Transferrin receptor binding polypeptides and uses thereof

Abstract

To provide a means for delivering to the brain an effector function positive therapeutic agent that does not cause reticulocyte loss.SOLUTION: The present disclosure generally provides an Fc polypeptide dimer comprising a non-native transferrin receptor (TfR) binding site, wherein the Fc polypeptide dimer does not substantially deplete reticulocytes in vivo, but retains binding to an Fey receptor (FcyR). The present disclosure also provides an Fc polypeptide dimer wherein one of the Fc polypeptides comprises a non-native site that specifically binds TfR and a modification (s) in the Fc polypeptide comprising the TfR binding site that reduces binding of an Fc γ R when bound to TfR, wherein the other Fc polypeptide does not comprise a TfR binding site but retains binding to an Fc γ R.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 615,914, filed January 10, 2018, U.S. Provisional Patent Application No. 62 / 631,281, filed February 15, 2018, U.S. Provisional Patent Application No. 62 / 682,639, filed June 8, 2018, and U.S. Provisional Patent Application No. 62 / 721,275, filed August 22, 2018, the disclosures of each of which are incorporated herein by reference in their entirety for all purposes.

[0002] Field The present disclosure relates to modified Fc polypeptide dimers that are capable of binding to the transferrin receptor (TfR) and inducing at least one effector function activity (e.g., antibody-dependent cellular cytotoxicity (ADCC)), but do not result in significant reduction of reticulocytes. [Background technology]

[0003] TfR has been proposed as a target for receptor-mediated transcytosis of therapeutic agents across the blood-brain barrier (BBB). TfR is expressed on endothelial cells that form the BBB, but TfR is also expressed on other cell types, including reticulocytes. Previous studies have shown that anti-TfR antibodies can deplete reticulocytes from the circulation.

[0004] Because reticulocyte reduction is mediated by effector function activity, this toxicity can be overcome by modifications that reduce or eliminate effector function, however, this approach precludes the use of therapeutic agents in which effector function is desirable or required.

[0005] Therefore, it would be useful to have a means for delivering effector function-positive therapeutic agents to the brain that does not cause reticulocyte depletion. Summary of the Invention

[0006] The present inventors have developed Fc polypeptides engineered to bind to TfR. These Fc polypeptides can be actively transported into the brain by receptor-mediated transcytosis through binding to TfR at the BBB. Because Fc polypeptides can induce effector function activities, including ADCC, through binding to Fcγ receptors (FcγR) on immune cells, and because TfR is expressed on reticulocytes, simultaneous binding of these polypeptides to reticulocytes and FcγR can lead to a reduction in reticulocytes. Effector function can be reduced or eliminated by introducing mutations into the Fc polypeptide, but this is undesirable for certain therapeutic applications.

[0007] The present disclosure is based on the development of modified Fc polypeptide dimers that bind to TfR, cross the BBB, and retain effector function activity, but do not cause significant TfR-dependent toxicity, including reticulocyte depletion. Such dimers can be engineered as described herein.

[0008] In one aspect, the present disclosure relates to a modified Fc polypeptide dimer, or a dimeric fragment thereof, that (a) comprises a TfR binding site that specifically binds to TfR, (b) is capable of binding to an Fcγ receptor (FcγR), and (c) does not substantially reduce reticulocytes in vivo.

[0009] In one aspect, the present disclosure relates to an engineered Fc polypeptide dimer, or a dimeric fragment thereof, comprising: (a) a first Fc polypeptide that specifically binds to TfR, comprising (i) a TfR-binding site and (ii) one or more amino acid modifications that, e.g., reduce FcγR binding when bound to TfR (e.g., limit or no reduction in FcγR binding when not bound to TfR); and (b) a second Fc polypeptide that does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0010] In some embodiments of this aspect, the TfR-binding site comprises a modified CH3 domain. In some embodiments, the modified CH3 domain is derived from a CH3 domain of human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the modified CH3 domain comprises 5, 6, 7, 8, or 9 substitutions at a set of amino acid positions including: 384, 386, 387, 388, 389, 390, 413, 416, and 421, according to EU numbering. In certain embodiments, the modified CH3 domain further comprises 1, 2, 3, or 4 substitutions at positions including: 380, 391, 392, and 415.

[0011] In some embodiments, the modified CH3 domain further comprises one, two, or three substitutions at positions 414, 424, and 426, inclusive.

[0012] In some embodiments, the modified Fc polypeptide dimer binds to the apical domain of the TfR. In some embodiments, the modified Fc polypeptide dimer binds to the TfR without inhibiting the binding of transferrin to the TfR. In certain embodiments, the modified Fc polypeptide dimer binds to an epitope comprising amino acid 208 of the TfR.

[0013] In some embodiments, the modified CH3 domain comprises a Trp at position 388. In some embodiments, the modified CH3 domain comprises an aromatic amino acid at position 421. In particular embodiments, the aromatic amino acid at position 421 is Trp or Phe.

[0014] In some embodiments of this aspect, the modified CH3 domain comprises at least one position selected from the following: Leu, Tyr, Met, or Val at position 384; Leu, Thr, His, or Pro at position 386; Val, Pro, or an amino acid at position 387; Trp at position 388; Val, Ser, or Ala at position 389; Glu, Ala, Ser, Leu, Thr, or Pro at position 413; Thr, or an amino acid at position 416; and Trp, Tyr, His, or Phe at position 421.

[0015] In some embodiments of this aspect, the modified CH3 domain comprises two, three, four, five, six, seven, or eight positions selected from the following: Leu, Tyr, Met, or Val at position 384; Leu, Thr, His, or Pro at position 386; Val, Pro, or an amino acid at position 387; Trp at position 388; Val, Ser, or Ala at position 389; Glu, Ala, Ser, Leu, Thr, or Pro at position 413; Thr, or an amino acid at position 416; and Trp, Tyr, His, or Phe at position 421.

[0016] In some embodiments of this aspect, the CH3 domain comprises Leu or Met at position 384, Leu, His, or Pro at position 386, Val at position 387, Trp at position 388, Val or Ala at position 389, Pro at position 413, Thr at position 416, and / or Trp at position 421.

[0017] In some embodiments, the modified CH3 domain further comprises Ser, Thr, Gln, or Phe at position 391. In some embodiments, the modified CH3 domain further comprises Trp, Tyr, Leu, or Gln at position 380. In some embodiments, the modified CH3 domain further comprises Gln, Phe, or His at position 392. In some embodiments, the modified CH3 domain further comprises Trp at position 380 and / or Gln at position 392.

[0018] In some embodiments, the modified CH3 domain further comprises one, two, or three positions selected from the following: Lys, Arg, Gly, or Pro at position 414, Ser, Thr, Glu, or Lys at position 424, and Ser, Trp, or Gly at position 426.

[0019] In some embodiments, the modified CH3 domain comprises a Tyr at position 384, a Thr at position 386, a Glu or Val at position 387, a Trp at position 388, a Ser at position 389, a Ser or Thr at position 413, a Glu at position 416, and / or a Phe at position 421. In some embodiments, the modified CH3 domain further comprises a Trp, Tyr, Leu, or Gln at position 380. In some embodiments, the modified CH3 domain further comprises a Glu at position 415. In some embodiments, the modified CH3 domain further comprises a Trp at position 380 and / or a Glu at position 415. In some embodiments, the modified CH3 domain comprises an Asn at position 390.

[0020] In some embodiments, the modified CH3 domain comprises one or more of the following substitutions: Trp at position 380, Thr at position 386, Trp at position 388, Val at position 389, Ser or Thr at position 413, Glu at position 415, and / or Phe at position 421.

[0021] In some embodiments, the modified CH3 domain has 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: 4-29 and 64-127. In certain embodiments, the modified CH3 domain has 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: 66, 68, 94, 107-109, 119, and 268-270.

[0022] In some embodiments, the modified CH3 domain has at least 85% identity to amino acids 111-217 of SEQ ID NO:1, provided that the percent identity does not include the set of positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 according to EU numbering.

[0023] In some embodiments, the modified CH3 domain comprises amino acids 154-160 and / or 183-191 of any one of SEQ ID NOs: 4-29 and 125-127.

[0024] In some embodiments, the modified CH3 domain comprises at least one position selected from the following: Trp, Leu, or Glu at position 380, Tyr or Phe at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser, Ala, Val, or Asn at position 389, Ser or Asn at position 390, Thr or Ser at position 413, Glu or Ser at position 415, Glu at position 416, and Phe at position 421. In some embodiments, the modified CH3 domain comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from the following: Trp, Leu, or Glu at position 380, Tyr or Phe at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser, Ala, Val, or Asn at position 389, Ser or Asn at position 390, Thr or Ser at position 413, Glu or Ser at position 415, Glu at position 416, and Phe at position 421.

[0025] In some embodiments, the modified CH3 domain comprises the following 11 positions: Trp, Leu, or Glu at position 380, Tyr or Phe at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser, Ala, Val, or Asn at position 389, Ser or Asn at position 390, Thr or Ser at position 413, Glu or Ser at position 415, Glu at position 416, and Phe at position 421.

[0026] In some embodiments, the modified CH3 domain has 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: 4-29 and 64-127. In certain embodiments, the modified CH3 domain has 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: 66, 68, 94, 107-109, 119, and 268-270.

[0027] In some embodiments, residues at at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the positions corresponding to 380, 384, 386, 384, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424, and 426 according to the EU numbering scheme are not deleted or substituted.

[0028] In some embodiments, the modified CH3 domain comprises the sequence of any one of SEQ ID NOs: 38-61 and 131-173.

[0029] In some embodiments of this aspect, the modified CH3 domain further comprises (i) a Trp at position 366, or (ii) a Ser at position 366, an Ala at position 368, and a Val at position 407 according to the EU numbering scheme.

[0030] In some embodiments of this aspect, the corresponding unmodified CH3 domain is a CH3 domain of human IgG1, IgG2, IgG3, or IgG4.

[0031] In some embodiments of this aspect, for example, the amino acid modification that reduces FcγR binding when bound to TfR comprises Ala at positions 234 and 235 according to the EU numbering scheme. In some embodiments of this aspect, for example, the amino acid modification that reduces FcγR binding when bound to TfR further comprises Gly at position 329 according to the EU numbering scheme.

[0032] In some embodiments of this aspect, the first Fc polypeptide and / or the second Fc polypeptide comprises an amino acid modification that increases serum stability (e.g., serum half-life). In some embodiments, the amino acid modification that increases serum stability (e.g., serum half-life) comprises Tyr at position 252, Thr at position 254, and Glu at position 256, according to the EU numbering scheme. In some embodiments, the amino acid modification that increases serum stability (e.g., serum half-life) comprises (i) Leu at position 428 and Ser at position 434, or (ii) Ser or Ala at position 434, according to the EU numbering scheme.

[0033] In some embodiments of this aspect, the modified Fc polypeptide dimer is further fused to a Fab, hi some embodiments, the first Fc polypeptide and / or the second Fc polypeptide is further fused to a Fab.

[0034] In some embodiments, the first Fc polypeptide comprises knob mutation T366W and the second Fc polypeptide comprises hole mutations T366S, L368A, and Y407V according to the EU numbering scheme. In some embodiments, the first Fc polypeptide comprises hole mutations T366S, L368A, and Y407V and the second Fc polypeptide comprises knob mutation T366W according to the EU numbering scheme.

[0035] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and amino acid modifications L234A and L235A according to the EU numbering scheme and knob mutation T366W; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and not comprising a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 178, 190, 202, 214, 226, 238, 238, 252, 286, 298, and 310. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 397.

[0036] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme and knob mutation T366W; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and not comprising a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 179, 191, 203, 215, 227, 239, 275, 287, 299, and 311. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 397.

[0037] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and amino acid modifications L234A and L235A according to the EU numbering scheme, knob mutation T366W, and amino acid modifications M252Y, S254T, and T256E, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and not comprising a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 181, 193, 205, 217, 229, 241, 277, 289, 301, and 313. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 397.

