naxitamab, ipilimumab, tebentafusp, glembatumumab, margetuximab-cmkb, enhertu, trastuzumab, pertuzumab, patritumab, seribantumab, lumretuzumab, elgemtumab, U3-1402, AV-203, KTN3379, AVE1642, MK-0646, cixutumumab, ladiratuzumab,gemtuzumab, pembrolizumab, sacituzumab, samrotamab, amivantamab-vmjw, TEPMETKO, lifastuzumab,177lutetium-PSMA-617, cofetuzumab, Zt / g4-MMAE, VLS-101, brexucabtagene, CS5001, tisotumab, sacituzumab, teclistamab, atezolizumab, avelumab, cosibelimab, durvalumab, belantamab, benralizumab, tafasitamab, loncastuximab, obinutuzumab, ofatumumab, rituximab, MEN1309 / OBT076, inotuzumab, brentuximab, and polatuzumab.BRIEF DESCRIPTION OF THE DRAWINGS

[0073] FIGS. 1A-1C: The plasma (FIG 1A), brain, (FIG. IB), and CSF (FIG. 1C) PK of the clones and control with a 50 mg / kg dose in huTfRapicalknock-in mice.

[0074] FIG. ID: The brain and plasma PK ratios of the clones and control.

[0075] FIG. IE: The brain PD evaluation of the clones and control with a 50 mg / kg dose in huTfRapicalknock-in mice.

[0076] FIGS. 2A and 2B: The effect of the clones on circulating reticulocytes (FIG. 2A) and red blood cells (FIG. 2B) with a 50 mg / kg dose in huTfRapicalknock-in mice.

[0077] FIG. 3A: The plasma PK of the clones and control at different dose levels in TREM2 BAC transgenic (tg) mouse model expressing human TREM2 in the CNS and periphery.

[0078] FIG. 3B: Target engagement (TE) of the clones in the brain.

[0079] FIG. 4A: Detection of antibody concentrations showed matched brain exposure of three clones with different TfR-binding affinities dosed at different levels TREM2 BAC transgenic (tg) mouse model expressing human TREM2 in the CNS and periphery.

[0080] FIG. 4B and 4C: The effect of the antibodies on CSF1R levels in the brain (FIG. 4B) and CSF (FIG. 4C) using TREM2 BAC transgenic (tg) mouse model expressing human TREM2 in the CNS and periphery.DETAILED DESCRIPTION OF THE DISCLOSUREI. INTRODUCTION

[0081] The disclosure provides various methods and compositions related to transferrin receptor (TfR)-binding molecules. We have developed methods for optimizing the activity of therapeutic molecules to bind to both a therapeutic target and also to the TfR, as well as usesin target discovery. In particular, we have discovered that the desired TfR-binding affinity for transporting a therapeutic agent across the blood-brain barrier (BBB) depends on the therapeutic target of the therapeutic agent, as well as the mechanism of action that drives efficacy in treating the disease.

[0082] In particular, it has been discovered that using TfR-binding molecules (e.g., TfR- binding polypeptides having a modified CH3 domain) that have relatively lower TfR-binding affinities result in slower clearance, which leads to prolonged exposure. For some therapies, achieving prolonged brain exposure of the therapeutic agent is desired over the dosing window in order to engage targets for longer periods of time. In some embodiments, for achieving prolonged or sustained brain exposure to a therapeutic agent, using TfR-binding molecules that have a TfR-binding affinity range of, e.g., from about 2 pM to about 10 pM, is particularly useful.II. DEFINITIONS

[0083] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an antibody” optionally includes a combination of two or more such molecules, and the like.

[0084] As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art, for example ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value.

[0085] 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:74. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee, accession number XP_003310238.1; rhesus monkey, NP_001244232.1; dog, NP-001003111.1; cattle, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP 990587.1). The term “transferrin receptor” also encompasses allelic variants of exemplary reference sequences, e.g., human sequences, that are encoded by a gene at a transferrin receptor protein 1 chromosomal locus. Full-length transferrin receptor protein includes a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a proteaselike domain, a helical domain, and an apical domain.

[0086] As used herein, the terms “CH3 domain” and “CH2 domain” refer to immunoglobulin constant region domain polypeptides. For purposes of this application, a CH3 domain polypeptide refers to the segment of amino acids from about position 341 to about position 447 as numbered according to the EU numbering scheme, and a CH2 domain polypeptide refers to the segment of amino acids from about position 231 to about position 340 as numbered according to the EU numbering scheme and does not include hinge region sequences. CH2 and CH3 domain polypeptides may also be numbered by the IMGT (ImMunoGeneTics) numbering scheme in which the CH2 domain numbering is 1-110 and the CH3 domain numbering is 1-107, according to the IMGT Scientific chart numbering (IMGT website). CH2 and CH3 domains are part of the Fc region of an immunoglobulin. An Fc region refers to the segment of amino acids from about position 231 to about position 447 as numbered according to the EU numbering scheme, but as used herein, can include at least a part of a hinge region of an antibody. An illustrative hinge region sequence is the human IgGl hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO:4).

[0087] As used herein, the terms “wild-type,” “native,” and “naturally occurring” as used with reference to a CH3 or CH2 domain, refer to a domain that has a sequence that occurs in nature.

[0088] As used herein, the term “mutant,” as used with reference to a mutant polypeptide or mutant polynucleotide, is used interchangeably with “variant.” A variant with respect to a given wild-type CH3 or CH2 domain reference sequence can include naturally occurring allelic variants. A “non-naturally” occurring CH3 or CH2 domain refers to a variant or mutant domain that is not present in a cell in nature and that is produced by genetic modification, e.g., using genetic engineering technology or mutagenesis techniques, of a native CH3 domain or CH2 domain polynucleotide or polypeptide. A “variant” includes any domain comprising at least one amino acid mutation with respect to wild-type. Mutations may include substitutions, insertions, and deletions.

[0089] As used herein, the term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate and O-phosphoserine. Naturally occurring a-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu),phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (He), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Vai), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of a naturally occurring a-amino acids include, without limitation, D- alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D- phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D- lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D- tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof. “Amino acid analogs” refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0090] As used herein, the terms “polypeptide” and “peptide” are used interchangeably to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L and D amino acids.

[0091] As used herein, the term “constant domain” refers to a domain in the constant region of an immunoglobulin molecule (e.g., CHI, CH2, CH3, CH4, Ckappa, Clambda).

[0092] As used herein, the term “modified constant domain” refers to a constant domain that has at least one mutation, e.g., a substitution, deletion or insertion, as compared to a wildtype immunoglobulin constant domain sequence, but retains the overall Ig fold or structure of the native constant domain.

[0093] As used herein, the term “Fc polypeptide” refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide that is characterized by an Ig fold as a structural domain. An Fc polypeptide contains constant region sequences including at least the CH2 domain and / or the CH3 domain and may contain at least part of the hinge region, but does not contain a variable region.

[0094] As used herein, the term “protein” refers to either a polypeptide or a dimer (i.e, two) or multimer (i.e., three or more) of single chain polypeptides. The single chain polypeptides of a protein may be joined by a covalent bond, e.g., a disulfide bond, or non-covalent interactions.

[0095] As used herein, the term “therapeutic agent” refers to any molecule, drug, or agent that is used in the treatment and / or prevention of a disease. A therapeutic agent can be an organic small molecule or compound, a polypeptide, a protein, a nucleic acid, and / or a combination of any of the above. In some embodiments, a therapeutic agent can be a known molecule, drug, or agent. In some embodiments, the therapeutic agent is a polypeptide containing an antigen-binding domain, e.g., an antibody variable domain polypeptide having one or more complimentarity determining regions (CDRs), or an antigen-binding fragment thereof. In particular embodiments, a therapeutic agent can be a Fab (e.g., a Fab that binds to a target that is not a TfR). In some embodiments, depending on the disease to be treated, a therapeutic agent can bind to a target (e.g., a biological target, a therapeutic target, a target that is not a TfR) to treat and / or prevent the disease.

[0096] As used herein, the term “therapeutic molecule” refers to a molecule comprising a therapeutic agent and a TfR-binding molecule (e.g., a TfR-binding polypeptide) described herein. In some embodiments, a therapeutic molecule binds to the ectodomain, i.e., the extracellular portion of a therapeutic target (e.g., a cell surface receptor). In certain embodiments, the therapeutic molecule modulates (e.g., increase or decrease) the level of the therapeutic target (e.g., a cell surface receptor).

[0097] As used herein, the term “therapeutic target” refers to a biological target whose activity can be modulated by a therapeutic agent in the treatment and / or prevention of a disease. In some embodiments, a therapeutic target can be a biological target implicated in a neurodegnerative disease, e.g., Alzheimer’s disease (AD). Examples of therapeutic targets are provided further herein. In some embodiments, a therapeutic target is a knowntherapeutic target implicated in a disease, e.g., a neurodegnerative disease (e.g., Alzheimer’s disease (AD)).

[0098] As used herein, the term “modulate” refers to changing or altering one or more properties of a protein (e.g., a therapeutic target, a biological target) or a cell. Properties of a cell can be altered as a result of altering one or more properties of a protein (e.g., a therapeutic target, a biological target) of the cell. In some embodiments, one or more properties of a protein can be modulated by a therapeutic agent binding to the protein. Properties of a protein that can be modulated include, but are not limited to, biological activities of the protein (e.g., protein signaling, ligand binding activities, interactions of the protein with other molecules or cells), half-life, and structure. Properties of a cell that can be modulated include, but are not limited to, cell growth, migration, survival, signaling, phagocytosis, and biomarker secretion.

[0099] As used herein, the term “enhanced target engagement” refers to the increase in binding of the biological target or therapeutic target by a molecule (e.g., a therapeutic molecule) that binds to a TfR and also to the target, relative to a reference molecule that binds to the target but not the TfR or binds to the TfR with a TfR-binding affinity that is higher than the used molecule. In certain embodiments, the target engagement by the molecule is increased by at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 5-fold, or more, as compared to the reference molecule.

[0100] As used herein, the terms “identical” or percent “identity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues, e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater, that are identical over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0101] For sequence comparison of polypeptides, typically one amino acid sequence acts as a reference sequence, to which a candidate sequence is compared. Alignment can be performed using various methods available to one of skill in the art, e.g., visual alignment or using publicly available software using known algorithms to achieve maximal alignment. Such programs include the BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters employed for an alignment toachieve maximal alignment can be determined by one of skill in the art. For sequence comparison of polypeptide sequences for purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two proteins sequence with the default parameters is used.

[0102] 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 described herein (or a target-binding portion thereof) and a target. Thus, for example, the term may refer to 1: 1 interactions between a Fab or scFv and an antigen or between a polypeptide described herein (or a target-binding portion thereof) and a target, unless otherwise indicated or clear from context. Binding affinity may be quantified by measuring an equilibrium dissociation constant (KD), which refers to the dissociation rate constant (kd, time'1) divided by the association rate constant (ka, time'1M'1). KD can be determined by measurement of the kinetics of complex formation and dissociation, e.g., using Surface Plasmon Resonance (SPR) methods, e.g., a Biacore™ system; kinetic exclusion assays such as KinExA®; and BioLayer interferometry (e.g., using the ForteBio® Octet platform). As used herein, “binding affinity” includes not only formal binding affinities, such as those reflecting 1 : 1 interactions between a Fab or scFv and an antigen or between a polypeptide described herein (or a target-binding portion thereof) and a target, but also apparent affinities for which KD’S are calculated that may reflect avid binding.

[0103] As used herein, the term “specifically binds” refers to a molecule (e.g., a Fab, an scFv, or a polypeptide described herein (or a target-binding portion thereof) that binds to an epitope or target with greater affinity, greater avidity, and / or greater duration to that epitope or target in a sample than it binds to another epitope or non-target compound (e.g., a structurally different antigen). In some embodiments, a Fab, scFv, or polypeptide described herein (or a target-binding portion thereof) that specifically binds to an epitope or target is a Fab, scFv, or polypeptide described herein (or a target-binding portion thereof) that binds to the epitope or target with at least 5-fold greater affinity than other epitopes or non-target compounds, e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 100-fold, 1000-fold, 10,000-fold, or greater affinity. The term “specific binding,” “specifically binds to,” or “is specific for” a particular epitope or target, as used herein, can be exhibited, for example, by a molecule having an equilibrium dissociation constant KD for the epitope or target to which it binds of, e.g., 10'4M or smaller, e.g., 10'5M, 10'6M, 10'7M, 10'8M, 10'9M, IO'10M, 10'11M, or 10'12M. It will be recognized by one of skill that a Fab or scFv thatspecifically binds to a target from one species may also specifically bind to orthologs of that target.

[0104] As used herein, the terms “subject,” “individual,” and “patient” are used interchangeably to refer to a mammal, 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.

[0105] As used herein, the terms “treatment,” “treating,” and the like generally mean obtaining a desired pharmacologic and / or physiologic effect. “Treating” or “treatment” may refer to any indicia of success in the treatment or amelioration of a neurodegenerative disease (e.g., Alzheimer’s disease or another neurodegenerative disease described herein), including any objective or subjective parameter such as abatement, remission, improvement in patient survival, increase in survival time or rate, diminishing of symptoms or making the disease more tolerable to the patient, slowing in the rate of degeneration or decline, or improving a patient’s physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment.

[0106] As used herein, the term “pharmaceutically acceptable excipient” refers to a nonactive pharmaceutical ingredient that is biologically or pharmacologically compatible for use in humans or animals, such as, but not limited to a buffer, carrier, or preservative.

[0107] As used herein, a “therapeutic amount” or “therapeutically effective amount” of an agent is an amount of the agent (e.g., any of the proteins described herein) that treats a disease in a subject.

[0108] As used herein, term “administer” refers to a method of delivering agents, compounds, or compositions to the desired site of biological action. These methods include, but are not limited to, topical delivery, oral delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, a protein as described herein is administered intravenously.III. METHODSMethods of Screening For a Biological Target

[0109] A TfR-binding molecule described herein can also be used to screen for a biological target as a therapeutic target for treating a brain disease. To screen for a biological target as the therapeutic target for treating a brain disease, a molecule that comprises a biological target-binding moiety and a TfR-binding moiety can be used. The molecule can be used to test whether there is enhanced modulation of the particular biological target, as compared to a reference molecule that comprises the biological target-binding moiety but lacking the TfR- binding moiety or a reference molecule that comprises the biological target-binding moiety and also a TfR-binding moiety, in which the TfR-binding moiety binds to the TfR with a different (e.g., higher) binding affinity relative to the used molecule. If the molecule showed enhanced modulation of the particular biological target, relative to the reference molecule, then the particular biological target can be chosen as the therapeutic target for treating a brain disease using a therapeutic molecule that binds both TfR and the particular biological target.

[0110] In some embodiments, enhanced modulation of the biological target when using a molecule having both the biological target-binding moiety and the TfR-binding moiety refers to changes in one or more activities of the biological target, relative to the activities of the biological target when using a molecule only having the biological target-binding moiety but not the TfR-binding moiety, in a manner such that it is beneficial in treating or preventing the disease (e.g., a brain disease). For example, when considering TREM2 as a therapeutic target, one of the activities of TREM2 that can be examined is TREM2-dependent pSyk signaling. To show enhanced modulation of TREM2-dependent pSyk signaling, a molecule having both the TREM2 -binding moiety and the TfR-binding moiety would show increased TREM2-dependent pSyk signaling relative to a molecule with only the TREM2-binding moiety. In some embodiments, the reference molecule can be a molecule that has a biological target-binding moiety and a TfR-binding moiety, in which the TfR-binding moiety has a different (e.g., higher) TfR-binding affinity compared to the tested molecule.[OHl] A number of different biological targets can be tested in this manner to screen for the biological target whose activity can be modulated by the molecule having both the biological target-binding moiety and the TfR-binding moiety (e.g., a TfR-binding moiety can be an Fc polypeptide that binds to the TfR as described herein). In some embodiments, a biological target is a therapeutic target implicated in a neurodegenerative disease. In someembodiments, the biological target is on the cell surface of a cell e.g., a brain cell). In some embodiments, the biological target is expressed on the cell surface of a cell (e.g., a brain cell) that also expresses a TfR. In some embodiments, the biological target is expressed on the cell surface of a cell e.g., a brain cell) that is adjacent to another cell that expresses a TfR. In particular embodiments, the biological target is not a TfR. In further embodiments, the biological target is a soluble target, e.g., a soluble target inside the brain. In some embodiments, the biological target is found near a cell that also expresses a TfR, for example, a biological target in the vasculature of the brain. In some embodiments, the biological target is expressed inside a cell that also expresses a TfR. In other embodiments, the biological target is expressed inside a cell that is adjacent to another cell that expresses a TfR.

[0112] In some embodiments, the cell is selected from the group consisting of a microglial cell, an astrocyte, an oligodendrocyte, and a neuron. In some embodiments, the biological target is on the surface of a microglial cell. In some embodiments, the cell is expressed by a cancer cell, e.g., a tumor cell e.g., a cell in a solid tumor) or a hematological cancer cell. In some embodiments, the biological target is on the surface of a tumor cell.

[0113] In some embodiments, the biological target is expressed by a microglial cell and examples of such biological targets include, but are not limited to, TREM2, PILRA, CD33, CR1, ABC Al, ABCA7, MS4A4A, MS4A6A, MS4A4E, HLA-DR5, HLA-DR1, IL1RAP, TREML2, IL-34, SORL1, ADAM17, and Siglecl l (see, e.g., Bellenguez et al., Nature Genetics, 54.412—436, 2022).

[0114] In some embodiments, the biological target is expressed by a tumor cell and examples of such biological targets include, but are not limited to, ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL-3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, ROR1, TF (tissue factor), and TROP2.

[0115] In some embodiments, the biological target is expressed by a hematological cancer cell and examples of such biological targets include, but are not limited to, B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b.

[0116] Methods for screening for a biological target for the treatment of a brain disease can include: (a) selecting a biological target in the brain; (a) testing whether a molecule comprising a biological target-binding moiety and TfR-binding moiety has enhanced modulation of the biological target, as compared to a reference molecule comprising the biological target-binding moiety but lacking the TfR-binding moiety or or a reference molecule that comprises the biological target-binding moiety and also a TfR-binding moiety, in which the TfR-binding moiety binds to the TfR with a different (e.g., higher) binding affinity relative to the used molecule; and (b) selecting the biological target based on whether the TfR-binding moiety enhances modulation of the target. In some embodiments, the method for screening for a biological target for the treatment of a brain disease is an in vitro cell based assay or is performed in a transgenic animal model. In some embodiments, detection and quantification of the biological target (e.g., a cell surface receptor) can be performed using FACS based cell binding (i.e., for surface detection). In other embodiments, detection and quantification of the biological target (e.g., a cell surface receptor) can also be performed using an orthogonal antibody that binds to the biological target (e.g., a cell surface receptor). In further embodiments, detection and quantification of the biological target (e.g., a cell surface receptor) can be performed using cell-based assays, i.e., for pSyk detection. In further embodiments, detection and quantification of the biological target (e.g., a cell surface receptor) can be performed using methods that detect and measure phosporylation, i.e., pSTAT3 (Y705) antibody based detection quantified by high content imaging.

[0117] In some embodiments, the reference molecule can also be a molecule that has a biological target-binding moiety and a TfR-binding moiety, in which the TfR-binding moiety has a different (e.g., higher) TfR-binding affinity compared to the tested molecule.

[0118] In further embodiments of the methods for screening for a biological target as a therapeutic target for treating a brain disease, the TfR-binding moiety in the molecule is a TfR-binding polypeptide as described herein, e.g., an Fc polypeptide with a modified CH3 domain. In some embodiments, the TfR-binding polypeptide binds to a TfR with a binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM).

[0119] The disclosure also provides a method of screening to identify a biological target for the treatment of a neurodegenerative disease, the method comprising: (a) selecting a biological target in the brain associated with a neurodegenerative disease; (b) providing a molecule comprising a biological target-binding moiety and a TfR-binding moiety; (c) testing whether the molecule comprising a biological target-binding moiety and a TfR-binding moiety has enhanced modulation of the biological target, as compared to a reference molecule comprising the biological target-binding moiety but lacking the TfR-binding moiety; and (d) selecting the biological target for treatment of the neurodegenerative disease based on whether the TfR-binding moiety enhances modulation of the target, wherein the biological target is not a TfR; and wherein the enhanced modulation is a greater modulating effect on the biological target as compared to the reference molecule.

[0120] In some embodiments, the molecule has a binding affinity to the TfR from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM).

[0121] In some embodiments of this aspect, the biological target is located on the cell surface of a brain cell selected from a microglial cell, an astrocyte, a neuron, and an oligodendrocyte. In particular embodiments, the cell surface receptor is located on a microglial cell. In certain embodiments, the cell surface receptor 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 Siglecl 1.

[0122] The disclosure provides a method of screening to identify a biological target for the treatment of a cancer in the brain of a patient, the method comprising: (a) selecting a biological target selected from the group consisting of ALK, AXL, B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, CD79b, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL-3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17,PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, ROR1, TF (tissue factor), and TROP2; (b) providing a molecule comprising a biological target-binding moiety and a TfR-binding moiety; (c) testing whether the molecule comprising a biological target-binding moiety and a TfR-binding moiety has enhanced modulation of the biological target, as compared to a reference molecule comprising the biological target-binding moiety but lacking the TfR-binding moiety; and (d) selecting the biological target for treatment of cancer in the brain of the patient based on whether the TfR-binding moiety enhances modulation of the target; wherein the biological target is not a TfR; and wherein the enhanced modulation is a greater modulating effect on the biological target as compared to the reference molecule.

[0123] In some embodiments of this aspect, the cancer is glioblastoma or a metastatic cancer in the brain. In some embodiments, the molecule has a binding affinity to the TfR from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM).Methods of Modulating a Biological Activity

[0124] A TfR-binding molecule can also be used to modulate a biological activity of a cell. In some embodiments of methods of modulating a biological activity of a cell, the methods use a molecule that can bind to a biological target expressed by the cell and also a TfR. In some embodiments, the TfR-binding moiety of the molecule binds to the TfR with a binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM). In some embodiments, the TfR-binding molecule is a TfR-binding polypeptide comprising a modified CH3 domain described herein.

[0125] Methods of modulating a biological activity of a cell can include contacting the cell with a molecule that binds to: (i) a biological target that is expressed by the cell; and (ii) a TfR (e.g., binding to the TfR with a binding affinity from about 2 pM to about 10 pM), in an amount sufficient to modulate at least one biological activity of the cell. In someembodiments, the molecule used in the methods of modulating a biological activity of a cell includes a TfR-binding polypeptide as described herein (e.g., an Fc polypeptide having a modified CH3 domain). By modulating the biological activity of a cell, one or more properties (e.g., cell growth, migration, survival, signaling, phagocytosis, biomarker secretion) of the cell can be changed or altered as a result of the molecule binding to the biological target of the cell. For example, in some embodiments, the biological target can be TREM2. By binding of the molecule to TREM2 and the TfR, TREM2 / DAP12 signaling and / or TREM2-dependent pSyk signaling can be modulated by the molecule. In the case of TREM2 / DAP12 signaling, the TREM2 / DAP12 signaling in turn modulates cellular activities such as phagocytosis, cell growth and survival, pro-inflammatory cytokine secretion, and the migration of cells such as microglia and macrophages. In further embodiments, the biological activity of a cell can be measured and quantified using cell-based assays, i.e., for pSyk detection. In further embodiments, the biological activity of a cell can be measured and quantified using methods that detect and measure phosporylation, i.e., pSTAT3 (Y705) antibody based detection quantified by high content imaging.

[0126] The disclosure includes methods of modulating a biological activity of a cell that include: (a) providing a molecule that binds to: (i) a biological target that is expressed by the cell; and (ii) a TfR (e.g., binding to TfR with a binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM)); and (b) contacting the cell with the molecule of step (a) in an amount sufficient to modulate at least one biological activity of the cell. In some embodiments, the molecule used in the methods has a greater modulating effect on the cell than a corresponding molecule that does not bind to the TfR or a corresponding molecule that binds to the TfR with a binding affinity stronger than 2 pM.

[0127] Also provided herein are methods for modulating a biological target of a subject by (a) selecting a molecule that binds to a TfR and to the biological target (e.g., a molecule that binds to the TfR with a binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM toabout 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM)); and (b) peripherally administering the molecule to the subject in an effective amount to modulate the biological target, wherein the biological target is not a TfR. In some embodiments, the biological target is in the brain of the subject.

[0128] In some embodiments of the methods of modulating a biological activity of a cell, the cell is in a subject, i.e., in the brain of the subject. In some embodiments, the biological target expressed by the cell is on the cell surface of the cell. In some embodiments, the biological target is expressed on the cell surface of a cell that also expresses a TfR. In some embodiments, the biological target is expressed on the cell surface of a cell that is adjacent to another cell that expresses a TfR. In particular embodiments, the biological target is not a TfR. In further embodiments, the biological target is a soluble target, e.g., a soluble target inside the brain. In some embodiments, the biological target is found near a cell that also expresses a TfR, for example, a biological target in the vasculature of the brain. In some embodiments, the biological target is expressed inside a cell that also expresses a TfR. In other embodiments, the biological target is expressed inside a cell that is adjacent to another cell that expresses a TfR. In some embodiments, the biological target is expressed by a brain cell, i.e., a brain cell of a subject. In some embodiments, the biological target is on the cell surface of the brain cell. In some embodiments, the cell is selected from the group consisting of a microglial cell, an astrocyte, an oligodendrocyte, a neuron, and a tumor cell. In further embodiments of the methods, the molecule that binds to the biological target and the TfR with a binding affinity from about 2 pM to about 10 pM is peripherally administered to the subject.Methods of Screening to Identify Enhanced Target Engagement

[0129] TfR-binding molecules described herein can also be used in methods for screening to identify enhanced target engagement of a biologicaly target (e.g., a cell surface receptor). In certain embodiments, the methods are performed in the brain of a model organism that expresses a TfR molecule. The method can include: (a) administering to the model organism a molecule that binds a TfR and a biological target (e.g., a cell surface receptor) that is not TfR; (b) measuring activity of the biological target; and (c) comparing the activity measured in step (b) with that of a reference molecule that binds to the biological target, wherein the reference molecule does not bind to the TfR, or binds to the TfR with a binding affinity that differs from the binding affinity of the molecule.

[0130] In some embodiments, the target engagement of the biological target (e.g., a cell surface receptor) by the moleucle is enhanced relative to that of the reference molecule. In certain embodiments, the target engagement by the molecule is increased by at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 5-fold, or more, as compared to that of the reference molecule. In some embodiments, target engagement of the biological target (e.g., a cell surface receptor) can be measured and quantified using FACS based cell binding (i.e., for surface detection). In other embodiments, target engagement of the biological target (e.g., a cell surface receptor) can be measured and quantified using an orthogonal antibody that binds to the biological target (e.g., a cell surface receptor). In further embodiments, target engagement of the biological target (e.g., a cell surface receptor) can be measured and quantified using cell-based assays, i.e., for pSyk detection. In further embodiments, target engagement of the biological target (e.g., a cell surface receptor) can be measured and quantified using methods that detect and measure phosporylation, i.e., pSTAT3 (Y705) antibody based detection quantified by high content imaging.

[0131] In certain embodiments, the molecule and the reference molecule have approximately equivalent brain exposure. In some embodiments, the molecule comprises a TfR-binding molecule linked to a biological target-binding moiety.

[0132] The methods of screening to identify enhanced target engagement of a biological target can also be used to select the appropriate or desired biological target. In some embodiments, the appropriate or desired biological target to serve as a therapeutic target in a method of treating a brain disease would demonstrate enhanced target engagement using a molecule comprising a TfR-binding molecule (e.g., a TfR-binding polypeptide comprising a modified CH3 domain) linked to a target-binding moiety, compared to the reference molecule, e.g., a reference molecule that does not bind to a TfR or binds to a TfR with a higher binding affinity. In certain embodiments, the TfR-binding molecule used in the methods of screening to identify enhanced target engagement is a TfR-binding polypeptide having a binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM).

[0133] In some embodiments, the biological target is not a TfR. In some embodiments, the biological target is in the brain of the subject. Multiple biological targets (e.g., cell surface receptors) can be tested or screened using the methods until one or more biological targets with enhanced target engagement using a molecule comprising a TfR-binding molecule and a target-binding moiety, relative to the reference molecule, is identified.

[0134] Also provided herein are methods of screening to identify enhanced target engagement of a biological target (e.g., a cell surface receptor) using molecules with different TfR-binding affinities. As shown herein, TfR-binding affinities can affect rates of clearance and duration of brain exposure, leading to different target engagements for different biological targets. Molecules with different TfR-binding affinities can be tested to find the molecule with the desirable TfR-binding affinitiy that leads to the most enhanced target engagement for a particular biological target. In some embodiments, the methods are performed in the brain of a model organism that expresses a TfR molecule. In some embodiments, the model organism expresses a TfR molecule that has a partially humanized or fully human extracellular domain. In some embodiments, the methods can include: (a) administering to the model organism a first molecule that bind a TfR and the biological target (e.g., a cell surface receptor) that is not a TfR; (b) measuring activity of the biological target; (c) repeating steps (a) and (b) using a second molecule that binds the TfR and the same biological target, wherein the first and second molecules bind to the TfR with different binding affinities; and (d) comparing the activities of the biological target measured using the first molecule and the second molecule. Further, the methods can include repeating steps (a) and (b) using other molecules one at a time, wherein the other molecules bind to the TfR with different binding affinities and bind to the same biological target.

[0135] In some embodiments, the TfR-binding moiety of the tested molecules have a TfR- binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM). In some embodiments, the TfR-binding moiety of the tested molecules is a TfR-binding polypeptide comprising a modified CH3 domain described herein.

[0136] In some embodiments of the methods, the first molecule and the second molecule have approximately equivalent brain exposure.Methods of Transporting a Therapeutic Agent Across the BBB

[0137] TfR-binding molecules (e.g., TfR-binding polypeptides) can also be used to transport a therapeutic agent across the blood-brain barrier (BBB) of a subject. In some embodiments, the therapeutic agent can be linked (i.e., covalently attached) to the N-terminal or C-terminal region of the TfR-binding molecule (e.g., TfR-binding polypeptides), or linked to any region of the molecule, so long as the therapeutic agent does not interfere with binding of the TfR-binding molecule (e.g., TfR-binding polypeptides) to the TfR. In some embodiments, the therapeutic agent is a polypeptide containing an antigen-binding domain, e.g., an antibody variable region having one or more complimentarity determining regions (CDRs), or an antigen-binding fragment thereof. In some embodiments, the therapeutic agent is a Fab. In some embodiments, the therapeutic agent can be a cytotoxic agent, a DNA or RNA molecule, an antisense oligonucloetide, a chemical moiety, and the like. In some embodiments, the therapeutic agent can bind to a therapeutic target in the subject (e.g., a therapeutic target in the brain of the subject). In some embodiments, the therapeutic target is a cell surface receptor. In some embodiments, the therapeutic target is a cell surface receptor of a cell that also expresses a TfR. In some embodiments, the therapeutic target is a cell surface receptor of a cell that is adjacent to another cell that expresses a TfR. In particular embodiments, the therapeutic target is not a TfR.

[0138] In further embodiments, the therapeutic target is a soluble target, e.g., a soluble target inside the brain. In some embodiments, the therapeutic target is found near a cell that also expresses a TfR, for example, a therapeutic target in the vasculature of the brain. In some embodiments, the therapeutic target is expressed inside a cell that also expresses a TfR. In other embodiments, the therapeutic target is expressed inside a cell that is adjacent to another cell that expresses a TfR.

[0139] In some embodiments, methods of transporting a therapeutic agent across the BBB of a subject can include (a) selecting a molecule that binds to a TfR of the subject with a binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM,from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM) to attach to the therapeutic agent; and (b) peripherally administering to the subject the molecule attached to the therapeutic agent in a therapeutically effective amount.

[0140] The disclosure also provides methods of transporting a therapeutic agent across the BBB of a subject by peripherally administering to the subject a molecule comprising the therapeutic agent linked (i.e.g, covalently attached) to a TfR-binding molecule (e.g., a TfR- binding polypeptide), in which the TfR-binding molecule (e.g., a TfR-binding polypeptide) has been selected as having a binding affinity to the TfR from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM).Methods of Treatment

[0141] Also provided herein are methods for treating a human subject having a brain disease by using a therapeutic molecule that comprises a TfR-binding polypeptide described herein. In some embodiments, the methods comprise: (a) selecting a therapeutic molecule for the treatment of the brain disease by (i) its modulation of a therapeutic target in the brain, wherein the therapeutic target is not a transferrin receptor (TfR), and (ii) its binding to a TfR (e.g, with a TfR-binding affinity of about 2 to about 10 pM (e.g, from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM)); and (b) peripherally administering the therapeutic molecule to the human subject in a therapeutically effective amount to treat the brain disease. In some embodiments, a therapeutically effective amount is a concentration that is sufficient to treat one or more signs or symptoms of the brain disease (e.g., a neurodegenerative disease).

[0142] Also provided herein are methods for treating a human subject having a brain disease by peripherally administering a therapeutic molecule to the human subject in a therapeutically effective amount to treat the brain disease, wherein the therapeutic molecule (i) modulates a therapeutic target in the brain, wherein the therapeutic target is not atransferrin receptor (TfR), and (ii) binds to a TfR (e.g., with a TfR-binding affinity of about 2 to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM)).

[0143] In some embodiments, the therapeutic molecules bind the ectodomain, i.e., the extracellular portion of a therapeutic target (e.g., a cell surface receptor). In certain embodiments, the therapeutic molecules modulate (e.g., increase or decrease) the level of the therapeutic target (e.g., a cell surface receptor). In some embodiments, the therapeutic molecules modulate the activity of a therapeutic target (e.g., a cell surface receptor). For example, an increase in ITAM activity can be detected via pSyk measurement (e.g., as described in Schlepckow et al., EMBO Mol Med el 1227, 2020). In another example, a decrease in ITIM activity can be detected via cell based pSTAT3 signaling.

[0144] In some embodiments, the therapeutic molecule comprises a moiety that binds to the therapeutic target and a moiety that binds to the TfR (e.g., a TfR-binding polypeptide described herein, e.g., a polypeptide that binds to a TfR with a binding affinity from about 2 to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM).

[0145] In some embodiments, the therapeutic molecules comprises a TfR-binding molecule (e.g., a TfR-binding polypeptide comprising a modified CH3 domain described herein, e.g., a polypeptide that binds to a TfR with a binding affinity from about 2 to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM)) covalently linked to a therapeutic agent.

[0146] The activity of the therapeutic molecule can also be optimized in order to choose the most optimal therapeutic molecule for the methods described herein. The disclosure alsoprovides a method for optimizing the activity of a therapeutic molecule that binds to both a therapeutic target and a TfR, the method comprising: (a) selecting a therapeutic target in the brain associated with a neurodegenerative disease; (b) providing the therapeutic molecule comprising a therapeutic target-binding moiety and a TfR-binding moiety; (c) testing whether the therapeutic molecule enhances modulation and / or target engagement of the therapeutic target relative to a reference molecule comprising the the therapeutic target-binding moiety but lacking the TfR-binding moiety; (d) modifying the binding affinity of the therapeutic target-binding moiety and / or the TfR-binding moiety to achieve the desired acivity of the therapeutic molecule. In some embodiments, the method comprises repeating steps (c) and (d) until the desired activity of the therapeutic molecule is achieved. In some embodiments, the reference molecule binds to the TfR with a higher binding affinity than that of the used molecule.

[0147] In some embodiments, the brain disease is a neurodegenerative disease. Nonlimiting examples of suitable neurodegenerative diseases include Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and a combination thereof. In some embodiments, the therapeutic target is implicated in a neurodegenerative disease. In some embodiments, the brain disease is cancer. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is a metastatic cancer in the brain.

[0148] In some embodiments, the therapeutic target is on the cell surface of a cell (e.g., a brain cell). In some embodiments, the therapeutic target is expressed on the cell surface of a cell (e.g., a brain cell) that also expresses a TfR. In some embodiments, the therapeutic target is expressed on the cell surface of a cell (e.g., a brain cell) that is adjacent to another cell that expresses a TfR. In particular embodiments, the therapeutic target is not a TfR. In further embodiments, the therapeutic target is a soluble target, e.g., a soluble target inside the brain. In some embodiments, the therapeutic target is found near a cell that also expresses a TfR, for example, a therapeutic target in the vasculature of the brain. In some embodiments, the therapeutic target is expressed inside a cell that also expresses a TfR. In other embodiments, the therapeutic target is expressed inside a cell that is adjacent to another cell that expresses a TfR.