[0038] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A and L235A according to the EU numbering scheme, the knob mutation T366W, and the amino acid modification N434S with or without M428L; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and the second Fc polypeptide does not comprise a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 323, 330, 337, 344, 351, 358, 365, 372, 379, and 386. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 397.

[0039] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, knob mutation T366W, and amino acid modifications M252Y, S254T, and T256E; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and not comprising a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 182, 194, 206, 218, 230, 242, 278, 290, 302, and 314. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 397.

[0040] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, the knob mutation T366W, and the amino acid modification N434S, with or without M428L; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and the second Fc polypeptide does not comprise a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs:324, 331, 338, 345, 352, 359, 366, 373, 380, and 387. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:397.

[0041] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and amino acid modifications L234A and L235A according to the EU numbering scheme and knob mutation T366W; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V and amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme, and the second Fc polypeptide does not comprise a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 178, 190, 202, 214, 226, 238, 252, 286, 298, and 310. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 400.

[0042] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A and L235A according to the EU numbering scheme and the knob mutation T366W; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme and the amino acid modification N434S, with or without M428L, and the second Fc polypeptide does not comprise a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 178, 190, 202, 214, 226, 238, 252, 286, 298, and 310. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 407.

[0043] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme and knob mutation T366W; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V and amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme, and not comprising a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 179, 191, 203, 215, 227, 239, 275, 287, 299, and 311. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 400.

[0044] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme and the knob mutation T366W; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme and the amino acid modification N434S, with or without M428L, and the second Fc polypeptide does not comprise a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 179, 191, 203, 215, 227, 239, 275, 287, 299, and 311. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 407.

[0045] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and amino acid modifications L234A and L235A according to the EU numbering scheme, knob mutation T366W, and amino acid modifications M252Y, S254T, and T256E; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme and amino acid modifications M252Y, S254T, and T256E, and does not comprise a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 181, 193, 205, 217, 229, 241, 277, 289, 301, and 313. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:400.

[0046] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A and L235A according to the EU numbering scheme, the knob mutation T366W, and the amino acid modification N434S, with or without M428L; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and the amino acid modification N434S, with or without M428L, and the second Fc polypeptide does not comprise a TfR-binding site or any modification that reduces FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs:323, 330, 337, 344, 351, 358, 365, 372, 379, and 386. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:407.

[0047] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, knob mutation T366W, and amino acid modifications M252Y, S254T, and T256E, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme and the amino acid modifications M252Y, S254T, and T256E, and does not comprise a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 182, 194, 206, 218, 230, 242, 278, 290, 302, and 314. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:400.

[0048] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, the knob mutation T366W, and the amino acid modification N434S, with or without M428L; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme and the amino acid modification N434S, with or without M428L, and the second Fc polypeptide does not comprise a TfR-binding site or any modification that reduces FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs:324, 331, 338, 345, 352, 359, 366, 373, 380, and 387. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:407.

[0049] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and amino acid modifications L234A and L235A according to the EU numbering scheme and hole mutations T366S, L368A, and Y407V; and (b) a second Fc polypeptide comprising knob mutation T366W according to the EU numbering scheme, and not comprising a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 184, 196, 208, 220, 232, 244, 280, 292, 304, and 316. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 391.

[0050] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, and hole mutations T366S, L368A, and Y407V, and (b) a second Fc polypeptide comprising the knob mutation T366W according to the EU numbering scheme, and not comprising the TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 185, 197, 209, 221, 233, 245, 281, 293, 305, and 317. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 391.

[0051] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and amino acid modifications L234A and L235A according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and amino acid modifications M252Y, S254T, and T256E; and (b) a second Fc polypeptide comprising knob mutation T366W according to the EU numbering scheme, and not comprising a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 187, 199, 211, 223, 235, 247, 283, 295, 307, and 319. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 391.

[0052] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A and L235A according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and the amino acid modification N434S, with or without M428L; and (b) a second Fc polypeptide comprising a knob mutation T366W according to the EU numbering scheme, and the second Fc polypeptide does not comprise a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 326, 333, 340, 347, 354, 361, 368, 375, 382, ​​and 389. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 391.

[0053] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and amino acid modifications L234A, L235A, and P329G, hole mutations T366S, L368A, and Y407V, and amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme; and (b) a second Fc polypeptide comprising knob mutation T366W according to the EU numbering scheme, and not comprising a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 188, 200, 212, 224, 236, 248, 284, 296, 308, and 320. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 391.

[0054] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and the amino acid modification N434S, with or without M428L; and (b) a second Fc polypeptide comprising a knob mutation T366W according to the EU numbering scheme, and the second Fc polypeptide does not comprise a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs:327, 334, 341, 348, 355, 362, 369, 376, 383, and 390. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:391.

[0055] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and amino acid modifications L234A and L235A according to the EU numbering scheme and hole mutations T366S, L368A, and Y407V; and (b) a second Fc polypeptide comprising knob mutation T366W and amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme, and not comprising a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 184, 196, 208, 220, 232, 244, 280, 292, 304, and 316. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 394.

[0056] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A and L235A according to the EU numbering scheme and the hole mutations T366S, L368A, and Y407V; and (b) a second Fc polypeptide comprising the knob mutation T366W according to the EU numbering scheme and the amino acid modification N434S, with or without M428L, and the second Fc polypeptide does not comprise the TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 184, 196, 208, 220, 232, 244, 280, 292, 304, and 316. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 404.

[0057] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, and hole mutations T366S, L368A, and Y407V; and (b) a second Fc polypeptide comprising knob mutation T366W and amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme, and not comprising a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 185, 197, 209, 221, 233, 245, 281, 293, 305, and 317. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 394.

[0058] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, and hole mutations T366S, L368A, and Y407V; and (b) a second Fc polypeptide comprising the knob mutation T366W according to the EU numbering scheme and the amino acid modification N434S, with or without M428L, and the second Fc polypeptide does not comprise the TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 185, 197, 209, 221, 233, 245, 281, 293, 305, and 317. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 404.

[0059] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A and L235A according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme; and (b) a second Fc polypeptide comprising a knob mutation T366W and the amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme, and not comprising a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 187, 199, 211, 223, 235, 247, 283, 295, 307, and 319. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:394.

[0060] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A and L235A according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and the amino acid modification N434S, with or without M428L; and (b) a second Fc polypeptide comprising a knob mutation T366W according to the EU numbering scheme and the amino acid modification N434S, with or without M428L, and does not comprise a TfR-binding site or any modification that reduces FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs:326, 333, 340, 347, 354, 361, 368, 375, 382, ​​and 389. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:404.

[0061] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A, L235A, and P329G, hole mutations T366S, L368A, and Y407V, and amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme; and (b) a second Fc polypeptide comprising a knob mutation T366W and the amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme, and not comprising a TfR-binding site or any modifications that reduce FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 188, 200, 212, 224, 236, 248, 284, 296, 308, and 320. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:394.

[0062] In another aspect, the disclosure relates to an engineered Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR-binding site and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and the amino acid modification N434S, with or without M428L; and (b) a second Fc polypeptide comprising a knob mutation T366W according to the EU numbering scheme and the amino acid modification N434S, with or without M428L, and does not comprise a TfR-binding site or any modification that reduces FcγR binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs:327, 334, 341, 348, 355, 362, 369, 376, 383, and 390. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO:404.

[0063] In any aspect of the modified Fc polypeptide dimers described herein, the modified Fc polypeptide dimer does not substantially reduce reticulocytes (e.g., circulating reticulocytes). In some embodiments, the amount of reticulocytes reduced after administration of the modified Fc polypeptide dimer is less than the amount of reticulocytes reduced after administration of a control. In some embodiments, the amount of reticulocytes reduced after administration of the modified Fc polypeptide dimer is less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1% of the amount of reticulocytes reduced after administration of a control. In some embodiments, the amount of reticulocytes remaining after administration of the modified Fc polypeptide dimer is greater than the amount of reticulocytes remaining after administration of a control. In some embodiments, the amount of reticulocytes remaining after administration of the modified Fc polypeptide dimer is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater than the amount of reticulocytes remaining after administration of the control.

[0064] In any aspect of the modified Fc polypeptide dimers described herein, the modified Fc polypeptide dimer does not substantially reduce reticulocytes in the bone marrow. The amount of reticulocytes reduced in the bone marrow after administration of the modified Fc polypeptide dimer is less than the amount of reticulocytes reduced in the bone marrow after administration of a control. In some embodiments, the amount of reticulocytes reduced in the bone marrow after administration of the modified Fc polypeptide dimer is less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1% of the amount of reticulocytes reduced in the bone marrow after administration of a control. In some embodiments, the amount of reticulocytes remaining in the bone marrow after administration of the modified Fc polypeptide dimer is greater than the amount of reticulocytes remaining in the bone marrow after administration of a control. In some embodiments, the amount of reticulocytes remaining in the bone marrow after administration of the modified Fc polypeptide dimer is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater than the amount of reticulocytes remaining in the bone marrow after administration of the control.

[0065] In some embodiments, the control is a corresponding TfR-binding Fc dimer (i.e., having the same mutations that result in the same binding to TfR as the modified Fc polypeptide dimer described above) that has full effector function and / or does not contain mutations that reduce FcγR binding.

[0066] In another aspect, the present disclosure provides: (a) an antibody variable region, or an antigen-binding fragment thereof, capable of binding to an antigen; (b)(i) a first Fc polypeptide that specifically binds to TfR, comprising a TfR-binding site and, e.g., one or more amino acid modifications that reduce FcγR binding when bound to TfR (provided, e.g., that the reduction in FcγR binding is limited or not reduced when not bound to TfR); (ii) a second Fc polypeptide that does not contain a TfR binding site or any modification that reduces FcγR binding; and a modified Fc polypeptide dimer comprising The present invention features an Fc polypeptide dimer-Fab fusion protein capable of being actively transported across the BBB, comprising:

[0067] In some embodiments of this aspect, for example, the amino acid modification that reduces FcγR binding when bound to TfR comprises Ala at positions 234 and 235 according to the EU numbering scheme. In certain embodiments, for example, the amino acid modification that reduces FcγR binding when bound to TfR further comprises Gly at position 329 according to the EU numbering scheme.

[0068] In some embodiments of this aspect, the first Fc polypeptide and / or the second Fc polypeptide comprises an amino acid modification that increases serum stability (e.g., serum half-life). In some embodiments, the amino acid modification that increases serum stability (e.g., serum half-life) comprises Tyr at position 252, Thr at position 254, and Glu at position 256, according to the EU numbering scheme. In some embodiments, the amino acid modification that increases serum stability (e.g., serum half-life) comprises (i) Leu at position 428 and Ser at position 434, or (ii) Ser or Ala at position 434, according to the EU numbering scheme.

[0069] In some embodiments of this aspect, the antibody variable region sequence comprises a Fab domain, hi some embodiments, the antibody variable region sequence comprises two antibody variable heavy chains and two antibody variable light chains, or fragments of each thereof.

[0070] In some embodiments, the Fc polypeptide or Fc polypeptide dimer is fucose-deficient or hypofucosylated (eg, as described herein).

[0071] In another aspect, the present disclosure relates to a pharmaceutical composition comprising a modified Fc polypeptide dimer described herein and a pharmaceutically acceptable carrier.

[0072] In another aspect, the present disclosure relates to a pharmaceutical composition comprising an Fc polypeptide dimer-Fab fusion protein described herein and a pharmaceutically acceptable carrier.

[0073] In another aspect, the present disclosure relates to a method for transcytosing a composition across an endothelium, the method comprising contacting the endothelium with a composition comprising a modified Fc polypeptide dimer described herein. In some embodiments, the endothelium is the BBB.