[0149] In some embodiments, the cell is selected from the group consisting of a microglial cell, an astrocyte, an oligodendrocyte, and a neuron. In some embodiments, the therapeutic target is on the surface of a microglial cell. In some embodiments, the cell is expressed by acancer cell, e.g., a tumor cell e.g., a cell in a solid tumor) or a hematological cancer cell. In some embodiments, the therapeutic target is on the surface of a tumor cell.

[0150] In some embodiments, the therapeutic target is expressed by a microglial cell and examples of such therapeutic targets include, but are not limited to, TREM2, PILRA, CD33, CR1, ABC Al, ABCA7, MS4A4A, MS4A6A, MS4A4E, HLA-DR5, HLA-DR1, IL1RAP, TREML2, IL-34, SORL1, ADAM17, and Siglecl l (see, e.g., Bellenguez et al., Nature Genetics, 54:412-436, 2022).

[0151] In some embodiments, the therapeutic target is expressed by a tumor cell and examples of such therapeutic targets include, but are not limited to, ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL-3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, ROR1, TF (tissue factor), and TROP2.

[0152] In some embodiments, the therapeutic target is expressed by a hematological cancer cell and examples of such therapeutic targets include, but are not limited to, B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b.

[0153] In some embodiments of the methods of treatment described herein, TfR-binding polypeptides described herein (e.g., polypeptides that bind to TfR with a binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM)) can be used to prolong brain exposure to a theraueptic agent in a subject and / or to sustain a modulating effect of a therapeutic agent. Methods for prolonging brain exposure to a therapeutic agent in a subject can include administering to the subject a molecule that binds to a TfR with a binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM toabout 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM), wherein the molecule is linked (e.g, covalently attached) to the therapeutic agent. In some embodiments, the molecule prolongs brain exposure to the therapeutic agent as compared to a corresponding molecule that does not bind to the TfR or a corresponding molecule that binds to the TfR with a binding affinity stronger than 2 pM.

[0154] In some embodiments, brain exposure is quantified by plotting brain exposure (e.g., concentration of the therapeutic agent in the brain) as a function of time and calculating the area under the curve (AUC). Increased AUC can represent increased or prolonged brain exposure. In some embodiments of the methods, duration of brain exposure to the therapeutic agent at a therapeutically effective concentration is increased.

[0155] In some embodiments, the TfR-binding moiety of the therapeutic molecule prolongs brain exposure to the therapeutic agent at a therapeutically effective concentration (e.g., a concentration that is sufficient to treat one or more signs or symptoms of a neurodegenerative disease) in the mammal as compared to the therapeutic agent linked to a reference molecule, e.g., a reference molecule that does not bind to TfR or binds to the TfR with a stronger affinity.

[0156] In some embodiments, brain exposure (e.g, to the therapeutic agent) is prolonged by at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3- fold, 5-fold, or more, as compared to a reference molecule, e.g., a reference molecule that does not bind to TfR or binds to the TfR with a stronger affinity.

[0157] For evaluation of brain and / or plasma concentration or exposure of the therapeutic agent (e.g., a therapeutic agent linked to a TfR-binding molecule (e.g., a TfR-binding polypeptide), the linked therapeutic agent can be administered to a mouse model e.g., hTfRapical+ / +). Plasma samples can be obtained from the mouse after a suitable period of time, followed by perfusion of the vasuclar system with a suitable solution. Following perfusion, brains (or portions thereof) can be extracted and homogenized and lysed. Concentrations of the therapeutic agent in the plasma and / or brain lysate can then be determined using standard methods that will be known to one of ordinary skill in the art. By administering a range of doses to the knock-in mouse model, a standard curve can be generated. By administering to the knock-in mouse model a therapeutic agent linked to different TfR-binding polypeptides (e.g., having different TfR-binding affinities), comparisons can be made regarding the effectsof the TfR-binding polypeptides on brain exposure to the therapeutic agent and / or Cmax values of the therapeutic agent in the brain.

[0158] Also provided herein are methods of sustaining a modulating effect of a therapeutic agent in the brain of a subject by administering to the subject a molecule that binds to a transferrin receptor (TfR) with a binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM), wherein the molecule is linked (e.g., covalently attached) to the therapeutic agent.

[0159] In certain embodiments, the molecule prolongs brain exposure to the therapeutic agent and / or sustains a modulating effect of the therapeutic agent as compared to a corresponding molecule that does not bind to the TfR or a corresponding molecule that binds to the TfR with a binding affinity stronger than 2 pM. In certain cases, the molecule increases the duration of the modulating effect of the therapeutic agent as compared to a corresponding molecule that does not bind to the TfR or a corresponding molecule that binds to the TfR with a binding affinity stronger than 2 pM. In further embodiments, the therapeutic agent binds to a therapeutic target (e.g., a therapeutic target expressed on the cell surface of a cell, e.g., a brain cell) and the modulating effect is a change (e.g., increase or decrease) in the level of the therapeutic target.

[0160] In some embodiments of all methods described herein, the therapeutic agent is a known therapeutic agent. In some embodiments, the therapeutic target or biological target is a known therapeutic target or known biological target. In some embodiments, the therapeutic agent is a known therapeutic agent that binds to a known therapeutic target.

[0161] In some embodiments, the known therapeutic agent binds to a therapeutic target selected from the group consisting of ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL-3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, ROR1, TF (tissue factor), andTR0P2. In some embodiments, the known therapeutic agent binds to a therapeutic target selected from the group consisting of B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b.

[0162] In some embodiments, the known therapeutic agent is selected from the group consisting of lorlatinib, crizotinib, cabozantinib, basiliximab, daclizumab, bivatuzumab, promiximab, lorvotuzumab, polatuzumab, tusamitamab, sunitinib, cetuximab, panitumumab, nimotuzumab, necitumumab, rindopepimut (CDX-110), amivantamab, pemigatinib, erdafitinib, STRO-002, bevacizumab, naxitamab, ipilimumab, tebentafusp, glembatumumab, margetuximab-cmkb, enhertu, trastuzumab, pertuzumab, patritumab, seribantumab, lumretuzumab, elgemtumab, U3-1402, AV-203, KTN3379, AVE1642, MK-0646, cixutumumab, ladiratuzumab, gemtuzumab, pembrolizumab, sacituzumab, samrotamab, amivantamab-vmjw, TEPMETKO, lifastuzumab,177lutetium-PSMA-617, cofetuzumab, Zt / g4-MMAE, VLS-101, brexucabtagene, CS5001, tisotumab, sacituzumab, teclistamab, atezolizumab, avelumab, cosibelimab, durvalumab, belantamab, benralizumab, tafasitamab, loncastuximab, obinutuzumab, ofatumumab, rituximab, MEN1309 / OBT076, inotuzumab, brentuximab, and polatuzumab.

[0163] Also provided herein are improvements to a method of modulating a biological activity of a cell by providing a molecule that binds to the biological target that is expressed by the cell, wherein the biological target is not a TfR, the improvement comprising: (a) providing a molecule that binds to a TfR with a binding affinity from about 2 pM to about 10 pM; and (b) contacting the cell with the molecule of step (a) in an amount sufficient to modulate at least one biological activity of the cell. In certain embodiments, the cell is a microglial cell, an astrocyte, an oligodendrocyte, a neuron, and a tumor cell. In some embodiments, the cell is a microglial cell and the cell surface receptor 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 Siglecl 1. In some embodiments, the cell is a solid tumor cancer cell and the cell surface receptor is selected from the group consisting of ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL-3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, ROR1, TF (tissue factor), andTR0P2. 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.

[0164] Further provided herein are improvements to a method of selecting a biological target for treatment of a disease, wherein the biological target in the brain is located on a cell surface that also contains a TfR, the improvement comprising: (a) testing whether a molecule comprising a biological target-binding moiety and a TfR-binding moiety has enhanced modulation of the biological target, as compared to a molecule comprising the biological target-binding moiety but lacking the TfR-binding moiety; and (b) selecting the biological target for treatment based on the enhanced modulation observed in step (a), wherein the disease is selected from the group consisting of Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and brain cancer; and wherein said treatment of disease is by the administration of a molecule comprising a TfR-binding moiety and target-binding moiety to said biological target.

[0165] Further provided herein are improvements to a method of producing a therapeutic molecule that has optimized activity towards a known therapeutic target, the improvement comprising: (a) providing a molecule that binds (i) a transferrin receptor (TfR) and (ii) the known therapeutic target; (b) generating one or more variants of the molecule that have different binding affinities to the TfR; and (c) measuring at least one biological activity of the molecule that is associated with the known therapeutic target to determine whether the changes in the TfR binding affinity of the molecule alter the biological activity of the molecule, thereby producing a therapeutic molecule with optimized activity against the therapeutic target. In some embodiments, the known therapeutic target is selected from the group consisting of ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL- 3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, ROR1, TF (tissue factor), and TROP2. In some embodiments, the known therapeutic target is selected from the group consisting of B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b. In certain embodiments, the portion of the therapeutic molecule that binds the known therapeutic target is selected from the group consisting of lorlatinib, crizotinib, cabozantinib,basiliximab, daclizumab, bivatuzumab, promiximab, lorvotuzumab, polatuzumab, tusamitamab, sunitinib, cetuximab, panitumumab, nimotuzumab, necitumumab, rindopepimut (CDX-110), amivantamab, pemigatinib, erdafitinib, STRO-002, bevacizumab, naxitamab, ipilimumab, tebentafusp, glembatumumab, margetuximab-cmkb, enhertu, trastuzumab, pertuzumab, patritumab, seribantumab, lumretuzumab, elgemtumab, U3-1402, AV-203, KTN3379, AVE1642, MK-0646, cixutumumab, ladiratuzumab, gemtuzumab, pembrolizumab, sacituzumab, samrotamab, amivantamab-vmjw, TEPMETKO, lifastuzumab,177lutetium-PSMA-617, cofetuzumab, Zt / g4-MMAE, VLS-101, brexucabtagene, CS5001, tisotumab, sacituzumab, teclistamab, atezolizumab, avelumab, cosibelimab, durvalumab, belantamab, benralizumab, tafasitamab, loncastuximab, obinutuzumab, ofatumumab, rituximab, MEN1309 / OBT076, inotuzumab, brentuximab, and polatuzumab.Methods of Engineering and Producing

[0166] The disclosure also provides methods for producing a TfR-binding polypeptide (e.g., an Fc polypeptide with a modified CH3 domain) with a desirable binding affinity by making one or more amino acid substitutions, insertions, or deletions in a TfR-binding polypeptide with a binding affinity that is outside of the desirable range. Methods for producing a polypeptide for transporting a therapeutic agent across the BBB of a subject can include: (a) providing a polypeptide that binds to a TfR with a binding affinity stronger than 2 pM; (b) making one or more amino acid substitutions, insertions, or deletions in the polypeptide, thereby creating one or more modified polypeptides, each having an altered binding affinity to the TfR; and (c) selecting a polypeptide of step (b) having a binding affinity to the TfR from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM) to therefore produce the polypeptide for transporting the therapeutic agent across the BBB. In certain embodiments, the polypeptide is a part of a dimer that is monovalent for TfR binding. In some embodiments, once the polypeptide is selected from step (c), the polypeptide can be linked (e.g., covalently attached) to a therapeutic agent to produce a therapeutic molecule that can be used to transport the therapeutic agent across the BBB of the subject. In particular embodiments, the therapeutic molecule can be peripherally administered to the subject.

[0167] Also provided herein are methods of producing a therapeutic molecule that has optimized activity towards a therapeutic target. The methods can include (a) providing a molecule that binds (i) a TfR and (ii) the therapeutic target; (b) generating one or more variants of the molecule that have different binding affinities to the TfR; and (c) measuring at least one biological activity of the molecule that is associated with the therapeutic target to determine whether the changes in the TfR binding affinity of the molecule alter the biological activity of the molecule, thereby producing a therapeutic molecule with optimized activity against the therapeutic target. In some embodiments, the therapeutic target is expressed on the cell surface of a cell. In some embodiments, the therapeutic target is expressed on the cell surface of a cell that also expresses a TfR. In some embodiments, the therapeutic target is expressed on the cell surface of a cell that is adjacent to another cell that expresses a TfR. In particular embodiments, the therapeutic target is not a TfR. In further embodiments, the therapeutic target is a soluble target, e.g., a soluble target inside the brain. In some embodiments, the therapeutic target is found near a cell that also expresses a TfR, for example, a therapeutic target in the vasculature of the brain. In some embodiments, the therapeutic target is expressed inside a cell that also expresses a TfR. In other embodiments, the therapeutic target is expressed inside a cell that is adjacent to another cell that expresses a TfR.

[0168] In certain embodiments of the methods, the biological activity is measured in a cell or model organism (e.g., a mouse, a mouse that expresses a human TfR, a huTfRapicalknock- in mouse). In some cases, the portion of the molecule that binds to the TfR can include: (a) antibody variable regions that bind to the TfR; (b) a polypeptide that has been engineered to bind the TfR; or (c) a polypeptide comprising an immunoglobulin or a portion thereof; or (d) a fibronectin type III domain. In some embodiments, the portion of the molecule that binds to the TfR can include a TfR-binding polypeptide described herein (e.g., a polypeptide that binds to TfR with a binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM).

[0169] In a further aspect, methods of engineering a modified CH3 domain to bind TfR are provided. In some embodiments, modification of a CH3 domain comprises substitutingvarious amino acids relative to amino acids 111-217 of the sequence of SEQ ID NO: 68 or to amino acids 111-217 of the sequence of SEQ ID NO: 1. In some embodiments, the method comprises modifying a polynucleotide that encodes the modified CH3 domain polypeptide to incorporate amino acid changes, relative to amino acids 111-217 of the sequence of SEQ ID NO: 68 or to amino acids 111-217 of the sequence of SEQ ID NO: 1.

[0170] In some embodiments of engineering polypeptides to bind TfR, the method comprises modifying a polynucleotide that encodes the modified CH3 domain comprising a sequence having one of the following options: (a) F at position 384 and S at position 413; (b) E at position 380 and the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389; (c) E at position 380, N at position 386, and E at position 387; (d) E at position 380, E at position 387, and Y at position 421; (e) E at position 380, V at position 389, and T at position 413; or (f) Y at position 384, V at position 389, and S at position 413, wherein the positions are determined according to EU numbering. In some embodiments, the method further comprises expressing and recovering a polypeptide comprising the modified CH3 domain; and determining whether the polypeptide binds to TfR.

[0171] The amino acids introduced into the desired positions may be generated by randomization or partial randomization to generate a library of CH3 domain polypeptides with amino acid substitutions at the 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 part of, or all of, a full hinge region.

[0172] A polypeptide of the present disclosure (e.g., an Fc polypeptide comprising a modified CH3 domain described herein) may be expressed using any number of systems. For example, in some embodiments, polypeptides are expressed in a display system. In other illustrative embodiments, mutant polypeptides are expressed as soluble polypeptides that are 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 polysomal display system. The library is screened using known methodology to identify TfR binders, which may be further characterized to determine binding kinetics. Additional mutations may then be introduced into selected clones.

[0173] Methods for analyzing binding affinity, binding kinetics, and cross-reactivity are known in the art. These methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assay), immunoprecipitation, surface plasmon resonance (e.g., Biacore™ (GEHealthcare, Piscataway, NJ)), kinetic exclusion assays (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), BioLayer interferometry (e.g., Octet® (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to determine binding affinity and / or cross-reactivity. Methods for performing ELISA assays are known in the art. 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 crossreactivity. In some embodiments, BioLayer interferometry assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity.IV. TRANSFERRIN RECEPTOR-BINDING MOLECULES

[0174] This section describes molecules that bind to a transferrin receptor (TfR). In some embodiments, the molecules bind to a primate TfR. In some embodiments, the primate TfR is a human TfR. In some embodiments, the molecules bind to a TfR apical domain. These molecules are capable of being transported across the blood-brain barrier (BBB).Modified CH3 Domains That Bind TfR

[0175] A TfR-binding molecule can be a TfR-binding polypeptide. In some embodiments, a TfR-binding polypeptide (e.g., an Fc polypeptide) of the present disclosure may include, for example, a modified CH3 domain as described herein. As described herein, when describing a polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain comprising amino acids 111-217 of certain SEQ ID NO(S), or a modified CH3 domain comprising amino acid substitutions and / or deletions relative to amino acids 111-217 of certain SEQ ID NO(S), or a modified CH3 domain comprising a sequence having a percent identity to amino acids 111-217 of certain SEQ ID NO(S), such descriptions are directed to the sequence of the CH3 domain, and are not to be construed as limiting the polypeptide to contain amino acids 1-113 of the recited SEQ ID NO(S). In some embodiments, the polypeptide binds to a primate TfR. In some embodiments, the primate TfR is a human TfR. In some embodiments, the polypeptide binds to a TfR apical domain.

[0176] One of skill understands that the CH3 domains of other immunoglobulin isotypes, e.g., IgM, IgA, IgE, IgD, etc. may be similarly modified by identifying the amino acids in those domains that correspond to the amino acid substitutions at the positions described herein. Modifications may also be made to corresponding domains from immunoglobulinsfrom other species, e.g., non-human primates, monkey, mouse, rat, or other non-human mammals.TfR-Binding Site Modifications

[0177] 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 one of the following options: (a) F at position 384 and S at position 413; (b) E at position 380 and the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389; (c) E at position 380, N at position 386, and E at position 387; (d) E at position 380, E at position 387, and Y at position 421; (e) E at position 380, V at position 389, and T at position 413; or (f) Y at position 384, V at position 389, and S at position 413, wherein the positions are determined according to EU numbering.

[0178] In some embodiments, the polypeptides described herein bind to a TfR with a binding affinity from about 2 pM to about 10 pM (e.g., from about 2 pM to about 9 pM, from 2 pM to about 8 pM, from 2 pM to about 7 pM, from 2 pM to about 6 pM, from 2 pM to about 5 pM, from 2 pM to about 4 pM, from 2 pM to about 3 pM, from 3 pM to about 10 pM, from 4 pM to about 10 pM, from 5 pM to about 10 pM, from 6 pM to about 10 pM, from 7 pM to about 10 pM, from 8 pM to about 10 pM, or from 9 pM to about 10 pM).

[0179] 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 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 SEQ ID NO:68 and one of the following options: (a) F at position 384 and S at position 413; (b) E at position 380 and the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389; (c) E at position 380, N at position 386, and E at position 387; (d) E at position 380, E at position 387, and Y at position 421; (e) E at position 380, V at position 389, and T at position 413; or (f) Y at position 384, V at position 389, and S at position 413, wherein the positions are determined according to EU numbering.Modified CH 3 Domains with F at Position 384 and S at Position 413

[0180] Provided herein are polypeptides that comprise a modified CH3 domain having F at position 384 and S at position 413, according to EU numbering. In addition to F at position 384 and S at position 413, the modified CH3 domain can further comprise one or more (e.g., one, two, three, four, five, six, or seven) amino acid substitutions in a set of amino acidpositions comprising 386-390, 416, and 421. In addition to F at position 384 and S at position 413, the modified CH3 domain can further comprise one or more (e.g., one, two, three, four, five, six, or seven) amino acid substitutions in a set of amino acid positions comprising 386-390, 416, and 421, such as (i) N or T at position 386; (ii) E at position 387; (iii) W at position 388; (iv) a polar amino acid or V at position 389; (v) S or N at position 390; (vi) E at position 416; and / or (vii) an aromatic amino acid at position 421. In some embodiments, the polypeptides bind to the TfR with a binding affinity between about 1 pM and about 36 pM (e.g., between about 1 pM and about 30 pM, between about 1 pM and about 20 pM, between about 1 pM and about 10 pM, between about 1 pM and about 5 pM, between about 1 pM and about 3 pM, between about 5 pM and about 36 pM, between about 10 pM and about 36 pM, between about 20 pM and about 36 pM, or between about 30 pM and about 36 pM).

[0181] In some embodiments, the modified CH3 domain comprises N or T at position 386, in addition to F at position 384 and S at position 413. In some embodiments, the modified CH3 domain comprises E at position 387, in addition to F at position 384 and S at position 413. In some embodiments, the modified CH3 domain comprises W at position 388, in addition to F at position 384 and S at position 413. In some embodiments, the modified CH3 domain comprises a polar amino acid (e.g., S or T) or V at position 389, in addition to F at position 384 and S at position 413. In some embodiments, the modified CH3 domain comprises S or N at position 390, in addition to F at position 384 and S at position 413. In some embodiments, the modified CH3 domain comprises E at position 416, in addition to F at position 384 and S at position 413. In some embodiments, the modified CH3 domain comprises an aromatic amino acid (e.g., F or Y) at position 421, in addition to F at position 384 and S at position 413.

[0182] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising F at position 384; N or T at position 386; E at position 387; W at position 388; a polar amino acid (e.g, S or T) or V at position 389; S or N at position 390; S at position 413; E at position 416; and an aromatic amino acid (e.g, F or Y) at position 421. In particular embodiments, the polypeptides comprise a modified CH3 domain comprising a sequence having at least 85% identity, at least 90% identity, or at least 95% identity (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identity) to amino acids 111-217 of any one of SEQ ID NOS:31, 39, and 43-46 and F at position 384; N or T at position 386; E at position 387; W at position388; a polar amino acid (e.g., S or T) or V at position 389; S or N at position 390; S at position 413; E at position 416; and an aromatic amino acid (e.g., F or Y) at position 421.

[0183] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:31 and F at position 384; T at position 386; E at position 387; W at position 388; S at position 389; S at position 390; S at position 413; E at position 416; and F at position 421.

[0184] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:39 and F at position 384; T at position 386; E at position 387; W at position 388; S at position 389; N at position 390; S at position 413; E at position 416; and F at position 421.

[0185] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:43 and F at position 384; N at position 386; E at position 387; W at position 388; S at position 389; N at position 390; S at position 413; E at position 416; and F at position 421.

[0186] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:44 and F at position 384; T at position 386; E at position 387; W at position 388; T at position 389; N at position 390; S at position 413; E at position 416; and F at position 421.

[0187] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:45 and F at position 384; T at position 386; E at position 387; W at position 388; S at position 389; N at position 390; S at position 413; E at position 416; and Y at position 421.

[0188] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQID NO:46 and F at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; S at position 413; E at position 416; and Y at position 421.

[0189] The disclosure provides polypeptides comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR). In some embodiments, the polypeptide 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 SEQ ID NO:31 and F at position 384; T at position 386; E at position 387; W at position 388; S at position 389; S at position 390; S at position 413; E at position 416; and F at position 421, according to EU numbering.

[0190] In some embodiments, the polypeptide 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 SEQ ID NO:39 and F at position 384; T at position 386; E at position 387; W at position 388; S at position 389; N at position 390; S at position 413; E at position 416; and F at position 421, according to EU numbering.

[0191] In some embodiments, the polypeptide 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 SEQ ID NO:43 and F at position 384; N at position 386; E at position 387; W at position 388; S at position 389; N at position 390; S at position 413; E at position 416; and F at position 421, according to EU numbering.

[0192] In some embodiments, the polypeptide 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 SEQ ID NO:44 and F at position 384; T at position 386; E at position 387; W at position 388; T at position 389; N at position 390; S at position 413; E at position 416; and F at position 421, according to EU numbering.

[0193] In some embodiments, the polypeptide 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 SEQ ID NO:45 and F at position 384; T at position 386; E at position 387; W at position 388; S at position 389; N at position 390; S at position 413; E at position 416; and Y at position 421, according to EU numbering.

[0194] In some embodiments, the polypeptide comprises a sequence having at least 85% identity, at least 90% identity, or at least 95% identity (e.g., 95%, 96%, 97%, 98%, 99%, or100% identity) to SEQ ID NO:46 and F at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; S at position 413; E at position 416; and Y at position 421, according to EU numbering.Modified CH 3 Domains with E at Position 380 and the sequence of TEWT (SEQ ID NO:73) from Position 386 to Position 389

[0195] Provided herein are polypeptides that comprise a modified CH3 domain having E at position 380 and the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389, according to EU numbering. In addition to E at position 380 and the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389, the modified CH3 domain can further comprise one or more (e.g., one, two, three, four, five, or six) amino acid substitutions in a set of amino acid positions comprising 384, 390, 413, 415, 416, and 421, such as (i) Y at position 384; (ii) S or N at position 390; (iii) a polar amino acid (e.g., S or T) at position 413; (iv) S or E at position 415; (v) E at position 416; and / or (vi) F at position 421, according to EU numbering. In some embodiments, the polypeptides bind to the TfR with a binding affinity between about 3.5 pM and about 12 pM (e.g., between about 3.5 pM and about 10 pM, between about 3.5 pM and about 5 pM, between about 5 pM and about 12 pM, or between about 10 pM and about 12 pM).

[0196] In some embodiments, in addition to E at position 380 and the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389, the modified CH3 domain comprises Y at position 384. In some embodiments, the modified CH3 domain comprises S or N at position 390. In some embodiments, the modified CH3 domain comprises a polar amino acid (e.g., S or T) at position 413. In some embodiments, the modified CH3 domain comprises S or E at position 415. In some embodiments, the modified CH3 domain comprises E at position 416. In some embodiments, the modified CH3 domain comprises F at position 421.

[0197] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising E at position 380; Y at position 384; the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389; S or N at position 390; a polar amino acid (e.g., S or T) at position 413; S or E at position 415; E at position 416; and F at position 421. In particular embodiments, the polypeptides comprise a modified CH3 domain comprising a sequence having at least 85% identity, at least 90% identity, or at least 95% identity (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identity) to amino acids 111-217 of any one of SEQ ID NOS:29, 33, 36, 41, and 50 and E at position 380; Y at position 384; the sequence of TEWT (SEQ IDNO:73) from position 386 to position 389; S or N at position 390; a polar amino acid (e.g., S or T) at position 413; S or E at position 415; E at position 416; and F at position 421.

[0198] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:29 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; S at position 390; T at position 413; S at position 415; E at position 416; and F at position 421.

[0199] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:33 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; S at position 390; S at position 413; S at position 415; E at position 416; and F at position 421.

[0200] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:36 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; N at position 390; T at position 413; S at position 415; E at position 416; and F at position 421.

[0201] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:41 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; N at position 390; S at position 413; S at position 415; E at position 416; and F at position 421.

[0202] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:50 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; N at position 390; S at position 413; E at position 415; E at position 416; and F at position 421.

[0203] In some embodiments, the polypeptide 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 SEQ ID NO:29 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; S at position 390; T at position 413; S at position 415; E at position 416; and F at position 421, according to EU numbering.

[0204] In some embodiments, the polypeptide 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 SEQ ID NO:33 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; S at position 390; S at position 413; S at position 415; E at position 416; and F at position 421, according to EU numbering.

[0205] In some embodiments, the polypeptide 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 SEQ ID NO:36 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; N at position 390; T at position 413; S at position 415; E at position 416; and F at position 421, according to EU numbering.

[0206] In some embodiments, the polypeptide 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 SEQ ID NO:41 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; N at position 390; S at position 413; S at position 415; E at position 416; and F at position 421, according to EU numbering.

[0207] In some embodiments, the polypeptide 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 SEQ ID NO:50 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; N at position 390; S at position 413; E at position 415; E at position 416; and F at position 421, according to EU numbering.Modified CH 3 Domains with E at Position 380, N at Position 386, and E at Position 387

[0208] Provided herein are polypeptides that comprise a modified CH3 domain having E at position 380, N at position 386, and E at position 387, according to EU numbering. In addition to E at position 380, N at position 386, and E at position 387, the modified CH3 domain can further comprise one or more (e.g., one, two, three, four, five, six, seven, or eight) amino acid substitutions in a set of amino acid positions comprising 384, 388-390, 413,415, 416, and 421, such as (i) Y at position 384; (ii) W at position 388; (iii) S at position 389; (iv) S or N at position 390; (v) a polar amino acid (e.g., S or T) at position 413; (vi) S or E at position 415; (vii) E at position 416; and / or (viii) F at position 421, according to EU numbering. In some embodiments, the polypeptides bind to the TfR with a binding affinity between about 5 pM and about 19 pM (e.g., between about 5 pM and about 15 pM, between about 5 pM and about 10 pM, between about 5 pM and about 8 pM, between about 8 pM and about 19 pM, between about 10 pM and about 19 pM, or between about 15 pM and about 19 pM).

[0209] In some embodiments, in addition to E at position 380, N at position 386, and E at position 387, the modified CH3 domain comprises Y at position 384. In some embodiments, the modified CH3 domain comprises W at position 388. In some embodiments, the modified CH3 domain comprises S at position 389. In some embodiments, the modified CH3 domain comprises S or N at position 390. In some embodiments, the modified CH3 domain comprises a polar amino acid (e.g., S or T) at position 413. In some embodiments, the modified CH3 domain comprises S or E at position 415. In some embodiments, the modified CH3 domain comprises E at position 416. In some embodiments, the modified CH3 domain comprises F at position 421.

[0210] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising E at position 380; Y at position 384; N at position 386; E at position 387; W at position 388; S at position 389; S or N at position 390; a polar amino acid (e.g., S or T) at position 413; S or E at position 415; E at position 416; and F at position 421. In particular embodiments, the polypeptides comprise a modified CH3 domain comprising a sequence having at least 85% identity, at least 90% identity, or at least 95% identity (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identity) to amino acids 111-217 of any one of SEQ ID NOS:28, 32, 35, 40, and 49 and E at position 380; Y at position 384; N at position 386; E at position 387; W at position 388; S at position 389; S or N at position 390; a polar amino acid (e.g., S or T) at position 413; S or E at position 415; E at position 416; and F at position 421.

[0211] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:28 and E at position 380; Y at position 384; N at position 386; E at position 387; W atposition 388; S at position 389; S at position 390; T at position 413; S at position 415; E at position 416; and F at position 421.

[0212] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:32 and E at position 380; Y at position 384; N at position 386; E at position 387; W at position 388; S at position 389; S at position 390; S at position 413; S at position 415; E at position 416; and F at position 421.

[0213] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:35 and E at position 380; Y at position 384; N at position 386; E at position 387; W at position 388; S at position 389; N at position 390; T at position 413; S at position 415; E at position 416; and F at position 421.

[0214] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:40 and E at position 380; Y at position 384; N at position 386; E at position 387; W at position 388; S at position 389; N at position 390; S at position 413; S at position 415; E at position 416; and F at position 421.

[0215] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:49 and E at position 380; Y at position 384; N at position 386; E at position 387; W at position 388; S at position 389; N at position 390; S at position 413; E at position 415; E at position 416; and F at position 421.

[0216] In some embodiments, the polypeptide 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 SEQ ID NO:28 and E at position 380; Y at position 384; N at position 386; E at position 387; W at position 388; S at position 389; S at position 390; T at position 413; S at position 415; E at position 416; and F at position 421, according to EU numbering.

[0217] In some embodiments, the polypeptide 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 SEQ ID NO:32 and E at position 380; Y at position 384; N at position 386; E at position 387; W at position 388; S at position 389; S at position 390; S at position 413; S at position 415; E at position 416; and F at position 421, according to EU numbering.

[0218] In some embodiments, the polypeptide 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 SEQ ID NO:35 and E at position 380; Y at position 384; N at position 386; E at position 387; W at position 388; S at position 389; N at position 390; T at position 413; S at position 415; E at position 416; and F at position 421, according to EU numbering.

[0219] In some embodiments, the polypeptide 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 SEQ ID NO:40 and E at position 380; Y at position 384; N at position 386; E at position 387; W at position 388; S at position 389; N at position 390; S at position 413; S at position 415; E at position 416; and F at position 421, according to EU numbering.

[0220] In some embodiments, the polypeptide 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 SEQ ID NO:49 and E at position 380; Y at position 384; N at position 386; E at position 387; W at position 388; S at position 389; N at position 390; S at position 413; E at position 415; E at position 416; and F at position 421, according to EU numbering.Modified CH 3 Domains with E at Position 380, E at Position 387, and Y at Position 421

[0221] Provided herein are polypeptides that comprise a modified CH3 domain having E at position 380, E at position 387, and Y at position 421, according to EU numbering. In addition to E at position 380, E at position 387, and Y at position 421, the modified CH3 domain can further comprise one or more (e.g., one, two, three, four, five, six, seven, or eight) amino acid substitutions in a set of amino acid positions comprising 384, 386, 388-390, 413, 415, and 416, such as (i) Y at position 384; (ii) T at position 386; (iii) W at position 388; (iv) S or V at position 389; (v) S or N at position 390; (vi) a polar amino acid at position 413; (vii) S or E at position 415; and / or (viii) E at position 416, according to EU numbering. In some embodiments, the polypeptides bind to the TfR with a binding affinity between about 1 pM and about 15 pM (e.g., between about 1 pM and about 10 pM, between about 1 pM andabout 8 pM, between about 1 pM and about 5 pM, between about 5 pM and about 15 pM, between about 8 pM and about 15 pM, or between about 10 pM and about 15 pM).

[0222] In some embodiments, in addition to E at position 380, E at position 387, and Y at position 421, the modified CH3 domain comprises Y at position 384. In some embodiments, the modified CH3 domain comprises T at position 386. In some embodiments, the modifiedCH3 domain comprises W at position 388. In some embodiments, the modified CH3 domain comprises S or V at position 389. In some embodiments, the modified CH3 domain comprises S or N at position 390.some embodiments, the modified CH3 domain comprises a polar amino acid (e.g., S or T) at position 413. In some embodiments, the modified CH3 domain comprises S or E at position 415. In some embodiments, the modified CH3 domain comprises E at position 416.

[0223] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; S or V at position 389; S or N at position 390; a polar amino acid (e.g., S or T) at position 413; S or E at position 415; E at position 416, and Y at position 421. In particular embodiments, the polypeptides comprise a modified CH3 domain comprising a sequence having at least 85% identity, at least 90% identity, or at least 95% identity (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identity) to amino acids 111-217 of any one of SEQ ID NOS:30, 34, 38, 42, 48, and 51 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; S or V at position 389; S or N at position 390; a polar amino acid (e.g., S or T) at position 413; S or E at position 415; E at position 416, and Y at position 421.

[0224] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:30 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; S at position 389; S at position 390; T at position 413; S at position 415; E at position 416, and Y at position 421.

[0225] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:34 and E at position 380; Y at position 384; T at position 386; E at position 387; W atposition 388; S at position 389; S at position 390; S at position 413; S at position 415; E at position 416, and Y at position 421.

[0226] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:38 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; S at position 389; N at position 390; T at position 413; S at position 415; E at position 416, and Y at position 421.

[0227] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:42 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; S at position 389; N at position 390; S at position 413; S at position 415; E at position 416, and Y at position 421.

[0228] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:48 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; S at position 413; S at position 415; E at position 416, and Y at position 421.

[0229] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO: 51 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; S at position 389; N at position 390; S at position 413; E at position 415; E at position 416, and Y at position 421.

[0230] In some embodiments, the polypeptide 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 SEQ ID NO:30 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; S at position 389; S at position 390; T at position 413; S at position 415; E at position 416, and Y at position 421, according to EU numbering.

[0231] In some embodiments, the polypeptide 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 SEQ ID NO:34 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; S at position 389; S at position 390; S at position 413; S at position 415; E at position 416, and Y at position 421, according to EU numbering.

[0232] In some embodiments, the polypeptide 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 SEQ ID NO:38 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; S at position 389; N at position 390; T at position 413; S at position 415; E at position 416, and Y at position 421, according to EU numbering.

[0233] In some embodiments, the polypeptide 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 SEQ ID NO:42 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; S at position 389; N at position 390; S at position 413; S at position 415; E at position 416, and Y at position 421, according to EU numbering.

[0234] In some embodiments, the polypeptide 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 SEQ ID NO:48 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; S at position 413; S at position 415; E at position 416, and Y at position 421, according to EU numbering.