[0074] In another aspect, the present disclosure relates to a method for transcytosing a composition across an endothelium, the method comprising contacting the endothelium with a composition comprising an Fc polypeptide dimer-Fab fusion protein described herein. In some embodiments, the endothelium is the BBB. [The present invention 1001] (a) specifically binds to TfR; (b) capable of binding to an Fcγ receptor (FcγR); and (c) does not substantially reduce reticulocytes in vivo , a modified Fc polypeptide dimer, or a dimeric fragment thereof. [The present invention 1002] (a) a first Fc polypeptide that specifically binds to TfR, comprising (i) a TfR-binding site and (ii) one or more amino acid modifications that reduce FcγR binding when bound to TfR; (b) a second Fc polypeptide that does not contain a TfR binding site or any modification that reduces FcγR binding; 10. A modified Fc polypeptide dimer, or a dimeric fragment thereof, comprising: [The present invention 1003] 1001 or 1002. The modified Fc polypeptide dimer of the present invention, wherein said TfR binding site comprises a modified CH3 domain. [The present invention 1004] 1003. The modified Fc polypeptide dimer of the present invention, wherein said modified CH3 domain is derived from the CH3 domain of human IgG1, IgG2, IgG3, or IgG4. [The present invention 1005] The modified Fc polypeptide dimer of the present invention 1003 or 1004, wherein the modified CH3 domain comprises 5, 6, 7, 8, or 9 substitutions at the set of amino acid positions consisting of 384, 386, 387, 388, 389, 390, 413, 416, and 421 according to EU numbering. [The present invention 1006] 1005. The modified Fc polypeptide dimer of the present invention, wherein said modified CH3 domain further comprises one, two, three, or four substitutions at positions 380, 391, 392, and 415, inclusive. [The present invention 1007] The modified Fc polypeptide dimer of claim 1005 or 1006, wherein said modified CH3 domain further comprises one, two or three substitutions at positions 414, 424 and 426, inclusive. [The present invention 1008] A modified Fc polypeptide dimer according to any one of claims 1001 to 1007 of the present invention, which binds to the apical domain of TfR. [The present invention 1009] 1008. The modified Fc polypeptide dimer of the present invention, which binds to TfR without inhibiting the binding of transferrin to TfR. [The present invention 1010] The modified Fc polypeptide dimer of the present invention 1008 or 1009, which binds to an epitope comprising amino acid 208 of TfR. [The present invention 1011] The modified Fc polypeptide dimer of any of claims 1005 to 1010, wherein the modified CH3 domain comprises Trp at position 388. [The present invention 1012] 1012. The modified Fc polypeptide dimer of any of claims 1005 to 1011, wherein the modified CH3 domain comprises an aromatic amino acid at position 421. [The present invention 1013] The modified Fc polypeptide dimer of the present invention 1012, wherein the aromatic amino acid at position 421 is Trp or Phe. [The present invention 1014] 1010. The modified Fc polypeptide dimer of any of claims 1005 to 1010, wherein said modified CH3 domain comprises at least one position selected from: Position 384 is Leu, Tyr, Met, or Val; position 386 is Leu, Thr, His, or Pro; position 387 is Val, Pro, or an acidic amino acid; position 388 is Trp; position 389 is Val, Ser, or Ala; position 413 is Glu, Ala, Ser, Leu, Thr, or Pro; position 416 is Thr or an acidic amino acid; and position 421 is Trp, Tyr, His, or Phe. [The present invention 1015] 1014. The modified Fc polypeptide dimer of the present invention, wherein said modified CH3 domain comprises two, three, four, five, six, seven, or eight positions selected from the following: Position 384 is Leu, Tyr, Met, or Val; position 386 is Leu, Thr, His, or Pro; position 387 is Val, Pro, or an acidic amino acid; position 388 is Trp; position 389 is Val, Ser, or Ala; position 413 is Glu, Ala, Ser, Leu, Thr, or Pro; position 416 is Thr or an acidic amino acid; and position 421 is Trp, Tyr, His, or Phe. [The present invention 1016] 10. The modified Fc polypeptide dimer of any of claims 1005 to 1015, wherein the modified CH3 domain comprises Leu or Met at position 384, Leu, His, or Pro at position 386, Val at position 387, Trp at position 388, Val or Ala at position 389, Pro at position 413, Thr at position 416, and / or Trp at position 421. [The present invention 1017] 1016. The modified Fc polypeptide dimer of the present invention, wherein said modified CH3 domain further comprises Ser, Thr, Gln, or Phe at position 391. [The present invention 1018] The modified Fc polypeptide dimer of the present invention 1016 or 1017, wherein the modified CH3 domain further comprises Trp, Tyr, Leu, or Gln at position 380. [The present invention 1019] The modified Fc polypeptide dimer of any of 1016 to 1018, wherein the modified CH3 domain further comprises Gln, Phe, or His at position 392. [The present invention 1020] The modified Fc polypeptide dimer of the present invention 1016 or 1017, wherein the modified CH3 domain further comprises Trp at position 380 and / or Gln at position 392. [The present invention 1021] The modified Fc polypeptide dimer of any of claims 1014 to 1020, wherein said modified CH3 domain further comprises one, two, or three positions selected from the following: Position 414 is Lys, Arg, Gly, or Pro; position 424 is Ser, Thr, Glu, or Lys; and position 426 is Ser, Trp, or Gly. [The present invention 1022] 10. The modified Fc polypeptide dimer of any of claims 1005 to 1015, wherein the modified CH3 domain comprises Tyr at position 384, Thr at position 386, Glu or Val at position 387, Trp at position 388, Ser at position 389, Ser or Thr at position 413, Glu at position 416, and / or Phe at position 421. [The present invention 1023] 1022. The modified Fc polypeptide dimer of the present invention, wherein said modified CH3 domain further comprises Trp, Tyr, Leu, or Gln at position 380. [The present invention 1024] The modified Fc polypeptide dimer of the present invention 1022 or 1023, wherein the modified CH3 domain further comprises Glu at position 415. [The present invention 1025] 1022. The modified Fc polypeptide dimer of the present invention, wherein said modified CH3 domain further comprises Trp at position 380 and / or Glu at position 415. [The present invention 1026] The modified Fc polypeptide dimer of any of 1022 to 1025, wherein the modified CH3 domain comprises Asn at position 390. [The present invention 1027] 10. The modified Fc polypeptide dimer of any of claims 1006 to 1010, wherein said modified CH3 domain comprises one or more of the following substitutions: Trp at position 380, Thr at position 386, Trp at position 388, Val at position 389, Ser or Thr at position 413, Glu at position 415, and / or Phe at position 421. [The present invention 1028] The modified Fc polypeptide dimer of any of claims 1005 to 1027, wherein the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 111 to 217 of any one of SEQ ID NOs: 4 to 29, and 64 to 127. [The present invention 1029] 1028. The modified Fc polypeptide dimer of the present invention, wherein the modified CH3 domain has 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: 66, 68, 94, 107-109, 119, and 268-270. [The present invention 1030] The modified Fc polypeptide dimer of any of claims 1005 to 1027, wherein the modified CH3 domain has at least 85% identity with amino acids 111 to 217 of SEQ ID NO:1, provided that the percent identity does not include the set of positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 according to EU numbering. [The present invention 1031] The modified Fc polypeptide dimer of any of claims 1005 to 1030, wherein the modified CH3 domain comprises amino acids 154 to 160 and / or 183 to 191 of any one of SEQ ID NOs: 4 to 29, and 125 to 127. [The present invention 1032] 1010. The modified Fc polypeptide dimer of any of claims 1006 to 1010, wherein said modified CH3 domain comprises at least one position selected from: Position 380 is Trp, Leu, or Glu, position 384 is Tyr or Phe, position 386 is Thr, position 387 is Glu, position 388 is Trp, position 389 is Ser, Ala, Val, or Asn, position 390 is Ser or Asn, position 413 is Thr or Ser, position 415 is Glu or Ser, position 416 is Glu, and position 421 is Phe. [The present invention 1033] 1032. The modified Fc polypeptide dimer of the present invention, wherein said modified CH3 domain comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from the following: Position 380 is Trp, Leu, or Glu, position 384 is Tyr or Phe, position 386 is Thr, position 387 is Glu, position 388 is Trp, position 389 is Ser, Ala, Val, or Asn, position 390 is Ser or Asn, position 413 is Thr or Ser, position 415 is Glu or Ser, position 416 is Glu, and position 421 is Phe. [The present invention 1034] 1033. The modified Fc polypeptide dimer of the present invention, wherein said modified CH3 domain comprises the following 11 positions: Position 380 is Trp, Leu, or Glu, position 384 is Tyr or Phe, position 386 is Thr, position 387 is Glu, position 388 is Trp, position 389 is Ser, Ala, Val, or Asn, position 390 is Ser or Asn, position 413 is Thr or Ser, position 415 is Glu or Ser, position 416 is Glu, and position 421 is Phe. [This invention 1035] The modified Fc polypeptide dimer of the present invention 1033 or 1034, wherein the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 111 to 217 of any one of SEQ ID NOs: 4 to 29, and 64 to 127. [The present invention 1036] 1035. The modified Fc polypeptide dimer of the present invention, wherein the modified CH3 domain has 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: 66, 68, 94, 107-109, 119, and 268-270. [This invention 1037] 1035. An engineered Fc polypeptide dimer of the invention, wherein residues at at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the positions corresponding to positions 380, 384, 386, 384, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424, and 426 according to the EU numbering scheme are not deleted or substituted. [The present invention 1038] 1003. The modified Fc polypeptide dimer of the present invention, wherein the modified CH3 domain comprises any one of the sequences of SEQ ID NOs: 38 to 61 and 131 to 173. [This invention 1039] A modified Fc polypeptide dimer of any of claims 1005 to 1037, wherein the modified CH3 domain further comprises (i) Trp at position 366, or (ii) Ser at position 366, Ala at position 368, and Val at position 407 according to the EU numbering scheme. [The present invention 1040] The modified Fc polypeptide dimer of any of claims 1005 to 1039, wherein the corresponding unmodified CH3 domain is a CH3 domain of human IgG1, IgG2, IgG3, or IgG4. [The present invention 1041] The modified Fc polypeptide dimer of any of claims 1002 to 1040, wherein the amino acid modifications that reduce FcγR binding when bound to TfR include Ala at positions 234 and 235 according to the EU numbering scheme. [The present invention 1042] The modified Fc polypeptide dimer of any of claims 1002 to 1041, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises an amino acid modification that increases serum half-life. [This invention 1043] 1042. A modified Fc polypeptide dimer of the present invention, wherein the amino acid modifications that increase serum half-life include (i) Leu at position 428 and Ser at position 434, or (ii) Ser or Ala at position 434, according to the EU numbering scheme. [This invention 1044] The modified Fc polypeptide dimer of any of claims 1001 to 1043, wherein the first Fc polypeptide and / or the second Fc polypeptide is further fused to Fab. [This invention 1045] 10. The modified Fc polypeptide dimer of any of claims 1002 to 1044, wherein the first Fc polypeptide comprises a knob mutation T366W and the second Fc polypeptide comprises hole mutations T366S, L368A, and Y407V according to the EU numbering scheme. [The present invention 1046] 10. The modified Fc polypeptide dimer of any of claims 1002 to 1044, wherein the first Fc polypeptide comprises hole mutations T366S, L368A, and Y407V, and the second Fc polypeptide comprises knob mutation T366W, according to the EU numbering scheme. [This invention 1047] (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR binding site and the amino acid modifications L234A and L235A according to the EU numbering scheme and the knob mutation T366W; (b) a second Fc polypeptide comprising the hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and which does not contain a TfR binding site or any modification that reduces FcγR binding; 1. A modified Fc polypeptide dimer comprising: [This invention 1048] 1047. The modified Fc polypeptide dimer of the present invention, wherein the first Fc polypeptide comprises any one of the sequences of SEQ ID NOs: 178, 190, 202, 214, 226, 238, 252, 286, 298, and 310. [This invention 1049] The modified Fc polypeptide dimer of the present invention 1047 or 1048, wherein said second Fc polypeptide comprises the sequence of SEQ ID NO:397. [The present invention 1050] (a) a first Fc polypeptide that specifically binds to TfR, comprising a TfR-binding site and the amino acid modifications L234A and L235A according to the EU numbering scheme, a knob mutation T366W, and an amino acid modification N434S with or without M428L; (b) a second Fc polypeptide comprising the hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and which does not contain a TfR binding site or any modification that reduces FcγR binding; 1. A modified Fc polypeptide dimer comprising: [This invention 1051] 1050. The modified Fc polypeptide dimer of the present invention, wherein the first Fc polypeptide comprises any one of SEQ ID NOs: 323, 330, 337, 344, 351, 358, 365, 372, 379, and 386. [This invention 1052] 1050 or 1051, wherein said second Fc polypeptide comprises the sequence of SEQ ID NO:397. [This invention 1053] (a) a first Fc polypeptide that specifically binds to TfR, the first Fc polypeptide comprising a TfR binding site and the amino acid modifications L234A and L235A according to the EU numbering scheme and the knob mutation T366W; (b) a second Fc polypeptide comprising the hole mutations T366S, L368A, and Y407V according to the EU numbering scheme and the amino acid modification N434S with or without M428L, and which does not contain a TfR binding site or any modification that reduces FcγR binding; 1. A modified Fc polypeptide dimer comprising: [This invention 1054] 1053. The modified Fc polypeptide dimer of the present invention, wherein the first Fc polypeptide comprises any one of the sequences of SEQ ID NOs: 178, 190, 202, 214, 226, 238, 252, 286, 298, and 310. [This invention 1055] The modified Fc polypeptide dimer of the present invention 1053 or 1054, wherein said second Fc polypeptide comprises the sequence of SEQ ID NO:407. [The present invention 1056] (a) a first Fc polypeptide that specifically binds to TfR, comprising a TfR-binding site and the amino acid modifications L234A and L235A according to the EU numbering scheme, a knob mutation T366W, and an amino acid modification N434S with or without M428L; (b) a second Fc polypeptide comprising the hole mutations T366S, L368A, and Y407V according to the EU numbering scheme and the amino acid modification N434S with or without M428L, and which does not contain a TfR binding site or any modification that reduces FcγR binding; 1. A modified Fc polypeptide dimer comprising: [This invention 1057] 1056. The modified Fc polypeptide dimer of the present invention, wherein the first Fc polypeptide comprises any one of the sequences of SEQ ID NOs: 323, 330, 337, 344, 351, 358, 365, 372, 379, and 386. [This invention 1058] The modified Fc polypeptide dimer of the present invention 1056 or 1057, wherein said second Fc polypeptide comprises the sequence of SEQ ID NO:407. [This invention 1059] (a) a first Fc polypeptide that specifically binds to TfR, comprising a TfR-binding site and amino acid modifications L234A and L235A according to the EU numbering scheme, and hole mutations T366S, L368A, and Y407V; (b) a second Fc polypeptide comprising the knob mutation T366W according to the EU numbering scheme and which does not comprise a TfR binding site or any modification that reduces FcγR binding; 1. A modified Fc polypeptide dimer comprising: [The present invention 1060] 1059. The modified Fc polypeptide dimer of the present invention, wherein the first Fc polypeptide comprises any one of the sequences of SEQ ID NOs: 184, 196, 208, 220, 232, 244, 280, 292, 304, and 316. [The present invention 1061] 1059 or 1060, wherein said second Fc polypeptide comprises the sequence of SEQ ID NO:391. [The present invention 1062] (a) a first Fc polypeptide that specifically binds to TfR, comprising a TfR-binding site and the amino acid modifications L234A and L235A according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and the amino acid modification N434S with or without M428L; (b) a second Fc polypeptide comprising the knob mutation T366W according to the EU numbering scheme and which does not comprise a TfR binding site or any modification that reduces FcγR binding; 1. A modified Fc polypeptide dimer comprising: [The present invention 1063] 1062. The modified Fc polypeptide dimer of the present invention, wherein the first Fc polypeptide comprises any one of SEQ ID NOs: 326, 333, 340, 347, 354, 361, 368, 375, 382, ​​and 389. [The present invention 1064] 1062 or 1063, a modified Fc polypeptide dimer of the present invention, wherein said second Fc polypeptide comprises the sequence of SEQ ID NO:391. [This invention 1065] (a) a first Fc polypeptide that specifically binds to TfR, comprising a TfR-binding site and amino acid modifications L234A and L235A according to the EU numbering scheme, and hole mutations T366S, L368A, and Y407V; (b) a second Fc polypeptide comprising the knob mutation T366W according to the EU numbering scheme and the amino acid modification N434S with or without M428L, and which does not contain a TfR binding site or any modification that reduces FcγR binding; 1. A modified Fc polypeptide dimer comprising: [The present invention 1066] 1065. The modified Fc polypeptide dimer of the present invention, wherein the first Fc polypeptide comprises any one of the sequences of SEQ ID NOs: 184, 196, 208, 220, 232, 244, 280, 292, 304, and 316. [This invention 1067] The modified Fc polypeptide dimer of the present invention 1065 or 1066, wherein said second Fc polypeptide comprises the sequence of SEQ ID NO:404. [The present invention 1068] (a) a first Fc polypeptide that specifically binds to TfR, comprising a TfR-binding site and the amino acid modifications L234A and L235A according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and the amino acid modification N434S with or without M428L; (b) a second Fc polypeptide comprising the knob mutation T366W according to the EU numbering scheme and the amino acid modification N434S with or without M428L, and which does not contain a TfR binding site or any modification that reduces FcγR binding; 1. A modified Fc polypeptide dimer comprising: [The present invention 1069] 1068. The modified Fc polypeptide dimer of the present invention, wherein the first Fc polypeptide comprises any one of SEQ ID NOs: 326, 333, 340, 347, 354, 361, 368, 375, 382, ​​and 389. [The present invention 1070] 1068 or 1069. The modified Fc polypeptide dimer of the present invention, wherein said second Fc polypeptide comprises the sequence of SEQ ID NO:404. [This invention 1071] The modified Fc polypeptide dimer of any of claims 1001 to 1070, which does not substantially reduce reticulocytes. [This invention 1072] The modified Fc polypeptide dimer of the present invention 1071, wherein the amount of reticulocytes reduced after administration of the modified Fc polypeptide dimer is less than the amount of reticulocytes reduced after administration of a control. [This invention 1073] A modified Fc polypeptide dimer of the present invention 1072, wherein the amount of reticulocytes reduced after administration of the modified Fc polypeptide dimer is less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1% of the amount of reticulocytes reduced after administration of a control. [This invention 1074] The modified Fc polypeptide dimer of the present invention 1071, wherein the amount of reticulocytes remaining after administration of the modified Fc polypeptide dimer is greater than the amount of reticulocytes remaining after administration of a control. [This invention 1075] A modified Fc polypeptide dimer of the present invention 1074, wherein the amount of reticulocytes remaining after administration of the modified Fc polypeptide dimer is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater than the amount of reticulocytes remaining after administration of a control. [This invention 1076] The modified Fc polypeptide dimer of any of claims 1001 to 1070, which does not substantially reduce reticulocytes in bone marrow. [This invention 1077] The modified Fc polypeptide dimer of the present invention 1076, wherein the amount of reticulocytes reduced in the bone marrow after administration of the modified Fc polypeptide dimer is less than the amount of reticulocytes reduced in the bone marrow after administration of a control. [This invention 1078] A modified Fc polypeptide dimer of the present invention 1077, wherein the amount of reticulocytes reduced in the bone marrow after administration of the modified Fc polypeptide dimer is less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1% of the amount of reticulocytes reduced in the bone marrow after administration of a control. [This invention 1079] The modified Fc polypeptide dimer of the present invention 1076, wherein the amount of reticulocytes remaining in the bone marrow after administration of the modified Fc polypeptide dimer is greater than the amount of reticulocytes remaining in the bone marrow after administration of a control. [The present invention 1080] A modified Fc polypeptide dimer of the present invention 1079, wherein the amount of reticulocytes remaining in the bone marrow after administration of the modified Fc polypeptide dimer is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater than the amount of reticulocytes remaining in the bone marrow after administration of a control. [This invention 1081] The control is a corresponding TfR-binding Fc dimer having full effector function and / or an altered Fc polypeptide dimer of any of items 1072 to 1075 and 1077 to 1080 of the present invention that does not contain a mutation that reduces FcγR binding. [This invention 1082] (a) an antibody variable region, or an antigen-binding fragment thereof, capable of binding to an antigen; (b)(i) a first Fc polypeptide that specifically binds to TfR, comprising a TfR-binding site and one or more amino acid modifications that reduce FcγR binding when bound to TfR; (ii) a second Fc polypeptide that does not contain a TfR binding site or any modification that reduces FcγR binding; and a modified Fc polypeptide dimer comprising: 1. An Fc polypeptide dimer-Fab fusion protein capable of being actively transported across the BBB, comprising: [This invention 1083] 1082. An Fc polypeptide dimer-Fab fusion protein of the present invention, wherein the amino acid modifications that reduce FcγR binding when bound to TfR include Ala at positions 234 and 235 according to the EU numbering scheme. [This invention 1084] The Fc polypeptide dimer-Fab fusion protein of the present invention 1082 or 1083, wherein said first Fc polypeptide and / or said second Fc polypeptide comprises an amino acid modification that increases serum half-life. [This invention 1085] 1084. The Fc polypeptide dimer-Fab fusion protein of the present invention, wherein the amino acid modifications that increase serum half-life include (i) Leu at position 428 and Ser at position 434, or (ii) Ser or Ala at position 434, according to the EU numbering scheme. [The present invention 1086] The Fc polypeptide dimer-Fab fusion protein of any one of 1082 to 1085, wherein the antibody variable region sequence comprises a Fab domain. [This invention 1087] The Fc polypeptide dimer-Fab fusion protein of any of 1082 to 1086, wherein the antibody variable region sequence comprises two antibody variable region heavy chains and two antibody variable region light chains, or fragments of each of them. [This invention 1088] A pharmaceutical composition comprising the modified Fc polypeptide dimer of any one of 1001 to 1081 of the present invention and a pharmaceutically acceptable carrier. [This invention 1089] A pharmaceutical composition comprising the Fc polypeptide dimer-Fab fusion protein of any one of the present inventions 1082 to 1087 and a pharmaceutically acceptable carrier. [The present invention 1090] A method for transcytosing a composition across an endothelium, the method comprising contacting the endothelium with a composition comprising a modified Fc polypeptide dimer according to any one of claims 1001 to 1081 of the present invention. [This invention 1091] A method for transcytosing a composition across an endothelium, the method comprising contacting the endothelium with a composition comprising an Fc polypeptide dimer-Fab fusion protein of any one of claims 1082 to 1087. [This invention 1092] 1092. The method of claim 1090 or 1091, wherein the endothelium is the BBB. [Brief explanation of the drawings]