[0235] In some embodiments, the polypeptide 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 SEQ ID NO:51 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; S at position 389; N at position 390; S at position 413; E at position 415; E at position 416, and Y at position 421, according to EU numbering.Modified CH 3 Domains with E at Position 380, V at Position 389, and T at Position 413

[0236] Provided herein are polypeptides that comprise a modified CH3 domain having E at position 380, V at position 389, and T at position 413, according to EU numbering. In addition to E at position 380, V at position 389, and T at position 413, the modified CH3 domain can further comprise one or more (e.g., one, two, three, four, five, six, or seven) amino acid substitutions in a set of amino acid positions comprising 384, 386-388, 390, 416,and 421, such as (i) Y at position 384; (ii) T at position 386; (iii) E at position 387; (iv) W at position 388; (v) N at position 390; (vi) E at position 416; and / or (vii) F at position 421, according to EU numbering. In some embodiments, the polypeptides bind to the TfR with a binding affinity about 2.5 pM (e.g., between about 2.34 pM and about 2.86 pM).

[0237] In some embodiments, in addition to E at position 380, V at position 389, and T at position 413, the modified CH3 domain comprises Y at position 384. In some embodiments, the modified CH3 domain comprises T at position 386. In some embodiments, the modified CH3 domain comprises E at position 387. In some embodiments, the modified CH3 domain comprises W at position 388. In some embodiments, the modified CH3 domain comprises N at position 390. In some embodiments, the modified CH3 domain comprises E at position 416. In some embodiments, the modified CH3 domain comprises F at position 421.

[0238] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; T at position 413; E at position 416; and F at position 421. In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:37 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; T at position 413; E at position 416; and F at position 421.

[0239] In some embodiments, the polypeptide 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 SEQ ID NO:37 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; T at position 413; E at position 416; and F at position 421, according to EU numbering.Modified CH 3 Domains with Y at Position 384, V at Position 389, and S at Position 413

[0240] Provided herein are polypeptides that comprise a modified CH3 domain having Y at position 384, V at position 389, and S at position 413, according to EU numbering. In addition to Y at position 384, V at position 389, and S at position 413, the modified CH3 domain can further comprise one or more (e.g., one, two, three, four, five, or six) amino acid substitutions in a set of amino acid positions comprising 386-388, 390, 416, and 421, such as(i) T at position 386; (ii) E at position 387; (iii) W at position 388; (vi) N at position 390; (v) E at position 416; and / or (vi) F at position 421, according to EU numbering. In some embodiments, the polypeptides bind to the TfR with a binding affinity about 3.6 pM (e.g., between about 3.24 pM and about 3.96 pM).

[0241] In some embodiments, in addition to Y at position 384, V at position 389, and S at position 413, the modified CH3 domain comprises T at position 386. In some embodiments, the modified CH3 domain comprises E at position 387. In some embodiments, the modified CH3 domain comprises W at position 388. In some embodiments, the modified CH3 domain comprises N at position 390. In some embodiments, the modified CH3 domain comprises E at position 416. In some embodiments, the modified CH3 domain comprises F at position 421.

[0242] In particular embodiments, the polypeptides comprise a modified CH3 domain comprising Y at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; S at position 413; E at position 416; and F at position 421. In particular embodiments, the polypeptides comprise a modified CH3 domain comprising 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 SEQ ID NO:47 and Y at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; S at position 413; E at position 416; and F at position 421.

[0243] In some embodiments, the polypeptide 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 SEQ ID NO:47 and Y at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; S at position 413; E at position 416; and F at position 421, according to EU numbering.Anti-TfR Antibodies

[0244] In some embodiments, a TfR-binding molecule can be an anti-TfR antibody or an antibody fragment thereof. An anti-TfR antibody refers to an antibody with an antibody variable domain (e.g, an antibody variable domain having one or more complimentarity determining regions (CDRs)) that binds to a TfR, or an antigen-binding fragment thereof. In particular embodiments, an anti-TfR antibody can include a Fab that binds to a TfR. Examples of antibody fragments can include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab’)2 fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and (v) a dAb fragment which has a VH domain. In some embodiments, an anti-TfR antibody can be a humanized antibody. In some embodiments, an anti-TfR antibody can be a human antibody. In some embodiments, an anti-TfR antibody can be a monoclonal or polyclonal antibody. Examples of anti-TfR antibodies are described in, e.g., Pardridge, Expert Opin Drug Deliv 12(2):207-22, 2015.TfR-Binding Scaffolds

[0245] In some embodiments, a TfR-binding molecule can be a peptide or protein scaffold having an immunoglobulin (Ig) fold and engineered to bind to a TfR. In some embodiments, the protein scaffold is a fibronectin type III domain engineered to bind to TfR (see, e.g., WO202 1076546).

[0246] In further embodiments, a protein scaffold can be an albumin (e.g., heme-albumin) or variants thereof, which is shown to bind to TfR, see, e.g., Brell et al., Nat. Communications Biology 3:621, 2020.

[0247] In further embodiments, a protein scaffold can be a dual-variable-domain immunoglobulin (DVD-Ig) engineered to bind TfR, see, e.g., Hanzatian et al., MAbs. 10(5): 765-777, 2018.

[0248] In further emodiments, a peptide scaffold can be a peptide engineered to bind TfR, see, e.g., Crook et al., J. Mol. Bio., 432(14):3989-4009, 2020.V. ADDITIONAL FC POLYPEPTIDE MODIFICATIONS

[0249] A TfR-binding polypeptide described herein, e.g., an Fc polypeptide comprising a modified CH3 domain, or an anti-TfR antibody can also comprise additional mutations, e.g., to provide for knob and hole heterodimerization, to modulate effector function, to extend serum half-life, to influence glyscosylation, and / or to reduce immunogenicity in humans.Fc Polypeptide Modifications for Heterodimerization

[0250] In some embodiments, the polypeptides (e.g., an Fc polypeptide) comprising a modified CH3 domain described herein include mutations to promote heterodimer formation and hinder homodimer formation. These modifications are useful, for example, where it is desired to have only one of the polypeptide of a dimer have a TfR binding site (i.e., a monovalent TfR binder).

[0251] The knobs-into-holes approach generally involves introducing a protuberance (“knob”) at the interface of a polypeptide (e.g., an Fc polypeptide) and a corresponding cavity (“hole”) in the interface of a second polypeptide (e.g., an Fc polypeptide), such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and thus hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide (e.g., an Fc polypeptide) with larger side chains (e.g., Tyr or Trp). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide (e.g, an Fc polypeptide) by replacing large amino acid side chains with smaller ones (e.g., Ala or Thr). In some embodiments, such additional mutations are at a position in the polypeptide (e.g., an Fc polypeptide) that does not have a negative effect on binding of the polypeptide to TfR.

[0252] In one illustrative embodiment of a knob and hole approach for dimerization, position 366 of one of the polypeptides (e.g., an Fc polypeptide) comprises a Trp in place of a native Thr. The other polypeptide in the dimer has a Vai at position 407 in place of the native Tyr. The other polypeptide (e.g., an Fc polypeptide) may further comprise a substitution in which the native Thr at position 366 is substituted with a Ser and a native Leu at position 368 is substituted with an Ala. Thus, one of the polypeptides (e.g., an Fc polypeptide) has the T366W knob mutation and the other polypeptide (e.g., an Fc polypeptide) has the Y407V hole mutation, which is typically accompanied by the T366S and L368A hole mutations. As indicated above, all positions are numbered per EU numbering.

[0253] In some embodiments, one or both polypeptides (e.g., Fc polypeptides) present in a polypeptide dimer (e.g., an Fc polypeptide dimer) can also be engineered to contain other modifications for heterodimerization, e.g., electrostatic engineering of contact residues within a CH3-CH3 interface that are naturally charged or hydrophobic patch modifications.

[0254] The knobs-into-holes approach (e.g., T366W knob substitution on one polypeptide (e.g., an Fc polypeptide) with the T366S, L368A, and Y407V hole substitution on the other polypeptide (e.g., an Fc polypeptide)) can be used with any of the polypeptides described herein (e.g., a TfR-binding polypeptide having a sequence of any one of SEQ ID NOS:28-51 (e.g., SEQ ID NO:36 or 37)).

[0255] 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 a sequence of any one of SEQ ID NOS:28-51 (e.g., SEQ ID NO:36 or 37)) while the other polypeptide (e.g., an Fcpolypeptide) does not contain a TfR-binding site. In particular embodiments, one of the polypeptides (e.g, an Fc polypeptide) is a TfR-binding polypeptide and contains a knob mutation (e.g, T366W), while the other polypeptide (e.g., an Fc polypeptide) does not bind to TfR and contains a hole mutation (e.g., T366S, L368A, and Y407V). In other embodiments, one of the polypeptides (e.g., an Fc polypeptide) is a TfR-binding polypeptide and contains a hole mutation e.g., T366S, L368A, and Y407V), while the other polypeptide e.g., an Fc polypeptide) does not bind to TfR and contains a knob mutation e.g., T366W).

[0256] In particular embodiments, a polypeptide dimer (e.g., an Fc polypeptide dimer) that specifically binds to TfR can have a first polypeptide e.g., an Fc polypeptide) having the T366W knob mutation and 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 a sequence of any one of SEQ ID NOS:28-51 (e.g., SEQ ID NO:36 or 37) wherein the sequence comprises substitutions listed for any one of the clones listed in Table 3, and a second polypeptide e.g., an Fc polypeptide) having the T366S, L368A, and Y407V hole mutations and 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:6. In other embodiments, a polypeptide dimer (e.g., an Fc polypeptide dimer) that specifically binds to TfR can have a first polypeptide (e.g., an Fc polypeptide) having the T366W knob mutation and 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:5, and a second polypeptide (e.g., an Fc polypeptide) having the T366S, L368A, and Y407V hole mutations and 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 a sequence of any one of SEQ ID NOS:28-51 (e.g., SEQ ID NO:36 or 37) wherein the sequence comprises substitutions listed for any one of the clones listed in Table 3. In other embodiments, an Fc polypeptide dimer e.g., an Fc polypeptide dimer) that specifically binds to TfR can have a first polypeptide (e.g., an Fc polypeptide) having the T366W knob mutation and 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 a sequence of any one of SEQ ID NOS:28-51 e.g., SEQ ID NO:36 or 37) wherein the sequence comprises substitutions listed for any one of the clones listed in Table 3, and a second polypeptide e.g., an Fc polypeptide) having the T366S, L368A, and Y407V hole mutations and 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 a sequenceof any one of SEQ ID NOS:28-51 e.g., SEQ ID NO:36 or 37) wherein the sequence comprises substitutions listed for any one of the clones listed in Table 3.Fc Polypeptide Modifications for Modulating Effector Function

[0257] In some embodiments, a polypeptide dimer described herein is an Fc polypeptide dimer comprising two Fc polypeptides. In some embodiments, both Fc polypeptides in the Fc polypeptide dimer can comprise modifications that reduce or eliminate effector function, i.e., having a reduced ability to induce certain biological functions upon binding to an Fc receptor expressed on an effector cell that mediates the 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, Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibodydependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptor), and B-cell activation.

[0258] Illustrative Fc polypeptide mutations that reduce effector function include, but are not limited to, substitutions in a CH2 domain, e.g., at positions 234 and 235 and / or at position 329, according to the EU numbering scheme. For example, in some embodiments, both Fc polypeptides comprise Ala residues at positions 234 and 235 (also referred to as “LALA” herein). In some embodiments, both Fc polypeptides comprise Gly residue at position 329 (also referred to as “P329G” or “PG” herein) or Ser residue at position 329 (also referred to as “P329S” or “PS” herein). In some embodiments, both Fc polypeptides comprise Ala residues at positions 234 and 235, and Gly residue at position 329 (also referred to as “LALA PG” herein). In some embodiments, both Fc polypeptides comprise Ala residues at positions 234 and 235, and Ser residue at position 329 (also referred to as “LALA PS” herein).

[0259] Additional Fc polypeptide mutations that modulate an effector function include, but are not limited to, the following: position 329 may have a mutation in which Pro is substituted with a Gly, Ala, Ser, or Arg or an amino acid residue large enough to destroy the Fc / Fcy receptor interface that is formed between proline 329 of the Fc and Trp residues Trp87 and Trpl lO of FcyRIII. Additional illustrative substitutions include S228P, E233P, L235E, N297A, N297D, and P331S, according to the EU numbering scheme. Multiple substitutions may also be present, e.g., L234A, L235A, and P329G of human IgGl; S228Pand L235E of human IgG4; L234A and G237A of human IgGl; L234A, L235A, and G237A of human IgGl; V234A and G237A of human IgG2; L235A, G237A, and E318A of human IgG4; and S228P and L236E of human IgG4, according to the EU numbering scheme.

[0260] In some embodiments, a polypeptide (e.g., an Fc polypeptide) that specifically binds to TfR comprises LALA 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 a sequence of any one of SEQ ID NOS:28-51 (e.g., SEQ ID NO:36 or 37) wherein the sequence comprises substitutions listed for any one of the clones listed in Table 3.

[0261] In some embodiments, a polypeptide (e.g., an Fc polypeptide) that specifically binds to TfR comprises 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 a sequence of any one of SEQ ID NOS:28-51 (e.g., SEQ ID NO:36 or 37) wherein the sequence comprises substitutions listed for any one of the clones listed in Table 3.Fc Polypeptide Modifications for Extending Serum Half-Life

[0262] In some embodiments, modifications to enhance serum half-life can be introduced into any polypeptides described herein. For example, in some embodiments, a polypeptide dimer described herein is an Fc polypeptide dimer comprising two Fc polypeptides. In some embodiments, both Fc polypeptides in the Fc polypeptide dimer can comprise M428L and N434S substitutions (also referred to as LS substitutions), as numbered according to the EU numbering scheme. Alternatively, both Fc polypeptides in an Fc polypeptide dimer can have an N434S or N434A substitution. Alternatively, both Fc polypeptides in an Fc polypeptide dimer can have an M428L substitution. In other embodiments, both Fc polypeptides in an Fc polypeptide dimer can comprise M252Y, S254T, and T256E substitutions.

[0263] In any of the embodiments described herein, a polypeptide (e.g., an Fc polypeptide) that specifically binds to TfR can further comprise LS substitutions. For example, in some embodiments, a polypeptide (e.g., an Fc polypeptide) that specifically binds to TfR comprises LS 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 a sequence of any one of SEQ ID NOS:28-51 (e.g., SEQ ID NO:36 or 37) wherein the sequence comprises substitutions listed for any one of the clones listed in Table 3.Fc Polypeptide with C-terminal Lysine Residue Removed

[0264] In some embodiments, one or both of the polypeptides (e.g., Fc polypeptides) can have its C-terminal lysine removed (e.g., the Lys residue at position 447 of the Fc polypeptide, according to EU numbering). The C-terminal lysine residue is highly conserved in immunoglobulins across many species and may be fully or partially removed by the cellular machinery during protein production. In some embodiments, removal of the C- terminal lysines in the Fc polypeptides can improve the stability of the proteins.“cis-LALA” or “trans-LALA” Configuration

[0265] In some embodiments of a polypeptide dimer, only one of the two polypeptides (but not both polypeptides) in the dimer is modified to both bind TfR and reduce effector function. The other polypeptide does not contain a TfR-binding site or any modifications that reduce effector function. This is referred to as the cis-LALA configuration.

[0266] In some embodiments of a polypeptide dimer, one of the two polypeptides (but not both polypeptides) in the dimer is modified to bind TfR and is not modified to reduce effector function. The other polypeptide does not contain a TfR-binding site but does contain modifications that reduce effector function. This is referred to as the trans-LALA configuration.

[0267] For example, in some embodiments, a polypeptide dimer having the cis-LALA configuration can include a first polypeptide having a TfR-binding site, the T366W knob mutation, and the LALA substitutions, and at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,) or 100% identical to any one of SEQ ID NOS:28-51 (e.g., SEQ ID NO:36 or 37), and a second Fc polypeptide having the T366S, L368A, and Y407V hole mutations and at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:6. In some embodiments, apolypeptide dimer having the cis-LALA configuration can have a first Fc polypeptide having a TfR-binding site, the T366S, L368A, and Y407V hole mutations, the LALA substitutions, and at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of any one of SEQ ID NOS:28-51 (e.g., SEQ ID NO:36 or 37), and a second polypeptide having the T366W knob mutation and at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 5.VI. ILLUSTRATIVE FC POLYPEPTIDES THAT BIND TO TFR

[0268] A polypeptide (e.g., an Fc polypeptide) of the present disclosure may include, for example, a CH3 domain as described herein. A CH3 domain of the present disclosure may be joined to a CH2 domain, which may be a naturally occurring CH2 domain or a variant CH2 domain, typically at the C-terminal end of the CH2 domain to form a polypeptide (e.g., an Fc polypeptide) that binds to TfR. In some embodiments, the polypeptide (e.g., an Fc polypeptide) further comprises a partial or full hinge region of an antibody, which is joined to the N-terminal end of the CH2 domain. The hinge region can be from any immunoglobulin subclass or isotype. An illustrative immunoglobulin hinge is an IgG hinge region, such as an IgGl hinge region, e.g., human IgGl hinge amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO:4).

[0269] In some embodiments, a polypeptide (e.g., an Fc polypeptide) can comprise a sequence from Table 1 and the polypeptide (e.g., an Fc polypeptide) can be further modified to contain a TfR binding site in the modified CH3 domain as described herein.Table 1. Fc Sequences For Further TfR-Binding Site Modifications

[0270] In further embodiments, the polypeptide (e.g., an Fc polypeptide) can be further joined to another moiety, for example, a Fab fragment, thus generating a TfR-binding Fc-Fab fusion. In some embodiments, the TfR-binding Fc-Fab fusion comprises a modified CH3 domain, a CH2 domain, a hinge region, and a Fab fragment. The Fab fragment may be to any target of interest, e.g., a therapeutic neurological target, where the Fab is delivered to the target by transcytosis across the BBB mediated by the binding of the modified CH3 domain polypeptide to TfR.