[0075] [Figure 1] Figures 1A and 1B are graphs showing that a TfR-binding Fc polypeptide dimer fused to an anti-BACE1 Fab modified with L234A and L235A (LALA) mutations (numbered according to the EU numbering scheme) on both Fc polypeptides of the dimer to reduce FcγR binding did not reduce reticulocytes in the blood (A) and bone marrow (B) in human TfR knock-in (TfRms / huKI) mice. [Figure 2A] 10 is a graph showing that a TfR-binding Fc polypeptide dimer fused to an anti-BACE1 Fab having a LALA mutation in cis relative to the TfR binding site ("cis-LALA") did not reduce blood reticulocytes at 25 mg / kg in human TfR knock-in (TfRms / huKI) mice, whereas a similarly modified Fc polypeptide dimer fused to an anti-BACE1 Fab having a LALA mutation in trans relative to the TfR binding site reduced blood reticulocytes. [Figure 2B]10 is a graph showing that a TfR-binding Fc polypeptide dimer fused to an anti-BACE1 Fab having a LALA mutation in cis relative to the TfR binding site ("cisLALA") did not reduce bone marrow reticulocytes at 25 mg / kg in human TfR knock-in (TfRms / huKI) mice, whereas a similarly modified Fc polypeptide dimer fused to an anti-BACE1 Fab having a LALA mutation in trans relative to the TfR binding site reduced bone marrow reticulocytes. [Figure 2C] 10 is a graph showing that a TfR-binding Fc polypeptide dimer fused to an anti-BACE1 Fab having a LALA mutation in cis relative to the TfR binding site ("cis-LALA") did not reduce blood reticulocytes at 50 mg / kg in human TfR knock-in (TfRms / huKI) mice, whereas a similarly modified Fc polypeptide dimer fused to an anti-BACE1 Fab having a LALA mutation in trans relative to the TfR binding site reduced blood reticulocytes. [Figure 2D] 10 is a graph showing that a TfR-binding Fc polypeptide dimer fused to an anti-BACE1 Fab having a LALA mutation in cis relative to the TfR binding site ("cisLALA") did not reduce bone marrow reticulocytes at 50 mg / kg in human TfR knock-in (TfRms / huKI) mice, whereas a similarly modified Fc polypeptide dimer fused to an anti-BACE1 Fab having a LALA mutation in trans relative to the TfR binding site reduced bone marrow reticulocytes. [Figure 3A]FIG. 10 is a graph showing that a cis LALA-modified Fc polypeptide dimer (CH3C.35.21) fused to anti-BACE1 Fab and a modified Fc polypeptide with LALA mutations in both Fc polypeptides did not induce TfR-mediated ADCC, whereas hIgG1 containing the TfR-binding site but without the LALA mutation induced ADCC in Ramos cells expressing endogenous TfR. [Figure 3B] FIG. 10 is a graph showing that a cis LALA-modified Fc polypeptide dimer (CH3C.35.23) fused to anti-BACE1 Fab and a modified Fc polypeptide with LALA mutations in both Fc polypeptides did not induce TfR-mediated ADCC, whereas hIgG1 containing the TfR-binding site but without the LALA mutation induced ADCC in Ramos cells expressing endogenous TfR. [Figure 4] 1 is a graph showing that the TfR-binding Fc polypeptide dimer (CH3C.35.21) had no TfR-mediated complement-dependent cytotoxicity (CDC) activity in CHO-hTfR cells, whereas the anti-TfR control antibody Ab204 induced CDC. [Figure 5] 10 is a graph showing that a cis-LALA modified Fc polypeptide dimer fused to an anti-BACE1 Fab induced pSyk protein levels in primary human microglial cells similar to those seen with TfR-binding polypeptides by wild-type hIgG1, whereas a modified Fc polypeptide dimer fused to an anti-BACE1 Fab with LALA mutations in both Fc polypeptides did not induce pSyk. [Figure 6] 6A and 6B are graphs showing that hIgG1 comprising a cis-LALA Fc polypeptide dimer and an mCD20 Fab-binding site induced ADCC similarly to anti-mCD20 antibody and hIgG1 comprising a TfR-binding site and an mCD20 Fab-binding site (FIG. 6A). Similarly, a graph showing that hIgG1 comprising a cis-LALA Fc polypeptide dimer and an hCD20 Fab-binding site induced Fab-mediated CDC similarly to anti-hCD20 and hIgG1 comprising a TfR-binding site and an hCD20 Fab-binding site (FIG. 6B). [Figure 7] 7A and 7B are graphs showing that a cis-LALA Fc polypeptide dimer and an hIgG1 containing the Fab-binding site of mCD20 induced stable B cell depletion, similar to anti-mCD20 antibody and an hIgG1 containing the TfR-binding site and the Fab-binding site of mCD20 (A and B). These results demonstrate that the cis-LALA-modified Fc polypeptide dimer retains its Fc function and has Fab-mediated effector function in vivo. [Figure 8A] These graphs show that mice treated with anti-Aβ (CH3C.35.23.4) containing a TfR-binding site containing cis-LALA Fc polypeptide dimer induced stable microglial recruitment to Aβ plaques (A: percentage of plaque area containing microglia overlap; B: same data normalized to control IgG) and reduced plaques between 30 and 125 μm in size (C). These results demonstrate that anti-Aβ containing cis-LALA Fc polypeptide dimer maintains stable effector function on microglial recruitment and the ability to reduce Aβ plaques to some extent, similar to anti-Aβ. [Figure 8B] These graphs show that mice treated with anti-Aβ (CH3C.35.23.4) containing a TfR-binding site containing cis-LALA Fc polypeptide dimer induced stable microglial recruitment to Aβ plaques (A: percentage of plaque area containing microglia overlap; B: same data normalized to control IgG) and reduced plaques between 30 and 125 μm in size (C). These results demonstrate that anti-Aβ containing cis-LALA Fc polypeptide dimer maintains stable effector function on microglial recruitment and the ability to reduce Aβ plaques to some extent, similar to anti-Aβ. [Figure 8C]These graphs show that mice treated with anti-Aβ (CH3C.35.23.4) containing a TfR-binding site containing cis-LALA Fc polypeptide dimer induced stable microglial recruitment to Aβ plaques (A: percentage of plaque area containing microglia overlap; B: same data normalized to control IgG) and reduced plaques between 30 and 125 μm in size (C). These results demonstrate that anti-Aβ containing cis-LALA Fc polypeptide dimer maintains stable effector function on microglial recruitment and the ability to reduce Aβ plaques to some extent, similar to anti-Aβ. DETAILED DESCRIPTION OF THE INVENTION