[0271] The TfR-binding polypeptide (e.g., a TfR-binding Fc polypeptide) may also be linked to a polypeptide of interest other than a Fab. For example, in some embodiments, theTfR-binding polypeptide (e.g., a TfR-binding Fc polypeptide) may be linked to a polypeptide that is desirable to target to a TfR-expressing cell or to deliver across an endothelium, e.g., the BBB, by transcytosis. In some embodiments, the TfR-binding polypeptide (e.g., a TfR- binding Fc polypeptide) is linked to a soluble protein. In still other embodiments, the TfR- binding polypeptide (e.g., a TfR-binding Fc polypeptide) may be linked to a peptide or protein useful in protein purification, e.g., polyhistidine, epitope tags, e.g., FLAG, c-Myc, hemagglutinin tags and the like, glutathione S transferase (GST), thioredoxin, protein A, protein G, or maltose binding protein (MBP). In some cases, the peptide or protein to which the TfR-binding polypeptide (e.g., a TfR-binding Fc polypeptide) is linked may comprise a protease cleavage site, such as a cleavage site for Factor Xa or Thrombin.Exemplary Fc Polypeptides That Bind to TfR35.dl.9

[0272] In certain embodiments, a polypeptide comprises the sequence of SEQ ID NO:36. Further, a polypeptide can comprise a sequence of any one of SEQ ID NOS:52-59. In one embodiment, a monovalent dimer (e.g., a monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; N at position 390; T at position 413; S at position 415; E at position 416; and F at position 421, wherein the positions are determined according to EU numbering. In one embodiment, a monovalent dimer (e.g., a monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) 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 SEQ ID NO:36 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; N at position 390; T at position 413; S at position 415; E at position 416; and F at position 421, wherein the positions are determined according to EU numbering. In one embodiment, the polypeptide (e.g., an Fc polypeptide) in the monovalent dimer (e.g., a monovalent Fc dimer) further comprises a T366W knob mutation. In another embodiment, the polypeptide (e.g., an Fc polypeptide) in the monovalent dimer (e.g., a monovalent Fc dimer) further comprises T366S, L368A, and Y407V hole mutations.

[0273] In another embodiment, a bivalent dimer (e.g., a bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each having E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; N at position 390; T at position 413; S at position 415; E at position 416; and F at position 421, wherein thepositions are determined according to EU numbering. In another embodiment, a bivalent dimer (e.g., a bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each 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 SEQ ID NO:36 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; N at position 390; T at position 413; S at position 415; E at position 416; and F at position 421, wherein the positions are determined according to EU numbering.35.dl.10

[0274] In certain embodiments, a polypeptide comprises the sequence of SEQ ID NO:37. Further, a polypeptide can comprise a sequence of any one of SEQ ID NOS:60-67. In one embodiment, a monovalent dimer (e.g., a monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) having E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; T at position 413; E at position 416; and F at position 421, wherein the positions are determined according to EU numbering. In one embodiment, a monovalent dimer (e.g., a monovalent Fc dimer) comprises a polypeptide (e.g., an Fc polypeptide) 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 SEQ ID NO:37 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; T at position 413; E at position 416; and F at position 421, wherein the positions are determined according to EU numbering. In one embodiment, the polypeptide (e.g., an Fc polypeptide) in the monovalent dimer (e.g., a monovalent Fc dimer) further comprises a T366W knob mutation. In another embodiment, the polypeptide (e.g., an Fc polypeptide) in the monovalent dimer (e.g., a monovalent Fc dimer) further comprises T366S, L368A, and Y407V hole mutations.

[0275] In another embodiment, a bivalent dimer (e.g., a bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each having E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; T at position 413; E at position 416; and F at position 421, wherein the positions are determined according to EU numbering. In another embodiment, a bivalent dimer (e.g., a bivalent Fc dimer) comprises two polypeptides (e.g., Fc polypeptides) each 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 SEQ ID NO:37 and E at position 380; Y at position 384; T at position 386; E atposition 387; W at position 388; V at position 389; N at position 390; T at position 413; E at position 416; and F at position 421, wherein the positions are determined according to EU numbering.VII. FC DIMERS FOR TFR-BINDING SITE MODIFICATION

[0276] In some embodiments, a TfR-binding polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain as described herein can form a dimer (e.g., an Fc dimer) comprising two polypeptides (e.g., Fc polypeptides). The dimer may be a heterodimer or a homodimer.Fc Dimer That Binds TfR Bivalently

[0277] In some embodiments, the dimer is an Fc dimer that comprises two polypeptides (e.g., Fc polypeptides) in which each contains a TfR binding site, i.e., binds TfR bivalently. In an Fc dimer that binds TfR bivalently, the first and second Fc polypeptides may comprise the same CH3 domain. In other embodiments, the second Fc polypeptide may comprise a different CH3 domain from that in the first Fc polypeptide to provide a second TfR-binding site.

[0278] In some embodiments, a bivalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2A and (i) each of the first and second Fc polypeptides are further modified to contain a TfR binding site in the modified CH3 domain as described herein or (ii) wherein the first and second Fc polypeptides contain a TfR binding site in the modified CH3 domain as described herein. In other embodiments, a bivalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2A, 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 2 A, 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 2 A, and wherein each of the first and second Fc polypeptides are further modified to contain a TfR binding site in the modified CH3 domain as described herein.

[0279] In one embodiment, a bivalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2A, 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 2A, 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 polylpeptide sequence from Table 2A, and wherein each of the first and second Fc polypeptides are further modified to contain a TfR binding site in the modified CH3 domain comprising one of the following: (a) F at position 384 and S at position 413; (b) E at position 380 and the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389; (c) E at position 380, N at position 386, and E at position 387; (d) E at position 380, E at position 387, and Y at position 421; (e) E at position 380, V at position 389, and T at position 413; or (f) Y at position 384, V at position 389, and S at position 413, wherein the positions are determined according to EU numbering.

[0280] In one embodiment, a bivalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2A, 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 2A, 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 polylpeptide sequence from Table 2A, and wherein each of the first and second Fc polypeptides are further modified to contain a TfR binding site in the modified CH3 domain comprising: E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; N at position 390; T at position 413; S at position 415; E at position 416; and F at position 421, according to EU numbering.

[0281] In one embodiment, a bivalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2A, 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 2A, 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 polylpeptide sequence from Table 2A, and wherein each of the first and second Fc polypeptides are further modified to contain a TfR binding site in the modified CH3 domain comprising: E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; T at position 413; E at position 416; and F at position 421, according to EU numbering.Fc Dimer That Binds TfR Monovalently

[0282] In some embodiments, the dimer is a monovalent Fc dimer that comprises two Fc polypeptides, in which only one of the two Fc polypeptides in the monovalent Fc dimer comprises a TfR-binding site, while the other Fc polypeptide does not bind to TfR. In addition, the Fc polypeptides can contain modifications for promoting heterodimzerization of the Fc dimer (e.g., T366W; and T366S, L368A, and Y407V). In some embodiments, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2B and the first Fc polypeptide is further modified to contain a TfR-binding site in the modified CH3 domain as described herein. In some embodiments, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2B and the second Fc polypeptide is further modified to contain a TfR-binding site in the modified CH3 domain as described herein.

[0283] In other embodiments, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2B, 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 from first Fc polypeptide sequence from Table 2B, 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 polypeptides is further modified to contain a TfR-binding site in the modified CH3 domain as described herein.

[0284] In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2B, wherein the first polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to the first Fc polypeptide sequence from Table 2B, 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 TfR-binding site in the modified CH3 domain comprising, and the modified CH3 domain comprises 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 SEQ ID NO:36 and E at position 380; Y at position 384; T at position 386; E at position 387;W at position 388; T at position 389; N at position 390; T at position 413; S at position 415; E at position 416; and F at position 421, wherein the positions are determined according to EU numbering.

[0285] In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2B, wherein the first polypeptide has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identity to the first Fc polypeptide sequence from Table 2B, 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 TfR-binding site in the modified CH3 domain comprising, and the modified CH3 domain comprises 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 SEQ ID NO:37 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; T at position 413; E at position 416; and F at position 421, wherein the positions are determined according to EU numbering.

[0286] In other embodiments, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2B, 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 from first Fc polypeptide sequence from Table 2B, 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 second Fc polypeptides is further modified to contain a TfR-binding site in the modified CH3 domain as described herein.

[0287] In other embodiments, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2B, 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 from first Fc polypeptide sequence from Table 2B, 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 thesecond Fc polypeptides is further modified to contain a TfR-binding site in the modified CH3 domain, and the modified CH3 domain comprises 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 SEQ ID NO:36 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; T at position 389; N at position 390; T at position 413; S at position 415; E at position 416; and F at position 421, wherein the positions are determined according to EU numbering.

[0288] In other embodiments, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2B, 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 from first Fc polypeptide sequence from Table 2B, 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 second Fc polypeptides is further modified to contain a TfR-binding site in the modified CH3 domain, and the modified CH3 domain comprises 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 SEQ ID NO:37 and E at position 380; Y at position 384; T at position 386; E at position 387; W at position 388; V at position 389; N at position 390; T at position 413; E at position 416; and F at position 421, wherein the positions are determined according to EU numbering.

[0289] In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2B, 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 2B, 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 2B, and wherein the first Fc polypeptide is further modified to contain a TfR-binding site in the modified CH3 domain comprising a set of modifications selected from a row from Table 3.

[0290] In one embodiment, a monovalent Fc dimer that specifically binds to TfR described herein comprises a first and second Fc polypeptide pair from Table 2B, wherein the first Fcpolypeptide 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 2B, 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 2B, and wherein the second Fc polypeptide is further modified to contain a TfR-binding site in the modified CH3 domain comprising a set of modifications selected from a row from Table 3.

[0291] In another aspect, the disclosure provides Fc polypeptide dimers having sequences of the first and second Fc polypeptides as listed in Tables 2A and 2B below:Table 2A. Dimer Combinations For Bivalent TfR Binding Site ModificationsTable 2B. Knob-Hole Dimer Combinations For Monovalent TfR Binding Site ModificationsVII. CONJUGATES

[0292] In some embodiments, a TfR-binding molecule (e.g, a TfR-binding polypeptide (e.g, an Fc polypeptide) comprising a modified CH3 domain) described herein is linked to an agent (e.g., a therapeutic agent), e.g., an agent that is to be internalized into a cell and / or for transcytosis across an endothelium, such as the BBB, via a linker. The linker may be any linker suitable for joining an agent to the molecule. In some embodiments, the linkage is enzymatically cleavable. In certain embodiments, the linkage is cleavable by an enzyme present in the central nervous system.

[0293] In some embodiments, the linker is a peptide linker. The peptide linker may allow for the rotation of the agent and the molecule relative to each other; and / or is 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 linkers are designed using known parameters. For example, the linker may have repeats, such as Gly- Ser repeats.

[0294] In various embodiments, the conjugates can be generated using well-known chemical cross-linking reagents and protocols. For example, the cross-linking agents are heterobifunctional cross-linkers, which can be used to link molecules in a stepwise manner. Heterobifunctional cross-linkers provide the ability to design more specific coupling methods for conjugating proteins, thereby reducing the occurrences of unwanted side reactions such as homo-protein polymers. A wide variety of heterobifunctional cross-linkers are known in the art, including N-hydroxysuccinimide (NHS) or its water soluble analog N- hydroxy sulfosuccinimide (sulfo-NHS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-l- carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxy succinimide ester (MBS); N- succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), succinimidyl 4-(p- maleimidophenyl)butyrate (SMPB), l-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 cross-linking agents having N- hydroxysuccinimide moieties can be obtained as the N-hydroxysulfosuccinimide analogs, which generally have greater water solubility. In addition, those cross-linking agents having disulfide bridges within the linking chain can be synthesized instead as the alkyl derivatives so as to reduce the amount of linker cleavage in vivo. In addition to the heterobifunctional cross-linkers, there exist a number of other cross-linking agents including homobifunctional and photoreactive cross-linkers. Disuccinimidyl subcrate (DSS), bismaleimidohexane (BMH) and dimethylpimelimidate.2HCl (DMP) are examples of useful homobifunctional cross-linking agents, and bis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) and N- succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) are examples of useful photoreactive cross-linkers.

[0295] The agent of interest may be a therapeutic agent, including a cytotoxic agent, a DNA or RNA molecule, an antisense oligonucloetide, a chemical moiety, 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 1000 Da, less than 750 Da, or less than 500 Da.

[0296] In some embodiments, the agent of interest may be linked to the N-terminal or C- terminal region of the TfR-binding molecule, or attached to any region of the molecule, so long as the agent does not interfere with binding of the TfR-binding molecule to TfR.IX. NUCLEIC ACIDS, VECTORS, AND HOST CELLS

[0297] The TfR-binding molecules (e.g., a TfR-binding polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain) as described herein are typically prepared using recombinant methods. Accordingly, in some aspects, the disclosure provides isolated nucleic acids comprising a nucleic acid sequence encoding any of the molecules as described herein, and host cells into which the nucleic acids are introduced that are used to replicate the nucleic acids and / or to express the molecules. In some embodiments, the host cell is eukaryotic, e.g., a human cell.

[0298] In another aspect, polynucleotides are provided that comprise a nucleotide sequence that encodes the TfR-binding molecules (e.g, a TfR-binding polypeptide (e.g, an Fc polypeptide) comprising a modified CH3 domain) described herein. The polynucleotides may be single-stranded or double-stranded. In some embodiments, the polynucleotide is DNA (e.g., cDNA). In some embodiments, the polynucleotide is RNA.

[0299] In some embodiments, the polynucleotide is included within a nucleic acid construct. In some embodiments, the construct is a replicable vector. In some embodiments, the vector is selected from a plasmid, a viral vector, a phagemid, a yeast chromosomal vector, and a non-episomal mammalian vector.

[0300] In some embodiments, the polynucleotide is operably linked to one or more regulatory nucleotide sequences in an expression construct. In one series of embodiments, the nucleic acid expression constructs are adapted for use as a surface expression library (e.g., yeast or phage). In another series of embodiments, the nucleic acid expression constructs are adapted for expression of the molecule in a system that permits isolation of the molecule in milligram or gram quantities. In some embodiments, the system is a mammalian cell or yeast cell expression system.

[0301] Expression vehicles for production of a recombinant molecule include plasmids and other vectors. Any appropriate plasmid or vector can be used for this purpose, including those suitable for transient expression of molecules in eukaryotic cells. In some embodiments, it may be desirable to express the recombinant molecule by the use of a baculovirus expression system using appropriate vectors. Additional expression systems include adenoviral, adeno-associated virus, and other viral expression systems.

[0302] Vectors may be transformed into any suitable host cell. In some embodiments, the host cells, e.g., bacteria or yeast cells, may be adapted for use as a surface expression library. In some cells, the vectors are expressed in host cells to express relatively large quantities of the molecule. Such host cells include mammalian cells, yeast cells, insect cells, and prokaryotic cells. In some embodiments, the cells are mammalian cells, such as Chinese Hamster Ovary (CHO) cell, baby hamster kidney (BHK) cell, NS0 cell, Y0 cell, HEK293 cell, COS cell, Vero cell, or HeLa cell.

[0303] A host cell transfected with an expression vector encoding a TfR-binding molecule can be cultured under appropriate conditions to allow expression of the molecule. The molecules may be secreted and isolated from a mixture of cells and medium containing the molecules. Alternatively, the molecule may be retained in the cytoplasm or in a membrane fraction and the cells harvested, lysed, and the molecule isolated using a desired method.X. PHARMACEUTICAL COMPOSITIONS AND KITS

[0304] In another aspect, pharmaceutical compositions and kits comprising a TfR-binding molecules (e.g, a TfR-binding polypeptide (e.g, an Fc polypeptide) comprising a modified CH3 domain) in accordance with the disclosure are provided.Pharmaceutical Compositions

[0305] Guidance for preparing formulations for use in the present disclosure can be found in any number of handbooks for pharmaceutical preparation and formulation that are known to those of skill in te art.

[0306] In some embodiments, a pharmaceutical composition comprises a molecule described herein and further comprises one or more pharmaceutically acceptable carriers and / or excipients. A pharmaceutically acceptable carrier includes any solvents, dispersion media, or coatings that are physiologically compatible and that preferably does not interfere with or otherwise inhibit the activity of the active agent. Various pharmaceutically acceptable excipients are well-known.

[0307] In some embodiments, the carrier is suitable for intravenous, intrathecal, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compounds that act, for example, to stabilize the composition or to increase or decrease the absorption of the molecule. Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and their formulations are also available in the art.

[0308] The pharmaceutical compositions described herein can be manufactured in a manner that is known to those of skill in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0309] Typically, a pharmaceutical composition for use in in vivo administration is sterile. Sterilization can be accomplished according to methods known in the art, e.g., heat sterilization, steam sterilization, sterile filtration, or irradiation.

[0310] Dosages and desired drug concentration of pharmaceutical compositions of the disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration can be determined by one of skill in the art.Kits

[0311] In some embodiments, kits comprising a TfR-binding molecules (e.g., a TfR- binding polypeptide (e.g., an Fc polypeptide) comprising a modified CH3 domain) described herein are provided. In some embodiments, the kits are for use in preventing or treating a neurological disorder such as a disease of the brain or central nervous system (CNS).

[0312] In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit comprises a molecule as described herein and further comprises one or more additional therapeutic agents for use in the treatment of a neurological disorder. In some embodiments, the kit further comprises instructional materials containing directions (i.e., protocols) for the practice of the methods described herein (e.g., instructions for using the kit for administering a composition across the BBB). While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD-ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.XI. EXAMPLES

[0313] The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the disclosure in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results. Efforts have been made to ensure accuracy with respect to numbers used (e.g, amounts, temperatures, etc.), but some experimental error and deviation may be present. The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. Additionally, it should be apparent to one of skill in the art that the methods forengineering as applied to certain libraries can also be applied to other libraries described herein.Example 1. Design and Affinity Measurement of Reduced Affinity TfR Binding Polypeptides

[0314] In an effort to reduce the affinity of clone CH3C.1.35, amino acid residues at positions 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 were mutated relative to CH3C.1.35 either as single point mutations or as combinations. Clones were expressed and purified from HEK293 cells, and their affinity to the human TfR apical domain was measured by Biacore (Table 3). Each molecule is a monovalent Fc-Fab (anti-BACEl) fusion polypeptide comprising a first Fc polypeptide containing the specified TfR-binding site mutations listed in a row in Table 3, LALA, and T366W knob mutations, and a second Fc polypeptide containing T366S, L368A, and Y407V hole mutations, LALA mutations, and not containing a TfR-binding site.Table 3Example 2. PK / PD Evaluation

[0315] Clones 35. dl .9, 35.dl.10, 35.dl.16, and 35.23.4 in monovalent forms were tested for their PK and PD in huTfRapicalknock-in mice to ensure that there are no PK / PD liabilities. Each molecule is a monovalent Fc-Fab fusion polypeptide comprising a first Fc polypeptide containing the specified TfR-binding site and T366W knob mutation, and a second Fc polypeptide containing T366S, L368A, and Y407V hole mutations and not containing a TfR- binding site, linked to the anti-BACEl Fab domain.