[0076] Detailed Description I. Introduction Modified Fc polypeptide dimers containing a TfR-binding site can not only cross the BBB but also transport therapeutic agents across the BBB. As described herein, because reticulocytes also express TfR, these Fc polypeptide dimers can also reduce reticulocytes in vivo unless engineered to reduce effector function. The reduction in reticulocytes can be avoided by introducing a modification that eliminates the effector function of the Fc polypeptide of the Fc polypeptide dimer, i.e., a modification that eliminates or reduces Fcγ receptor (FcγR) binding (e.g., L234A and L235A (LALA) substitutions when numbered according to the EU numbering scheme). However, this approach is disadvantageous when effector function is desired when the Fab portion of the molecule binds to its target (e.g., a therapeutic target protein).

[0077] The present disclosure provides modified Fc polypeptide dimers that maintain effector function but do not result in a substantial reduction in reticulocytes. These modified Fc polypeptide dimers are also referred to herein as "effector function-positive, TfR-binding Fc polypeptide dimers." In some embodiments, only one of the two Fc polypeptides (but not both Fc polypeptides) of an effector function-positive, TfR-binding Fc polypeptide dimer is modified to reduce effector function and bind to TfR. The other Fc polypeptide of the modified Fc polypeptide dimer does not contain a TfR-binding site or any modification that reduces effector function, but may contain a mutation that enhances effector function. An effector function-positive TfR-binding Fc polypeptide dimer in which only one of the two Fc polypeptides contains both a TfR-binding site and a modification that reduces FcγR binding upon binding to TfR, while the other Fc polypeptide contains neither a TfR-binding site nor any modification that reduces FcγR binding, is said to have a cis conformation. As described herein, these modified Fc polypeptide dimers having a cis conformation were tested for their effect on reticulocytes. These experiments demonstrated that by introducing both a TfR-binding site and a mutation that reduces FcγR binding upon binding to TfR into only one of the two polypeptides that form the modified Fc polypeptide dimer, it is possible to reduce the effector function upon binding to TfR, thereby allowing binding to TfR without significantly reducing reticulocytes.

[0078] As described in detail herein, modified Fc polypeptide dimers with different configurations were fused to a Fab targeted to a therapeutic target (e.g., CD20) to examine whether effector function (e.g., ADCC and CDC) can be maintained when the Fab binds to its target rather than TfR. As described in detail below, the specific configuration of (a) modifications that reduce FcγR binding when bound to, for example, TfR, and (b) modifications in the modified Fc polypeptide dimer that result in binding to TfR can result in an Fc polypeptide dimer-Fab fusion that still maintains effector function (e.g., ADCC or CDC) but does not reduce reticulocyte activity when the Fc polypeptide dimer is fused to the Fab. This approach enables the use of TfR-mediated transport across the BBB while maintaining effector function.

[0079] Thus, the present disclosure relates in part to modified Fc polypeptide dimers that bind to TfR and have reduced effector function (e.g., ADCC and CDC) when bound to TfR, but still maintain effector function (e.g., ADCC and CDC) when the Fc polypeptide dimer is fused to a therapeutic Fab and binds to the target antigen of the Fab.

[0080] II. Definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "polypeptide" may include two or more such molecules.

[0081] As used herein, the terms "about" and "approximately," when used to modify a quantity specified in a numerical value or range, indicate that the numerical value and reasonable deviations from that value known to those of ordinary skill in the art, such as ±20%, ±10%, or ±5%, are within the expected meaning of the stated value.

[0082] As used herein, the term "Fc polypeptide" refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide characterized by an Ig fold as a structural domain. An Fc polypeptide comprises a constant region sequence comprising at least a CH2 domain and / or a CH3 domain, and may include at least a portion of a hinge region. Generally, an Fc polypeptide does not comprise a variable region.

[0083] "Modified Fc polypeptide" refers to an Fc polypeptide that has at least one mutation, e.g., a substitution, deletion, or insertion, compared to a wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retains the overall Ig fold or structure of a native Fc polypeptide.

[0084] As used herein, "Fc polypeptide dimer" refers to a dimer consisting of two Fc polypeptides. In some embodiments, the Fc polypeptide dimer can bind to an Fc receptor (e.g., FcγR). In an Fc polypeptide dimer, two Fc polypeptides dimerize through interaction between the constant domains of two CH3 antibodies. In some embodiments, two Fc polypeptides can also dimerize through one or more disulfide bonds formed between the hinge regions of two dimerizing Fc domain monomers. The Fc polypeptide dimer may be a wild-type Fc polypeptide dimer or a modified Fc polypeptide dimer. A wild-type Fc polypeptide dimer is formed by the dimerization of two wild-type Fc polypeptides. The Fc polypeptide dimer may be a heterodimer or a homodimer.

[0085] As used herein, the term "modified Fc polypeptide dimer" refers to an Fc polypeptide dimer comprising at least one modified Fc polypeptide. In some embodiments, the modified Fc polypeptide dimer comprises two modified Fc polypeptides. The modified Fc polypeptide dimer may be a homodimer (i.e., comprising two identical modified Fc polypeptides) or a heterodimer (i.e., comprising two different Fc polypeptides, where at least one of the two Fc polypeptides is a modified Fc polypeptide).

[0086] As used herein, "transferrin receptor" or "TfR" refers to transferrin receptor protein 1. The polypeptide sequence of human transferrin receptor 1 is set forth in SEQ ID NO:63. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee (accession number XP_003310238.1), rhesus monkey (NP_001244232.1), dog (NP_001003111.1), cow (NP_001193506.1), mouse (NP_035768.1), rat (NP_073203.1), and chicken (NP_990587.1)). The term "transferrin receptor" also encompasses allelic variants of an exemplary reference sequence (e.g., a human sequence) encoded by a gene at the chromosomal locus of transferrin receptor protein 1. The full-length TfR protein contains a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain. The apical domain sequence of human transferrin receptor 1 is set forth in SEQ ID NO:31.

[0087] As used herein, the term "Fcγ receptor" or "FcγR" refers to one type of Fc receptor, classified based on the type of antibody recognized by the receptor. FcγRs include several members, FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b), which have different antibody affinities due to their different molecular structures. FcγRs bind to the Fc portion of IgG class antibodies and are crucial for inducing phagocytosis of opsonized microorganisms. FcγRs are found on the cell surface of immune system cells. FcγRs trigger effector functions of the immune system and are activated when the Fc portion of an antibody binds to the receptor. FcγRs mediate immune functions, such as binding to antibodies bound to infected cells or invading pathogens and stimulating phagocytes or cytotoxic cells to destroy microorganisms or infected cells via antibody-mediated phagocytosis or ADCC.

[0088] As used herein, the term "reduced FcγR binding" refers to a modified Fc polypeptide or modified Fc polypeptide dimer that has a mutation in the CH3 domain of the modified Fc polypeptide that reduces the affinity of the modified Fc polypeptide for FcγR by 0.01% to 90% (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%) compared to the affinity of an Fc polypeptide (e.g., a wild-type Fc polypeptide dimer) that does not have a mutation that reduces FcγR binding. FcγR binding can be measured, for example, using surface plasmon resonance (SPR) (e.g., a Biacore™ system). Alternatively, FcγR binding can be measured using a functional assay, such as, for example, an ADCC assay (e.g., an in vivo or in vitro cytotoxicity assay) such as those described herein. Reduction in FcγR binding can be measured when the modified Fc polypeptide or modified Fc polypeptide dimer binds to TfR. In some embodiments, the modified Fc polypeptide or modified Fc polypeptide dimer reduces FcγR binding when bound to TfR, but only slightly (e.g., less than a 25%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1% reduction) or not when not bound to TfR.

[0089] As further described herein, an altered Fc polypeptide dimer can comprise a first Fc polypeptide having both a TfR-binding site and a mutation that reduces FcγR binding when bound to TfR, and a second Fc polypeptide that has neither a TfR-binding site nor a modification that reduces FcγR binding. Thus, upon binding to TfR, the resulting asymmetric Fc polypeptide dimer having the first and second Fc polypeptides can have an overall reduced affinity for FcγR. In contrast, when not bound to TfR, the reduction in FcγR binding is limited (e.g., as described above) or not reduced.

[0090] The term "FcRn" refers to the neonatal Fc receptor. Binding of an Fc polypeptide to FcRn reduces clearance of the Fc polypeptide and increases its serum half-life. The human FcRn protein is a heterodimer consisting of a protein approximately 50 kDa in size that resembles major histocompatibility complex (MHC) class I proteins and β2-microglobulin, approximately 15 kDa in size.

[0091] As used herein, "FcRn-binding site" refers to the region of an Fc polypeptide that binds to FcRn. In human IgG, the FcRn-binding site includes L251, M252, I253, S254, R255, T256, M428, H433, N434, H435, and Y436 when numbered using the EU numbering scheme. These positions correspond to positions 21-26, 198, and 203-206 of SEQ ID NO:1.

[0092] As used herein, a "native FcRn-binding site" refers to a region of an Fc polypeptide that binds to FcRn and has the same amino acid sequence as a region of a naturally occurring Fc polypeptide that binds to FcRn.

[0093] As used herein, the term "does not substantially reduce reticulocytes in vivo" refers to the reduction in reticulocytes (e.g., reduction in bone marrow reticulocytes or circulating reticulocytes) caused by an effector function-positive TfR-binding Fc polypeptide dimer described herein, or an Fc polypeptide dimer-Fab fusion protein described herein comprising an effector function-positive TfR-binding Fc polypeptide dimer, compared to a control, e.g., a control with intact effector function and / or reduced FcγR binding. This means less than (e.g., less than 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1% of) the reduction in reticulocytes (e.g., bone marrow reticulocytes or circulating reticulocytes) caused by an antibody comprising a corresponding TfR-binding Fc dimer that does not contain a mutation or a corresponding TfR-binding Fc dimer that has full effector function and / or does not contain a mutation that reduces FcγR binding.

[0094] The term "does not substantially reduce reticulocytes in vivo" also refers to the amount or percentage (%) of reticulocytes remaining (e.g., reticulocytes remaining in the bone marrow or circulation) following administration of an effector function-positive TfR-binding Fc polypeptide dimer described herein, or an Fc polypeptide dimer-Fab fusion protein described herein comprising an effector function-positive TfR-binding Fc polypeptide dimer, compared to a control (e.g., an Fc polypeptide dimer-Fab fusion protein having full effector function and / or FcγR binding). "FcγR binding" means that the amount or percentage (%) of reticulocytes remaining after administration of a TfR-binding Fc dimer containing a corresponding TfR-binding Fc dimer that does not contain a mutation that reduces FcγR binding, or a corresponding TfR-binding Fc dimer that has full effector function and / or does not contain a mutation that reduces FcγR binding, is greater (e.g., at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater) than the amount or percentage (%) of reticulocytes remaining (e.g., reticulocytes remaining in the bone marrow or circulation) following administration of a TfR-binding Fc dimer containing a corresponding TfR-binding Fc dimer that does not contain a mutation that reduces FcγR binding.

[0095] The amount or percentage (%) of reticulocyte reduction (e.g., reduction in reticulocytes in the bone marrow or circulation) or the amount or percentage (%) of remaining reticulocytes (e.g., remaining reticulocytes in the bone marrow or circulation) can be measured using human TfR knock-in (TfR) mice engineered to replace the mouse TfR with a human apical domain / mouse chimeric TfR protein. ms / hu KI) mice (e.g., human TfR apical domain knock-in mice ("hTfR アピカル Measurements can be performed in mice (e.g., "knock-in mice") or in non-human primates such as cynomolgus monkeys. Measurements can be performed by combining the modified Fc dimer or a control (e.g., TfR ms / hu KI mice) can be administered intravenously at, for example, 25-50 mg / kg, and circulating reticulocytes can be measured 24 hours post-dose by cytochemistry using the Advia 120 Hematology System as described herein. Bone marrow reticulocytes can be measured by FACS sorting using Ter119 as described herein. + , hCD71 高 population, and FSC 低 This can be measured by determining the population.

[0096] As used herein, the terms "CH3 domain" and "CH2 domain" refer to immunoglobulin constant region domain polypeptides. In the context of an IgG antibody, a CH3 domain polypeptide refers to the segment of amino acids from about 341 to about 447 when numbered according to the EU numbering scheme, and a CH2 domain polypeptide refers to the segment of amino acids from about 231 to about 340 when numbered according to the EU numbering scheme. CH2 and CH3 domain polypeptides may also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, in which the CH2 domain is numbered 1 to 110 and the CH3 domain is numbered 1 to 107 according to the IMGT Scientific chart numbering (IMGT website). The CH2 and CH3 domains are part of the Fc region of an immunoglobulin. In the context of an IgG antibody, an Fc region refers to the segment of amino acids from about 231 to about 447 when numbered according to the EU numbering scheme. As used herein, the term "Fc region" may also include at least a portion of an antibody hinge region. An exemplary hinge region sequence is set forth in SEQ ID NO:62.

[0097] The term "variable region" refers to the domain within an antibody heavy or light chain that is derived from germline variable (V), diversity (D), or joining (J) genes (and not from constant (Cμ and Cδ) gene segments) and that confers to the antibody its specificity for binding to antigen. Antibody variable regions generally contain four conserved "framework" regions sandwiched between three hypervariable "complementarity-determining regions."

[0098] The terms "wild-type," "native," and "naturally occurring" with respect to CH3 and CH2 domains are used herein to refer to domains having sequences that occur in nature.

[0099] As used herein, the term "mutant" with respect to a mutant polypeptide or mutant polynucleotide is used interchangeably with "variant." With respect to a given wild-type CH3 or CH2 domain reference sequence, variants can include naturally occurring allelic variants. A "non-naturally occurring" CH3 or CH2 domain refers to a variant or mutant domain that does not naturally occur in cells and is generated by genetic modification (e.g., using genetic engineering techniques or mutagenesis) of a native CH3 or CH2 domain polynucleotide or polypeptide. A "variant" includes any domain that contains at least one amino acid mutation relative to the wild-type. Mutations can include substitutions, insertions, and deletions.

[0100] 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.

[0101] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analogs" refer to compounds (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, etc.) that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group). 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" refer to 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.

[0102] Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), and 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.

[0103] 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.

[0104] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a single chain of polymers of amino acid residues. Such terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. An amino acid polymer can contain entirely L-amino acids, entirely D-amino acids, or a mixture of L- and D-amino acids.

[0105] As used herein, a protein refers to a polypeptide or a dimer (i.e., two) or multimer (i.e., three or more) of single polypeptide chains. The single polypeptide chains of a protein can be linked by covalent bonds, such as disulfide bonds, or by non-covalent interactions.

[0106] The terms "conservative substitution," "conservative mutation," or "conservatively modified variant" refer to a change that results in the substitution of one amino acid with another amino acid that can be classified as having similar properties. Examples of categories of conservative amino acid groups defined in this manner include "charged / polar groups" including Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H); "aromatic groups" including Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and "aliphatic groups" including Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine ​​or C). Subgroups may also be identified within each group. For example, the group of charged or polar amino acids can be further divided into subgroups including a "positively charged subgroup" containing Lys, Arg, and His, a "negatively charged subgroup" containing Glu and Asp, and a "polar subgroup" containing Asn and Gln. In another example, aromatic or cyclic groups can be further divided into subgroups including a "nitrogen ring subgroup" containing Pro, His, and Trp, and a "phenyl subgroup" containing Phe and Tyr. In yet another example, aliphatic groups can be further divided into subgroups such as an "aliphatic non-polar subgroup" containing Val, Leu, Gly, and Ala, and an "aliphatic weakly polar subgroup" containing Met, Ser, Thr, and Cys.Examples of conservative mutation categories include amino acid substitutions of amino acids within the above subgroups, such as, but not limited to, Lys for Arg, or vice versa, to maintain a positive charge; Glu for Asp, or vice versa, to maintain a negative charge; Ser for Thr, or vice versa, to maintain a free -OH; Gln for Asn, or vice versa, to maintain a free -NH. In some embodiments, hydrophobicity is maintained, for example, by substituting a hydrophobic amino acid for a naturally occurring hydrophobic amino acid in the active site.

[0107] The terms "identical" or "identity" in the context of two or more polypeptide sequences refer to two or more sequences or subsequences that are the same over a specified region when compared and aligned for maximum correspondence over a comparison window, or designated region, as determined using a sequence comparison algorithm or by manual alignment and visual inspection, or that have a specified percentage (%) of amino acid residues that are identical, for example, at least 60% identical, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical or more.

[0108] In polypeptide sequence comparison, one amino acid sequence generally serves as a reference sequence to which candidate sequences are compared. Alignment can be performed using various methods available to those skilled in the art to achieve maximum alignment, such as visual alignment or using publicly available software with known algorithms. Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters used in alignment to achieve maximum alignment can be determined by those skilled in the art. For the purposes of this application, the standard protein BLAST of the BLASTP algorithm is used to align two protein sequences using default parameters.

[0109] When used in the context of identifying a given amino acid residue in a polypeptide sequence, the terms "corresponding to," "determined with respect to," or "numbered with respect to" refer to the position of the residue in a particular reference sequence when the given amino acid sequence is compared to the reference sequence for maximum alignment. Thus, for example, an amino acid residue in a polypeptide "corresponds to" an amino acid in a region of SEQ ID NO:1 when that residue is aligned with that amino acid in SEQ ID NO:1 when optimally aligned with SEQ ID NO:1. A polypeptide that is aligned with a reference sequence need not be the same length as the reference sequence.

[0110] The terms "specifically bind" or "selectively bind" to a target, e.g., TfR or FcγR, when referring to a polypeptide comprising a modified CH3 domain described herein, refer to a binding reaction in which the polypeptide binds to the target with greater affinity, greater avidity, and / or longer duration than it binds to a structurally different target. In typical embodiments, the polypeptide has at least 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more greater affinity for a particular target, e.g., TfR or FcγR, compared to an unrelated target when assayed under the same affinity assay conditions. As used herein, the terms "specific binding to," "specifically binds to," or "being specific for" a particular target (e.g., TfR or FcγR) refer to, for example, the equilibrium dissociation constant K for the bound target. D For example, 10 -4 M or less, e.g., 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M. In some embodiments, the modified CH3 domain polypeptide specifically binds to an epitope of the TfR that is conserved across species (e.g., structurally conserved across species), e.g., conserved between non-human primates and humans (e.g., structurally conserved between non-human primates and humans). In some embodiments, the polypeptide can bind only to human TfR.