[0316] The clones and the controls were dosed intravenously into huTfRapicalknock-in mice at 50 mg / kg. Plasma, brain, and CSF concentrations were measured at various timepoints (FIGS. 1A-1C). The study design is shown in Table 4. Plasma PK showed increasing plasma exposures with decreasing TfR-binding affinity (FIG. 1A). For Brain PK, Cmax decreased and exposures were durable with reduced affinity clones (FIG. IB). CSF concentrations were similar across treatment groups (FIG. 1C). Brain and plasma ratios of the clones are also shown in FIG. ID. Further, brain PD evaluation showed sustained amyloid beta 40 (Abeta40) reduction with clones with lower TfR-binding affinities (FIG. IE). Other measurements performed during the PK / PD evalution are showin in Table 5.

[0317] Further, the effect of the clones on circulating reticulocytes and red blood cells was also investigated. FIG. 2A shows the measurement of reticulocyte level. At 24 hrs, the data shows that clone with strong TfR-binding affinity (35.23.4) drove reticulocyte loss, which was not observed clones 35. dl.10 and 35. dl.9. FIG. 2B shows that there was no difference in red blood cell level across treatment groups.Table 4*Time points in parentheses are in-life bleeds. Times without are terminal bleeds and brain collection for PKTable 5Example 3. Effects on PD From Clones with Various Affinities

[0318] This example shows the impact of clones with different TfR-binding affinities on PD in human TREM2 BAC transgenic mouse model. A TREM2 BAC transgenic (tg) mouse model expressing human TREM2 in the CNS and periphery was generated. The study design is shown in Table 6 below. Each molecule is a monovalent Fc-Fab fusion polypeptide comprising a first Fc polypeptide comprising the specified TfR-binding site, T366W knob mutation, and LALA substitutions, and a second Fc polypeptide containing T366S, L368A, and Y407V hole mutations, and LALA substitutions, and not containing a TfR-binding site, linked to the anti-TREM2 Fab domain.Table 6

[0319] Plasma concentrations were measured at various timepoints (FIG. 3A). For the brain PK, 35.23.3 :TREM2 dosed at 10 mg / kg and 35.dl.9:TREM2 dosed at 30 mg / kg showed brain concentration at about 1 nM at 96-hr timepoint. The rest of the molecules were below the limit of detection at 96-hr. Moreover, molecules showed target engagement (TE) in the brain (FIG. 3B).Example 4. Effects on CSF1R From Clones with Various Affinities

[0320] Levels of CSF1R as a result of molecules 35.21 :anti-TREM2, 35.23.3 :anti-TREM2, and 35.dl.9:anti-TREM2, as described in the above example, were also measured. In the CNS, CSF1R is predominantly expressed on microglia and is essential for survival. Since soluble CSF1R (sCSFIR) has been shown to be elevated in CSF of healthy volunteers after TREM2 antibody dosing, it represents a potential pharmacodynamic biomarker of TREM2 pathway activation.

[0321] To elucidate affinity impacts of the TfR-binding clones on TREM2 antibody activity, WT; human TREM2 tg; TfRmu / humice were treated with the molecules with different TfR-binding affinities at variable doses selected to achieve similar brain exposures (FIG. 4A), then CSF1R was measured in brain and CSF 24 and 96 hrs post dose (FIGS. 4B and 4C). Molecule 35.dl.9:anti-TREM2 (TfR-binding affinity: 8000 nM) reached similar concentrations in brain as molecule 35.23.3:anti-TREM2 (TfR-binding affinity: 1100 nM), but did not induce CSF1R in brain or CSF. In contrast, molecule 35.21 : anti -TREM2 (TfR- binding affinity: 110 nM) induced similar CSF1R levels as molecule 35.23.3:anti-TREM2 (TfR-binding affinity: 1100 nM), suggesting the TfR-binding affinity requires a certain threshold to confer enhanced TREM2 Fab activity.XII. EXEMPLARY EMBODIMENTS

[0322] Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments:1. A method of screening to identify a biological target for the treatment of a neurodegenerative disease, the method comprising:(a) selecting a biological target in the brain associated with a neurodegenerative disease;(b) providing a molecule comprising a biological target-binding moiety and a TfR-binding moiety;(c) testing whether the molecule comprising a biological target-binding moiety and a TfR-binding moiety has enhanced modulation of the biological target, as compared to a reference molecule comprising the biological target-binding moiety but lacking the TfR- binding moiety; and(d) selecting the biological target for treatment of the neurodegenerative disease based on whether the TfR-binding moiety enhances modulation of the target, wherein the biological target is not a TfR; and wherein the enhanced modulation is a greater modulating effect on the biological target as compared to the reference molecule.2. The method of embodiment 1, wherein the biological target is located on the cell surface of a brain cell selected from a microglial cell, an astrocyte, an oligodendrocyte, and a neuron.3. The method of embodiment 2, wherein the cell surface receptor is located on a microglial cell.4. The method of embodiment 2 or 3, wherein the cell surface receptor 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, ADAM 17, and Siglecl 1.5. A method of screening to identify a biological target for the treatment of a cancer in the brain of a patient, the method comprising:(a) selecting a biological target selected from the group consisting of ALK, AXL, B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, CD79b, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL- 3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, R0R1, TF (tissue factor), and TROP2;(b) providing a molecule comprising a biological target-binding moiety and a TfR-binding moiety;(c) testing whether the molecule comprising a biological target-binding moiety and a TfR-binding moiety has enhanced modulation of the biological target, as compared to a reference molecule comprising the biological target-binding moiety but lacking the TfR- binding moiety; and(d) selecting the biological target for treatment of cancer in the brain of the patient based on whether the TfR-binding moiety enhances modulation of the target; wherein the biological target is not a TfR; and wherein the enhanced modulation is a greater modulating effect on the biological target as compared to the reference molecule.6. The method of embodiment 5, wherein the cancer is glioblastoma or a metastatic cancer in the brain.7. The method of any one of embodiments 1 to 6, wherein the molecule has a binding affinity to the TfR from about 2 pM to about 10 pM.8. A method for optimizing the activity of a therapeutic molecule that binds to both a therapeutic target and a TfR, the method comprising:(a) selecting a therapeutic target in the brain associated with a neurodegenerative disease;(b) providing the therapeutic molecule comprising a therapeutic target-binding moiety and a TfR-binding moiety;(c) testing whether the therapeutic molecule enhances modulation and / or target engagement of the therapeutic target relative to a reference molecule comprising the the therapeutic target-binding moiety but lacking the TfR-binding moiety;(d) modifying the binding affinity of the therapeutic target-binding moiety and / or the TfR-binding moiety to achieve the desired acivity of the therapeutic molecule.9. The method of embodiment 8, wherein the method further comprises repeating steps (c) and (d) until the desired activity of the therapeutic molecule is achieved.10. A method of modulating a biological activity of a cell, the method comprising contacting the cell with a molecule that binds to:(i) a biological target that is expressed by the cell; and(ii) a transferrin receptor (TfR) with a binding affinity from about 2 pM to about 10 pM, in an amount sufficient to modulate at least one biological activity of the cell.11. A method of modulating a biological activity of a cell the method comprising:(a) providing a molecule that binds to:(i) a biological target that is expressed by the cell; and(ii) a transferrin receptor (TfR) with a binding affinity from about 2 pM to about 10 pM; and(b) contacting the cell with the molecule of step (a) in an amount sufficient to modulate at least one biological activity of the cell.12. The method of embodiment 10 or 11, wherein the cell is an in vitro assay.13. The method of embodiment 10 or 11, wherein the cell is in a subject.14. The method of embodiment 13, wherein the cell is in the brain of the subject.15. The method of any one of embodiments 10 to 14, wherein the molecule has a greater modulating effect on the cell than a corresponding molecule that does not bind to the TfR or a corresponding molecule that binds to the TfR with a binding affinity stronger than 2 pM.16. The method of any one of embodiments 10 to 15, wherein the biological target is expressed on the cell surface of the cell.17. The method of embodiment 16, wherein the cell also expresses a TfR.18. The method of any one of embodiments 10 to 17, wherein the biological target is not a TfR.19. The method of any one of embodiments 10 to 18, wherein the cell is selected from the group consisting of a microglial cell, an astrocyte, an oligodendrocyte, a neuron, and a tumor cell.20. A method of modulating a biological target of a subject, the method comprising:(a) selecting a molecule that binds to a transferrin receptor (TfR) and to the biological target, wherein the molecule binds to the TfR with a binding affinity from about 2 pM to about 10 pM; and(b) peripherally administering the molecule to the subject in an effective amount to modulate the biological target, wherein the biological target is not a TfR.21. The method of embodiment 20, wherein the biological target is in the brain of the subject.22. A method of screening to identify enhanced target engagement of a biological target in the brain of a model organism that expresses a TfR molecule, the method comprising:(a) administering to the model organism a molecule that binds a TfR and the biological target that is not a TfR;(b) measuring activity of the biological target; and(c) comparing the activity measured in step (b) with that of a reference molecule that binds to the biological target, wherein the reference molecule does not bind to the TfR, or binds to the TfR with a binding affinity that differs from the binding affinity of the molecule.23. The method of embodiment 22, wherein the target engagement of the biological target by the moleucle is enhanced relative to that of the reference molecule.24. The method of embodiment 22 or 23, wherein the molecule and the reference molecule have approximately equivalent brain exposure.25. A method of screening to identify enhanced target engagement of a biological target in the brain of a model organism that expresses a TfR molecule, the method comprising:(a) administering to the model organism a first molecule that bind a TfR and the biological target that is not a TfR;(b) measuring activity of the biological target;(c) repeating steps (a) and (b) using a second molecule that binds the TfR and the same biological target, wherein the first and second molecules bind to the TfR with different binding affinities; and(d) comparing the activities of the biological target measured using the first molecule and the second molecule.26. The method of embodiment 25, further comprising repeating steps (a) and (b) using other molecules one at a time, wherein the other molecules bind to the TfR with different binding affinities and bind to the same biological target.27. The method of embodiment 25 or 26, wherein the molecules that bind to the TfR have a TfR-binding affinity from about 2 pM to about 10 pM.28. The method of any one of embodiments 25 to 27, wherein the first molecule and the second molecule have approximately equivalent brain exposure.29. The method of any one of embodiments 22 to 28, wherein the model organism expresses a TfR molecule that has a partially humanized or fully human extracellular domain.30. The method of any one of embodiments 22 to 29, wherein the biological target is a cell surface receptor.31. A method of transporting a therapeutic agent across the blood-brain barrier (BBB) of a subject, the method comprising:(a) selecting a molecule that binds to a transferrin receptor (TfR) of the subject with a binding affinity from about 2 pM to about 10 pM to attach to the therapeutic agent; and(b) peripherally administering to the subject the molecule attached to the therapeutic agent in a therapeutically effective amount.32. A method of transporting a therapeutic agent across the blood-brain barrier (BBB) of a subject, the method comprising peripherally administering to the subject a molecule comprising the therapeutic agent linked to a molecule that binds a transferrin receptor (TfR), wherein the molecule has been selected as having a binding affinity to the TfR from about 2 pM to about 10 pM.33. The method of embodiment 31 or 32, wherein the therapeutic agent is a polypeptide that binds to a cell surface receptor, wherein the cell surface receptor is located on a microglial cell or a cancer cell.34. The method of embodiment 33, wherein the cell surface receptor is located on a microglial cell.35. The method of embodiment 34, wherein the cell surface receptor 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, ADAM 17, and Siglecl l.36. The method of embodiment 33, wherein the cell surface receptor is located on a cancer cell.37. The method of embodiment 36, wherein the cancer cell is a solid tumor cancer cell.38. The method of embodiment 37, wherein the cell surface receptor is selected from the group consisting of ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL-3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, R0R1, TF (tissue factor), and TROP2.39. The method of embodiment 36, wherein the cancer cell is a hematological cancer cell.40. The method of embodiment 39, wherein 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.41. A method of producing a therapeutic molecule that has optimized activity towards a therapeutic target, the method comprising:(a) providing a molecule that binds (i) a transferrin receptor (TfR) and (ii) the therapeutic target;(b) generating one or more variants of the molecule that have different binding affinities to the TfR; and(c) measuring at least one biological activity of the molecule that is associated with the therapeutic target to determine whether the changes in the TfR binding affinity of the molecule alter the biological activity of the molecule, thereby producing a therapeutic molecule with optimized activity against the therapeutic target.42. The method of embodiment 41, wherein the therapeutic target is expressed on the cell surface of a cell that also expresses a TfR.43. The method of embodiment 42, wherein the cell is a microglia.44. The method of embodiment 42, wherein the cell is a cancer cell in the brain.45. The method of any one of embodiments 41 to 44, wherein the biological activity is measured in a cell or model organism.46. The method of embodiment 45, wherein the model organism is a mouse.47. The method of embodiment 46, wherein the mouse expresses a human TfR.48. The method of embodiment 46 or 47, wherein the mouse is a huTfRapicalknock-in mouse.49. The method of any one of embodiments 41 to 48, wherein the portion of the molecule that binds to the TfR comprises:(a) antibody variable regions that bind to the TfR; or(b) a polypeptide that has been engineered to bind the TfR; or(c) a polypeptide comprising an immunoglobulin or a portion thereof; or(d) a fibronectin type III domain.50. A method of producing a polypeptide for transport across the bloodbrain barrier (BBB) of a subject, the method comprising:(a) providing a polypeptide that binds to a transferrin receptor (TfR) with a binding affinity stronger than 2 pM;(b) making one or more amino acid substitutions, insertions, or deletions in the polypeptide, thereby creating one or more modified polypeptides, each having an altered binding affinity to the TfR; and(c) selecting a polypeptide of step (b) having a binding affinity to the TfR from about 2 pM to about 10 pM, thereby producing the polypeptide for transport across the BBB.51. The method of embodiment 50, wherein the polypeptide transports a therapeutic agent across the BBB.52. The method of embodiment 50 or 51, wherein the polypeptide is a part of a dimer that is monovalent for TfR binding.53. The method of any one of embodiments 50 to 52, further comprising producing a therapeutic molecule that comprises the polypeptide selected from step (c) and a therapeutic agent, wherein the polypeptide and the therapeutic agent are linked.54. A method of treating a human subject having a brain disease, the method comprising:(a) selecting a therapeutic molecule for the treatment of the brain disease by (i) its modulation of a biological target in the brain, wherein the biological target is not a transferrin receptor (TfR), and (ii) its binding to a TfR at an affinity of about 2 to about 10 pM; and(b) peripherally administering the therapeutic molecule to the human subject in a therapeutically effective amount to treat the brain disease.55. The method of embodiment 54, wherein the biological target is a cell surface receptor.56. A method of prolonging brain exposure to a therapeutic agent in a subject, the method comprising administering to the subject a molecule that binds to a transferrin receptor (TfR) with a binding affinity from about 2 pM to about 10 pM, wherein the molecule is linked to the therapeutic agent.57. The method of embodiment 56, wherein the molecule prolongs brain exposure to the therapeutic agent as compared to a corresponding molecule that does not bind to the TfR or a corresponding molecule that binds to the TfR with a binding affinity stronger than 2 pM.58. A method of sustaining a modulating effect of a therapeutic agent in the brain of a subject, the method comprising administering to the subject a molecule that binds to a transferrin receptor (TfR) with a binding affinity from about 2 pM to about 10 pM, wherein the molecule is linked to the therapetic agent.59. The method of embodiment 58, wherein the molecule increases the duration of the modulating effect of the therapeutic agent as compared to a correspondingmolecule that does not bind to the TfR or a corresponding molecule that binds to the TfR with a binding affinity stronger than 2 pM.60. The method of embodiment 58 or 59, wherein the therapeutic agent binds to a biological target and the modulating effect is a reduction in the level of the biological target.61. The method of any one of embodiments 56 to 60, wherein the therapeutic agent is a known therapeutic agent.62. The method of any one of embodiments 61, wherein the known therapeutic agent binds to a therapeutic target selected from the group consisting of ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL-3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, ROR1, TF (tissue factor), and TROP2.63. The method of any one of embodiments 61, wherein the known therapeutic agent binds to a therapeutic target selected from the group consisting of B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b.64. The method of any one of embodiments 61, wherein the known therapeutic agent is selected from the group consisting of lorlatinib, crizotinib, cabozantinib, basiliximab, daclizumab, bivatuzumab, promiximab, lorvotuzumab, polatuzumab, tusamitamab, sunitinib, cetuximab, panitumumab, nimotuzumab, necitumumab, rindopepimut (CDX-110), amivantamab, pemigatinib, erdafitinib, STRO-002, bevacizumab, naxitamab, ipilimumab, tebentafusp, glembatumumab, margetuximab-cmkb, enhertu, trastuzumab, pertuzumab, patritumab, seribantumab, lumretuzumab, elgemtumab, U3-1402, AV-203, KTN3379, AVE1642, MK-0646, cixutumumab, ladiratuzumab, gemtuzumab, pembrolizumab, sacituzumab, samrotamab, amivantamab-vmjw, TEPMETKO, lifastuzumab,177lutetium-PSMA-617, cofetuzumab, Zt / g4-MMAE, VLS-101, brexucabtagene, CS5001, tisotumab, sacituzumab, teclistamab, atezolizumab, avelumab, cosibelimab, durvalumab,belantamab, benralizumab, tafasitamab, loncastuximab, obinutuzumab, ofatumumab, rituximab, MEN1309 / OBT076, inotuzumab, and brentuximab.65. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises one of the following:(a) F at position 384 and S at position 413;(b) E at position 380 and the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389;(c) E at position 380, N at position 386, and E at position 387;(d) E at position 380, E at position 387, and Y at position 421;(e) E at position 380, V at position 389, and T at position 413; or(f) Y at position 384, V at position 389, and S at position 413, wherein the positions are determined according to EU numbering.66. The polypeptide of embodiment 65, wherein the modified CH3 domain comprises F at position 384 and S at position 413.67. The polypeptide of embodiment 66, wherein the modified CH3 domain further comprises one or more amino acid substitutions in a set of amino acid positions comprising 386-390, 416, and 421.68. The polypeptide of embodiment 66 or 67, wherein the modified CH3 domain comprises(i) N or T at position 386;(ii) E at position 387;(iii) W at position 388;(iv) a polar amino acid or V at position 389;(v) S or N at position 390;(vi) E at position 416; and / or(vii) an aromatic amino acid at position 421.69. The polypeptide of embodiment 65, wherein the modified CH3 domain comprises E at position 380 and the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389.70. The polypeptide of embodiment 69, wherein the modified CH3 domain further comprises one or more amino acid substitutions in a set of amino acid positions comprising 384, 390, 413, 415, 416, and 421.71. The polypeptide of embodiment 69 or 70, wherein the modified CH3 domain comprises:(i) Y at position 384;(ii) S or N at position 390;(iii) a polar amino acid at position 413;(iv) S or E at position 415;(v) E at position 416; and / or(vi) F at position 421.72. The polypeptide of embodiment 65, wherein the modified CH3 domain comprises E at position 380, N at position 386, and E at position 387.73. The polypeptide of embodiment 72, wherein the modified CH3 domain further comprises one or more amino acid substitutions in a set of amino acid positions comprising 384, 388-390, 413, 415, 416, and 421.74. The polypeptide of embodiment 72 or 73, wherein the modified CH3 domain comprises:(i) Y at position 384;(ii) W at position 388;(iii) S at position 389;(iv) S or N at position 390;(v) a polar amino acid at position 413;(vi) S or E at position 415;(vii) E at position 416; and / or(viii) F at position 421.75. The polypeptide of embodiment 65, wherein the modified CH3 domain comprises E at position 380, E at position 387, and Y at position 421.76. The polypeptide of embodiment 75, wherein the modified CH3 domain further comprises one or more amino acid substitutions in a set of amino acid positions comprising 384, 386, 388-390, 413, 415, and 416.77. The polypeptide of embodiment 75 or 76, wherein the modified CH3 domain comprises:(i) Y at position 384;(ii) T at position 386;(iii) W at position 388;(iv) S or V at position 389;(v) S or N at position 390;(vi) a polar amino acid at position 413;(vii) S or E at position 415; and / or(viii) E at position 416.78. The polypeptide of embodiment 65, wherein the modified CH3 domain comprises E at position 380, V at position 389, and T at position 413.79. The polypeptide of embodiment 78, wherein the modified CH3 domain further comprises one or more amino acid substitutions in a set of amino acid positions comprising 384, 386-388, 390, 416, and 421.80. The polypeptide of embodiment 78 or 79, wherein the modified CH3 domain comprises:(i) Y at position 384;(ii) T at position 386;(iii) E at position 387;(iv) W at position 388;(v) N at position 390;(vi) E at position 416; and / or(vii) F at position 421.81. The polypeptide of embodiment 65, wherein the modified CH3 domain comprises Y at position 384, V at position 389, and S at position 413.82. The polypeptide of embodiment 81, wherein the modified CH3 domain further comprises one or more amino acid substitutions in a set of amino acid positions comprising 386-388, 390, 416, and 421.83. The polypeptide of embodiment 81 or 82, wherein the modified CH3 domain comprises:(i) T at position 386;(ii) E at position 387;(iii) W at position 388;(vi) N at position 390;(v) E at position 416; and / or(vi) F at position 421.84. The polypeptide of any one of embodiments 65 to 83, 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:28-51.85. The polypeptide of embodiment 84, wherein the modified CH3 domain comprises amino acids 111-217 of any one of SEQ ID NOS:28-51.86. The polypeptide of any one of embodiments 65 to 85, wherein the polypeptide comprises a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to a sequence of any one of SEQ ID NOS:28-51.87. The polypeptide of embodiment 86, wherein the polypeptide comprises a sequence of any one of SEQ ID NOS:28-51.88. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: F at position 384, N or T at position 386, E at position 387, W at position 388, a polar amino acid or V at position 389, S or N at position 390, S at position 413, E at position 416, and an aromatic amino acid at position 421, wherein the positions are determined according to EU numbering.89. The polypeptide of embodiment 88, wherein the aromatic amino acid is F or Y.90. The polypeptide of embodiment 88 or 89, wherein the polypeptide binds to the TfR with a binding affinity between about 1 pM and about 36 pM.91. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: E at position 380, Y at position 384, the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389, S or N at position 390, a polar amino acid at position 413, S or E at position 415, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering.92. The polypeptide of embodiment 91, wherein the polypeptide binds to the TfR with a binding affinity between about 3.5 pM and about 12 pM.93. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: E at position 380, Y at position 384, N at position 386, E at position 387, W at position 388, S at position 389, S or N at position 390, a polar amino acid at position 413, S or E at position 415, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering.94. The polypeptide of embodiment 93, wherein the polypeptide binds to the TfR with a binding affinity between about 5 pM and about 19 pM.95. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: E at position 380, Y at position 384, T at position 386, E at position 387, W at position 388, S or V at position 389, S or N at position 390, a polar amino acid at position 413, S or E at position 415, E at position 416, and Y at position 421, wherein the positions are determined according to EU numbering.96. The polypeptide of any one of embodiments 95, wherein the polar amino acid is S or T.97. The polypeptide of embodiment 95 or 96, wherein the polypeptide binds to the TfR with a binding affinity between about 1 pM and about 15 pM.98. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: E at position 380, Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, N at position 390, and T at position 413, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering.99. The polypeptide of embodiment 98, wherein the polypeptide binds to the TfR with a binding affinity about 2.5 pM.100. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, N at position 390, and S at position 413, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering.101. The polypeptide of embodiment 100, wherein the polypeptide binds to the TfR with a binding affinity about 3.6 pM.102. The polypeptide of any one of embodiments 65 to 101, wherein the polypeptide comprises at least 85% identity to the sequence of SEQ ID NO:68.103. The polypeptide of any one of embodiments 65 to 102, wherein the modified CH3 domain further comprises at least one modification that promotes heterodimerization.104. The polypeptide of embodiment 103, wherein the at least one modification that promotes heterodimerization comprises a T366W substitution, according to EU numbering.105. The polypeptide of embodiment 103, wherein the at least one modification that promotes heterodimerization comprises T366S, L368A, and Y407V substitutions, according to EU numbering.106. The polypeptide of any one of embodiments 65 to 105, wherein the polypeptide further comprising L at position 428 and S at position 434.107. The polypeptide of any one of embodiments 65 to 106, wherein the polypeptide further comprises a CH2 domain.108. The polypeptide of embodiment 107, wherein the CH2 and CH3 domains form an Fc polypeptide.109. The polypeptide of embodiment 107 or 108, wherein the CH2 domain comprises modifications that reduce effector function.110. The polypeptide of embodiment 109, wherein the modifications that reduce effector function comprise Ala at position 234 and Ala at position 235, according to EU numbering.111. The polypeptide of embodiment 109 or 110, wherein the CH2 domain comprises Gly or Ser at position 329, according to EU numbering.112. The polypeptide of embodiment 107 or 108, wherein the CH2 domain does not comprise modifications that reduce effector function.113. The polypeptide of any one of embodiments 107 to 112, wherein the CH2 domain is derived from a human IgGl, IgG2, IgG3, or IgG4 CH2 domain.114. The polypeptide of any one of embodiments 65 to 113, wherein the polypeptide is part of a dimer.115. The polypeptide of embodiment 114, wherein the dimer is an Fc dimer.116. The polypeptide of any one of embodiments 65 to 115, wherein the polypeptide is further joined to a Fab.117. The polypeptide of any one of embodiments 114 to 116, wherein the polypeptide is a first polypeptide of a dimer such that the dimer is monovalent for TfR binding.118. The polypeptide of any one of embodiments 114 to 116, wherein the polypeptide is a first polypeptide of a dimer such that the dimer is bivalent for TfR binding.119. The polypeptide of any one of embodiments 65 to 118, wherein the C- terminal lysine of the polypeptide is absent.120. A polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of embodiments 65 to 119.121. A vector comprising the polynucleotide of embodiment 120.122. A host cell comprising the polynucleotide of embodiment 120.123. A method for producing a polypeptide comprising a modified CH3 domain, comprising culturing a host cell under conditions in which the polypeptide encoded by the polynucleotide of embodiment 120 is expressed.124. A pharmaceutical composition comprising the polypeptide of any one of embodiments 65 to 119 and a pharmaceutically acceptable carrier.125. A method of transcytosis of a therapeutic agent across an endothelium, the method comprising contacting the endothelium with a composition comprising a polypeptide of any one of embodiments 65 to 119 linked to the therapeutic agent.126. The method of embodiment 125, wherein the endothelium is the bloodbrain barrier (BBB).127. An improvement to a method of modulating a biological activity of a cell by providing a molecule that binds to the biological target that is expressed by the cell, wherein the biological target is not a TfR, the improvement comprising:(a) providing a molecule that binds to a TfR with a binding affinity from about 2 pM to about 10 pM; and(b) contacting the cell with the molecule of step (a) in an amount sufficient to modulate at least one biological activity of the cell.128. The improvement of embodiment 127, wherein the cell is a microglial cell, an astrocyte, an oligodendrocyte, a neuron, and a tumor cell.129. The improvement of embodiment 128, wherein the cell is a microglial cell.130. The improvement of of embodiment 129, wherein the cell surface receptor 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, ADAM 17, and Siglecl l.131. The improvement of embodiment 127, wherein the cell is a cancer cell.132. The improvement of embodiment 131, wherein the cancer cell is a solid tumor cancer cell.133. The improvement of embodiment 132, wherein the cell surface receptor is selected from the group consisting of ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL-3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, ROR1, TF (tissue factor), and TROP2.134. The improvement of embodiment 131, wherein the cancer cell is a hematological cancer cell.135. The improvement of embodiment 134, wherein 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.136. An improvement to a method of selecting a biological target for treatment of a disease, wherein the biological target in the brain is located on a cell surface that also contains a TfR, the improvement comprising:(a) testing whether a molecule comprising a biological target-binding moiety and a TfR-binding moiety has enhanced modulation of the biological target, as compared to a molecule comprising the biological target-binding moiety but lacking the TfR-binding moiety; and(b) selecting the biological target for treatment based on the enhanced modulation observed in step (a), wherein the disease is selected from the group consisting of Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and brain cancer; and wherein said treatment of disease is by the administration of a molecule comprising a TfR-binding moiety and target-binding moiety to said biological target.137. An improvement to a method of producing a therapeutic molecule that has optimized activity towards a known therapeutic target, the improvement comprising: (a) providing a molecule that binds (i) a transferrin receptor (TfR) and (ii) the known therapeutic target;(b) generating one or more variants of the molecule that have different binding affinities to the TfR; and(c) measuring at least one biological activity of the molecule that is associated with the known therapeutic target to determine whether the changes in the TfR binding affinity of the molecule alter the biological activity of the molecule, thereby producing a therapeutic molecule with optimized activity against the therapeutic target.138. The improvement of embodiment 137, wherein the known therapeutic target is selected from the group consisting of ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL-3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, ROR1, TF (tissue factor), and TROP2.139. The improvement of embodiment 137, wherein the known therapeutic target is selected from the group consisting of B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b.140. The improvement of any one of embodiments 137 to 139, wherein the portion of the therapeutic molecule that binds the known therapeutic target is selected from the group consisting of lorlatinib, crizotinib, cabozantinib, basiliximab, daclizumab,bivatuzumab, promiximab, lorvotuzumab, polatuzumab, tusamitamab, sunitinib, cetuximab, panitumumab, nimotuzumab, necitumumab, rindopepimut (CDX-110), amivantamab, pemigatinib, erdafitinib, STRO-002, bevacizumab, naxitamab, ipilimumab, tebentafusp, glembatumumab, margetuximab-cmkb, enhertu, trastuzumab, pertuzumab, patritumab, seribantumab, lumretuzumab, elgemtumab, U3-1402, AV-203, KTN3379, AVE1642, MK- 0646, cixutumumab, ladiratuzumab, gemtuzumab, pembrolizumab, sacituzumab, samrotamab, amivantamab-vmjw, TEPMETKO, lifastuzumab,177lutetium-PSMA-617, cofetuzumab, Zt / g4-MMAE, VLS-101, brexucabtagene, CS5001, tisotumab, sacituzumab, teclistamab, atezolizumab, avelumab, cosibelimab, durvalumab, belantamab, benralizumab, tafasitamab, loncastuximab, obinutuzumab, ofatumumab, rituximab, MEN1309 / OBT076, inotuzumab, brentuximab, and polatuzumab.