[0111] As used herein, "binding affinity" refers to the strength of a non-covalent interaction between two molecules, e.g., between one binding site of a polypeptide and a target to which the polypeptide binds, e.g., TfR. Thus, for example, the term may refer to a 1:1 interaction between a polypeptide and its target unless otherwise indicated or clear from the context. Binding affinity is measured by the dissociation rate constant (k d ,time -1 ) as the binding rate constant (k a ,time -1 M -1 ) is the equilibrium dissociation constant (K D ) can be quantified by measuring the K D can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods such as the 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 affinity, such as that reflecting a 1:1 interaction between a polypeptide and its target, but also K values, which may reflect strong binding. D The apparent affinity calculated is also included.

[0112] III. TFR-binding FC polypeptides This section describes the generation of modified Fc polypeptides that are capable of binding to the transferrin receptor and being transported across the blood-brain barrier (BBB).

[0113] CH3 TfR-binding polypeptide In some embodiments, the modified Fc polypeptide comprises a modified human Ig CH3 domain, such as an IgG CH3 domain. The CH3 domain may be of any IgG subtype, i.e., IgG1, IgG2, IgG3, or IgG4. In the context of an IgG antibody, the CH3 domain refers to the segment of amino acids from about 341 to about 447 when numbered according to the EU numbering scheme. Positions within the CH3 domain for purposes of identifying a set of corresponding amino acid positions for TfR binding are determined based on amino acids 111-217 of the EU numbering scheme, SEQ ID NO:3, or SEQ ID NO:1, unless otherwise specified. Substitutions are also determined based on the EU numbering scheme or SEQ ID NO:1, i.e., an amino acid is considered a substitution for the amino acid at the corresponding position in the EU numbering scheme or SEQ ID NO:1.

[0114] As noted above, the set of residues in the CH3 domain that can be modified are numbered herein with reference to the EU numbering scheme or SEQ ID NO: 1. For example, any CH3 domain, such as the CH3 domain of IgG1, IgG2, IgG3, or IgG4, can have a modification (e.g., an amino acid substitution) at one or more residue sets that correspond to residues at the recited positions in the EU numbering scheme or SEQ ID NO: 1. The respective positions in the IgG1, IgG2, IgG3, and IgG4 sequences that correspond to any particular position in the EU numbering scheme or SEQ ID NO: 1 can be readily determined.

[0115] Those skilled in the art will appreciate that CH3 domains of other immunoglobulin isotypes, such as IgM, IgA, IgE, and IgD, can be similarly modified by identifying amino acids within these domains that correspond to the amino acid positions described herein. Modifications can also be made to corresponding domains from immunoglobulins derived from other species, such as non-human primates, monkeys, mice, rats, rabbits, dogs, pigs, and chickens.

[0116] In one embodiment, a modified CH3 domain polypeptide that specifically binds to TfR binds to the apical domain of TfR at an epitope comprising position 208 of the full-length human TfR sequence (SEQ ID NO:63), which corresponds to position 11 of the apical domain sequence of human TfR set forth in SEQ ID NO:31. SEQ ID NO:31 corresponds to amino acids 198-378 of the human TfR-1 single protein sequence P02786 (SEQ ID NO:63). In some embodiments, a modified CH3 domain polypeptide binds to the apical domain of TfR at an epitope comprising positions 158, 188, 199, 207, 208, 209, 210, 211, 212, 213, 214, 215, and / or 294 of the full-length human TfR sequence (SEQ ID NO:63). The modified CH3 domain polypeptide may bind to TfR without blocking or otherwise inhibiting transferrin binding to the receptor. In some embodiments, binding of transferrin to TfR is not substantially inhibited. In some embodiments, binding of transferrin to TfR is inhibited by less than about 50% (e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%). In some embodiments, binding of transferrin to TfR is inhibited by less than about 20% (e.g., less than about 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%). Exemplary CH3 domain polypeptides that exhibit such binding specificity include polypeptides with amino acid substitutions at positions 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 according to the EU numbering scheme.

[0117] CH3, TfR-binding set: 384, 386, 387, 388, 389, 390, 413, 416, and 421 In some embodiments, the modified CH3 domain polypeptide contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions at a set of amino acid positions (set CH3C) including positions 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 according to the EU numbering scheme. Exemplary substitutions that can be introduced at these positions are shown in Table 3. Additional substitutions are shown in Table 4. In some embodiments, the amino acid at positions 388 and / or 421 is an aromatic amino acid (e.g., Trp, Phe, or Tyr). In some embodiments, the amino acid at position 388 is Trp. In some embodiments, the amino acid at position 388 is Gly. In some embodiments, the aromatic amino acid at position 421 is Trp or Phe.

[0118] In certain embodiments, the modified CH3 domain polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from the following: Glu, Leu, Ser, Val, Trp, Tyr, or Gln at position 380; Leu, Tyr, Phe, Trp, Met, Pro, or Val at position 384; Leu, Thr, His, Pro, Asn, Val, or Phe at position 386; Val, Pro, Ile, or an acidic amino acid at position 387; Trp at position 388; and an aliphatic amino acid at position 389. , Gly, Ser, Thr, or Asn, Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr at position 390, acidic amino acids Ala, Ser, Leu, Thr, Pro, Ile, or His at position 413, Glu, Ser, Asp, Gly, Thr, Pro, Gln, or Arg at position 415, Thr, Arg, Asn, or acidic amino acids at position 416, and / or aromatic amino acids His or Lys at position 421.

[0119] In some embodiments, modified CH3 domain polypeptides that specifically bind to TfR comprise at least one position having a substitution according to the EU numbering scheme: Leu, Tyr, Met, or Val at position 384; Leu, Thr, His, or Pro at position 386; Val, Pro, or an acidic amino acid at position 387; an aromatic amino acid, such as Trp or Gly (e.g., Trp), at position 388; Val, Ser, or Ala at position 389; an acidic amino acid, Ala, Ser, Leu, Thr, or Pro at position 413; Thr or an acidic amino acid at position 416; or Trp, Tyr, His, or Phe at position 421. In some embodiments, modified CH3 domain polypeptides comprise conservative substitutions of particular amino acids at one or more positions within the above sets, e.g., amino acids within the same charge group, hydrophobic group, side chain ring structure group (e.g., aromatic amino acids), or size group, and / or polar or nonpolar group. Thus, for example, Ile may be present at positions 384, 386, and / or 413. In some embodiments, the acidic amino acid at one, two, or each of positions 387, 413, and 416 is Glu. In other embodiments, the acidic amino acid at one, two, or each of positions 387, 413, and 416 is Asp. In some embodiments, two, three, four, five, six, seven, or all eight of positions 384, 386, 387, 388, 389, 413, 416, and 421 have an amino acid substitution as specified in this paragraph.

[0120] In some embodiments, a CH3 domain polypeptide having a modification in the set CH3C comprises a native Asn at position 390. In some embodiments, a modified CH3 domain polypeptide comprises Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, or Asp at position 390. In some embodiments, a modified CH3 domain polypeptide further comprises one, two, three, or four substitutions at positions including 380, 391, 392, and 415. In some embodiments, Trp, Tyr, Leu, or Gln may be present at position 380. In some embodiments, Ser, Thr, Gln, or Phe may be present at position 391. In some embodiments, Gln, Phe, or His may be present at position 392. In some embodiments, Glu may be present at position 415.

[0121] In certain embodiments, the modified CH3 domain polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions selected from the following: Trp, Leu, or Glu at position 380; Tyr or Phe at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser, Ala, Val, or Asn at position 389; Ser or Asn at position 390; Thr or Ser at position 413; Glu or Ser at position 415; Glu at position 416; and / or Phe at position 421. In some embodiments, the modified CH3 domain polypeptide contains all of the following 11 positions: Trp, Leu, or Glu at position 380; Tyr or Phe at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser, Ala, Val, or Asn at position 389; Ser or Asn at position 390; Thr or Ser at position 413; Glu or Ser at position 415; Glu at position 416; and / or Phe at position 421.

[0122] In certain embodiments, the modified CH3 domain polypeptide comprises Leu or Met at position 384, Leu, His, or Pro at position 386, Val at position 387, Trp at position 388, Val or Ala at position 389, Pro at position 413, Thr at position 416, and / or Trp at position 421. In some embodiments, the modified CH3 domain polypeptide further comprises Ser, Thr, Gln, or Phe at position 391. In some embodiments, the modified CH3 domain polypeptide further comprises Trp, Tyr, Leu, or Gln at position 380 and / or Gln, Phe, or His at position 392. In some embodiments, Trp is present at position 380 and / or Gln is present at position 392. In some embodiments, the modified CH3 domain polypeptide does not have a Trp at position 380.

[0123] In other embodiments, the modified CH3 domain polypeptide comprises a Tyr at position 384, a Thr at position 386, a Glu or Val at position 387, a Trp at position 388, a Ser at position 389, a Ser or Thr at position 413, a Glu at position 416, and / or a Phe at position 421. In some embodiments, the modified CH3 domain polypeptide comprises a native Asn at position 390. In certain embodiments, the modified CH3 domain polypeptide further comprises a Trp, Tyr, Leu, or Gln at position 380 and / or a Glu at position 415. In some embodiments, the modified CH3 domain polypeptide further comprises a Trp at position 380 and / or a Glu at position 415.

[0124] In a further embodiment, the modified CH3 domain further comprises one, two, or three positions selected from the following: Lys, Arg, Gly, or Pro at position 414, Ser, Thr, Glu, or Lys at position 424, and Ser, Trp, or Gly at position 426.

[0125] In some embodiments, the modified CH3 domain comprises one or more of the following substitutions: Trp at position 380, Thr at position 386, Trp at position 388, Val at position 389, Ser or Thr at position 413, Glu at position 415, and / or Phe at position 421.

[0126] In some embodiments, a modified CH3 domain polypeptide that specifically binds to TfR has at least 70% identity, at least 75% identity, at least 80% identity, 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:4-29, 64-127, and 268-274 (e.g., SEQ ID NOs:66, 68, 94, 107-109, 119, and 268-270). In some embodiments, such a modified CH3 domain polypeptide comprises amino acids 154-160 and / or 183-191 of any one of SEQ ID NOs:4-29, 64-127, and 268-274 (e.g., SEQ ID NOs:66, 68, 94, 107-109, 119, and 268-270). In some embodiments, such modified CH3 domain polypeptides comprise amino acids 150-160 and / or 183-191 of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270). In some embodiments, such modified CH3 domain polypeptides comprise amino acids 150-160 and / or 183-196 of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270).

[0127] In some embodiments, the modified CH3 domain polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 111-217 of SEQ ID NO:1, provided that the percent identity does not include the set of positions 154, 156, 157, 158, 159, 160, 183, 186, and 191 of SEQ ID NO:1 (positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 according to the EU numbering scheme). In some embodiments, the modified CH3 domain polypeptide comprises amino acids 154-160 and / or amino acids 183-191 set forth in any one of SEQ ID NOs:4-29, 64-127, and 268-274 (e.g., SEQ ID NOs:66, 68, 94, 107-109, 119, and 268-270).

[0128] In some embodiments, the engineered CH3 domain polypeptide is a polypeptide having a sequence corresponding to positions 150, 154, 156, 157, 158, 159, 160, 161, 162, 183, 184, 185, 186, 191, 194, and 196 (positions 380 and 380 according to the EU numbering scheme) of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270). , 384, 386, 384, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424, and 426) are not deleted or substituted, It has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270).

[0129] In some embodiments, the modified CH3 domain polypeptide has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270), and contains at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the following positions: Trp, Tyr, Leu, Gln, or Glu at position 380; Leu, Tyr, Met, or Val at position 384; Leu, Thr, His, or Pro at position 386; Val, Pro at position 387. or an acidic amino acid; an aromatic amino acid, such as Trp, at position 388; Val, Ser, or Ala at position 389; Ser or Asn at position 390; Ser, Thr, Gln, or Phe at position 391; Gln, Phe, or His at position 392; an acidic amino acid, such as Ala, Ser, Leu, Thr, or Pro at position 413; Lys, Arg, Gly, or Pro at position 414; Glu or Ser at position 415; Thr or an acidic amino acid at position 416; Trp, Tyr, His, or Phe at position 421; Ser, Thr, Glu, or Lys at position 424; and Ser, Trp, or Gly at position 426.