[0323] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. The sequences of the sequence accession numbers cited herein are hereby incorporated by reference.INFORMAL SEQUENCE LISTINGIll

Claims

WHAT IS CLAIMED IS:

1. A method of screening to identify a biological target for the treatment of a neurodegenerative disease, the method comprising:(a) selecting a biological target in the brain associated with a neurodegenerative disease;(b) providing a molecule comprising a biological target-binding moiety and a TfR-binding moiety;(c) testing whether the molecule comprising a biological target-binding moiety and a TfR-binding moiety has enhanced modulation of the biological target, as compared to a reference molecule comprising the biological target-binding moiety but lacking the TfR- binding moiety; and(d) selecting the biological target for treatment of the neurodegenerative disease based on whether the TfR-binding moiety enhances modulation of the target, wherein the biological target is not a TfR; and wherein the enhanced modulation is a greater modulating effect on the biological target as compared to the reference molecule.

2. The method of claim 1, wherein the biological target is located on the cell surface of a brain cell selected from a microglial cell, an astrocyte, an oligodendrocyte, and a neuron.

3. The method of claim 2, wherein the cell surface receptor 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 Siglecl 1.

4. A method of screening to identify a biological target for the treatment of a cancer in the brain of a patient, the method comprising:(a) selecting a biological target selected from the group consisting of ALK, AXL, B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, CD79b, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL- 3BP (galectin binidng protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeatcontaining 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, R0R1, TF (tissue factor), and TROP2;(b) providing a molecule comprising a biological target-binding moiety and a TfR-binding moiety;(c) testing whether the molecule comprising a biological target-binding moiety and a TfR-binding moiety has enhanced modulation of the biological target, as compared to a reference molecule comprising the biological target-binding moiety but lacking the TfR- binding moiety; and(d) selecting the biological target for treatment of cancer in the brain of the patient based on whether the TfR-binding moiety enhances modulation of the target; wherein the biological target is not a TfR; and wherein the enhanced modulation is a greater modulating effect on the biological target as compared to the reference molecule.

5. The method of claim 4, wherein the cancer is glioblastoma or a metastatic cancer in the brain.

6. The method of claim 1, wherein the molecule has a binding affinity to the TfR from about 2 pM to about 10 pM.

7. A method of screening to identify enhanced target engagement of a biological target in the brain of a model organism that expresses a TfR molecule, the method comprising:(a) administering to the model organism a first molecule that bind a TfR and the biological target that is not a TfR;(b) measuring activity of the biological target;(c) repeating steps (a) and (b) using a second molecule that binds the TfR and the same biological target, wherein the first and second molecules bind to the TfR with different binding affinities; and(d) comparing the activities of the biological target measured using the first molecule and the second molecule.

8. The method of claim 7, further comprising repeating steps (a) and (b) using other molecules one at a time, wherein the other molecules bind to the TfR with different binding affinities and bind to the same biological target.

9. The method of claim 7, wherein the molecules that bind to the TfR have a TfR-binding affinity from about 2 pM to about 10 pM.

10. The method of claim 7, wherein the first molecule and the second molecule have approximately equivalent brain exposure.

11. The method of claim 7, wherein the model organism expresses a TfR molecule that has a partially humanized or fully human extracellular domain.

12. The method of claim 7, wherein the biological target is a cell surface receptor.

13. A method of producing a therapeutic molecule that has optimized activity towards a therapeutic target, the method comprising:(a) providing a molecule that binds (i) a transferrin receptor (TfR) and (ii) the therapeutic target;(b) generating one or more variants of the molecule that have different binding affinities to the TfR; and(c) measuring at least one biological activity of the molecule that is associated with the therapeutic target to determine whether the changes in the TfR binding affinity of the molecule alter the biological activity of the molecule, thereby producing a therapeutic molecule with optimized activity against the therapeutic target.

14. The method of claim 13, wherein the therapeutic target is expressed on the cell surface of a cell that also expresses a TfR.

15. The method of claim 14, wherein the cell is a microglia.

16. The method of claim 14, wherein the cell is a cancer cell in the brain.

17. The method of claim 13, wherein the biological activity is measured in a cell or model organism.

18. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises one of the following:(a) F at position 384 and S at position 413;(b) E at position 380 and the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389;(c) E at position 380, N at position 386, and E at position 387;(d) E at position 380, E at position 387, and Y at position 421;(e) E at position 380, V at position 389, and T at position 413; or(f) Y at position 384, V at position 389, and S at position 413, wherein the positions are determined according to EU numbering.

19. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: F at position 384, N or T at position 386, E at position 387, W at position 388, a polar amino acid or V at position 389, S or N at position 390, S at position 413, E at position 416, and an aromatic amino acid at position 421, wherein the positions are determined according to EU numbering.

20. The polypeptide of claim 19, wherein the polypeptide binds to the TfR with a binding affinity between about 1 pM and about 36 pM.

21. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: E at position 380, Y at position 384, the sequence of TEWT (SEQ ID NO:73) from position 386 to position 389, S or N at position 390, a polar amino acid at position 413, S or E at position 415, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering.

22. The polypeptide of claim 21, wherein the polypeptide binds to the TfR with a binding affinity between about 3.5 pM and about 12 pM.

23. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: E at position 380, Y at position 384, N at position 386, E at position 387, W at position 388, S at position 389, S or N at position 390, a polar amino acid at position 413, S or E at position 415, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering.

24. The polypeptide of claim 23, wherein the polypeptide binds to the TfR with a binding affinity between about 5 pM and about 19 pM.

25. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: E at position 380, Y at position 384, T at position 386, E at position 387, W at position 388, S or V at position 389, S or N at position 390, a polar amino acid at position 413, S or E at position 415, E at position 416, and Y at position 421, wherein the positions are determined according to EU numbering.

26. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: E at position 380, Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, N at position 390, and T at position 413, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering.

27. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor (TfR), wherein the modified CH3 domain comprises: Y at position 384, T at position 386, E at position 387, W at position 388, V at position 389, N at position 390, and S at position 413, E at position 416, and F at position 421, wherein the positions are determined according to EU numbering.

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