[0130] In some embodiments, the TfR-binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:38-52. In other embodiments, the TfR-binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:38-52, but with one or two amino acid substitutions in the sequence. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:38-52, but with three amino acid substitutions in the sequence.

[0131] In some embodiments, the TfR-binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:53-61. In other embodiments, the TfR-binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:53-61, but with one or two amino acid substitutions in the sequence. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:53-61, but with three or four amino acid substitutions in the sequence.

[0132] In some embodiments, the TfR-binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:131-167. In other embodiments, the TfR-binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:131-167, but with one or two amino acid substitutions in the sequence. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:131-167, but with three amino acid substitutions in the sequence.

[0133] In some embodiments, the TfR-binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:58, 60, and 168-173. In other embodiments, the TfR-binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:58, 60, and 168-173, but with one or two amino acid substitutions in the sequence. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:58, 60, and 168-173, but with three or four amino acid substitutions in the sequence.

[0134] In further embodiments, the TfR-binding polypeptide comprises amino acids 157-194, amino acids 153-194, or amino acids 153-199 of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270). In further embodiments, the polypeptide comprises an amino acid sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 157-194 of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270), or to amino acids 153-194 or 153-199 of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270).

[0135] In some embodiments, the polypeptide comprises any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270). In further embodiments, the polypeptide can have at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270).

[0136] FcRn binding site The polypeptides described herein that are capable of being transported across the BBB may further comprise an FcRn binding site, hi some embodiments, the FcRn binding site is within the modified Fc polypeptide or fragment thereof.

[0137] In some embodiments, the FcRn-binding site comprises a native FcRn-binding site. In some embodiments, the FcRn-binding site does not contain amino acid changes relative to the amino acid sequence of the native FcRn-binding site. In some embodiments, the native FcRn-binding site is an IgG-binding site, e.g., a human IgG-binding site. In some embodiments, the FcRn-binding site comprises a modification that alters binding to FcRn.

[0138] In some embodiments, the FcRn-binding site has one or more amino acid residues mutated, e.g., substituted, such that the mutation(s) increase serum half-life or do not substantially decrease serum half-life (i.e., decrease serum half-life by 25% or less compared to the corresponding protein having wild-type residues at the mutated positions when assayed under the same conditions). In some embodiments, the FcRn-binding site has one or more substituted amino acid residues at positions 21-26, 198, and 203-206, where each position is determined with reference to SEQ ID NO:1.

[0139] In some embodiments, the FcRn-binding site comprises one or more mutations relative to a native human IgG sequence that increase the serum half-life of the modified polypeptide. In some embodiments, mutations, e.g., substitutions, are introduced at one or more of positions 14-27, 49-54, 77-87, 153-160, and 198-205 relative to SEQ ID NO:1 (these positions correspond to positions 244-257, 279-284, 307-317, 383-390, and 428-435 using EU numbering). In some embodiments, one or more mutations are introduced at positions 21, 22, 24, 25, 26, 77, 78, 79, 81, 82, 84, 155, 156, 157, 159, 198, 203, 204, or 206 relative to SEQ ID NO:1 (which correspond to positions 251, 252, 254, 255, 256, 307, 308, 309, 311, 312, 314, 385, 386, 387, 389, 428, 433, 434, or 436 using EU numbering). In some embodiments, mutations are introduced at one, two, or three of positions 22, 24, and 25 when numbered relative to SEQ ID NO:1 (these positions correspond to positions 252, 254, and 256 when numbered relative to SEQ ID NO:1). In some embodiments, the mutations are M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1. In some embodiments, the modified Fc polypeptides described herein further comprise the mutations M22Y, S24T, and T26E. In some embodiments, mutations are introduced at one or two of positions 198 and 204 when numbered relative to SEQ ID NO:1 (these positions correspond to positions 428 and 434 when numbered relative to SEQ ID NO:1). In some embodiments, the mutations are M198L and N204S when numbered relative to SEQ ID NO:1. In some embodiments, the modified Fc polypeptides described herein further comprise the mutation N204S, with or without M198L.In some embodiments, the modified Fc polypeptide comprises substitutions at one, two, or all three of positions T307, E380, and N434 according to EU numbering (these positions correspond to T77, E150, and N204 when numbered with reference to SEQ ID NO:1). In some embodiments, the mutations are T307Q and N434A (T77Q and N204A of SEQ ID NO:1). In some embodiments, the modified Fc polypeptide comprises mutations T307A, E380A, and N434A (T77A, E150A, and N204A of SEQ ID NO:1). In some embodiments, the modified Fc polypeptide comprises substitutions at positions T250 and M428 (these positions correspond to T20 and M198 when numbered with reference to SEQ ID NO:1). In some embodiments, the Fc polypeptide comprises the mutations T250Q and / or M428L (T20Q and M198L of SEQ ID NO:1). In some embodiments, the modified Fc polypeptide comprises substitutions at positions M428 and N434 (these positions correspond to M198 and N204 when numbered relative to SEQ ID NO:1). In some embodiments, the modified Fc polypeptide comprises substitutions M428L and N434S (these positions correspond to M198L and N204S when numbered relative to SEQ ID NO:1). In some embodiments, the modified Fc polypeptide comprises substitutions N434S or N434A (these substitutions correspond to N204S or N204A when numbered relative to SEQ ID NO:1).

[0140] IV. Mutations that reduce effector function or FcγR binding The Fc polypeptides provided herein that bind to TfR and initiate transport across the BBB can also contain additional mutations that reduce effector function. As described herein, by introducing both a TfR-binding site and a mutation that reduces FcγR binding into the same Fc polypeptide of an Fc polypeptide dimer, it was possible to reduce effector function upon binding to TfR, resulting in binding to TfR without a significant reduction in reticulocytes, while still maintaining effector function (e.g., ADCC and CDC) when the Fc polypeptide dimer is fused to a therapeutic Fab and binds to the Fab's target antigen.

[0141] In some embodiments, an Fc polypeptide comprising a modified CH3 domain has effector function (i.e., the ability to induce a specific biological function upon binding to an Fc receptor expressed on an effector cell that mediates that function. Effector cells include, but are not limited to, monocytes, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and cytotoxic T cells.

[0142] Examples of effector functions include, but are not limited to, C1q binding and CDC, Fc receptor binding, ADCC, antibody-dependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Effector functions can vary depending on the antibody class. For example, native human IgG1 and IgG3 antibodies can induce ADCC and CDC activity when bound to the appropriate Fc receptors present on immune system cells, and native human IgG1, IgG2, IgG3, and IgG4 antibodies can induce ADCP function when bound to the appropriate Fc receptors present on immune cells.

[0143] In some embodiments, the Fc polypeptides having a TfR-binding site described herein can have further modifications that reduce effector function, i.e., reduce effector function upon binding to TfR. Reduced effector function of an Fc polypeptide dimer upon binding to TfR is desirable because it leads to reduced reticulocyte reduction, since reticulocytes also have TfR on their cell surface. As described in detail herein, an Fc polypeptide dimer having a cis-configuration, i.e., an Fc polypeptide dimer having both a TfR-binding site and a mutation that reduces effector function on the same Fc polypeptide of the Fc polypeptide dimer, exhibits binding to TfR without significantly reducing reticulocyte reduction, while still maintaining effector function (e.g., ADCC and CDC) when the Fc polypeptide dimer is fused to a therapeutic Fab and binds to the target antigen of the Fab. Having effector function upon fusion to a therapeutic Fab bound to the target antigen of the Fab is desirable, for example, in cancer therapy (e.g., brain tumor therapy).

[0144] Exemplary Fc polypeptide mutations that modulate effector function include, but are not limited to, substitutions within the CH2 domain, e.g., at positions corresponding to positions 4 and 5 of SEQ ID NO:1 (positions 234 and 235 according to the EU numbering scheme). In some embodiments, the substitutions within the modified CH2 domain comprise Ala at positions 4 and 5 of SEQ ID NO:1. In some embodiments, the substitutions within the modified CH2 domain comprise Ala at positions 4 and 5 and Gly at position 99 of SEQ ID NO:1.

[0145] Additional Fc polypeptide mutations that modulate effector function include, but are not limited to, substitutions at one or more of positions 238, 265, 269, 270, 297, 327, and 329 (which, in the EU numbering scheme, correspond to positions 8, 35, 39, 40, 67, 97, and 99 when numbered based on SEQ ID NO:1). Exemplary substitutions (when numbered using the EU numbering scheme) include the following: Position 329 can be mutated to replace proline with glycine or arginine, or an amino acid residue large enough to disrupt the Fc / Fcγ receptor interface formed between proline 329 of the Fc and tryptophan residues Trp87 and Trp110 of the FcγRIII. Further exemplary substitutions include S228P, E233P, L235E, N297A, N297D, and P331S. Multiple substitutions may be present, such as L234A and L235A in the Fc region of human IgG1, L234A, L235A, and P329G in the Fc region of human IgG1, S228P and L235E in the Fc region of human IgG4, L234A and G237A in the Fc region of human IgG1, L234A, L235A, and G237A in the Fc region of human IgG1, V234A and G237A in the Fc region of human IgG2, L235A, G237A, and E318A in the Fc region of human IgG4, and S228P and L236E in the Fc region of human IgG4. In some embodiments, the Fc polypeptide can have one or more amino acid substitutions that modulate ADCC (e.g., substitutions at positions 298, 333, and / or 334 in the Fc region, according to the EU numbering scheme).

[0146] In some embodiments, the polypeptides described herein may have one or more amino acid substitutions that increase or decrease ADCC, or may have mutations that alter C1q binding and / or CDC.

[0147] In specific embodiments, an Fc polypeptide having a TfR-binding site can be modified to reduce effector function, i.e., reduce FcγR binding. In some embodiments, an Fc polypeptide having a TfR-binding site can comprise the mutations L234A and L235A (corresponding to positions 4 and 5 according to the EU numbering scheme in SEQ ID NO:1). In other embodiments, an Fc polypeptide having a TfR-binding site can comprise the mutations L234A, L235A, and P329G (corresponding to positions 4, 5, and 99 according to the EU numbering scheme in SEQ ID NO:1).

[0148] V. Effector Function-Positive TfR-Binding Fc Polypeptide Dimers In certain aspects, the present disclosure provides effector function-positive TfR-binding Fc polypeptide dimers that are modified to bind to TfR and exhibit reduced FcγR binding when bound to TfR, but only limited or no reduction in FcγR binding when not bound to TfR. These modified Fc polypeptide dimers can be fused to a therapeutic Fab to transport the Fab across the BBB. These modified Fc polypeptide dimers have been shown to have reduced effector function when bound to TfR. When the modified Fc polypeptide dimer is fused to a Fab, the Fc polypeptide dimer maintains effector function when the Fab binds to its target (e.g., a target on a cancer cell). Thus, the effector function-positive TfR-binding Fc polypeptide dimers described herein can transport the Fab across the BBB without significantly reducing reticulocytes (which also have TfR on their cell surface), and can fulfill their therapeutic purpose by exhibiting effector functions that enable the targeted destruction of extracellular aggregates (e.g., plaques) or specific diseased cells (e.g., cancer cells) in the brain when the Fab binds to its target.

[0149] The effector function-positive TfR-binding Fc polypeptide dimers described herein have a cis conformation, meaning that only one (but not both) of the Fc polypeptides of the Fc polypeptide dimer is modified to have a TfR-binding site and a modification that reduces FcγR binding when bound to TfR. The other Fc polypeptide of the Fc polypeptide dimer does not contain a TfR-binding site or a modification that substantially reduces FcγR binding. A trans conformation of a modified Fc polypeptide dimer refers to an Fc polypeptide dimer in which one of the two Fc polypeptides contains a TfR-binding site and the other Fc polypeptide contains a modification that reduces FcγR binding, for example, when bound to TfR. As shown herein, modified Fc polypeptide dimers having a cis conformation but not a trans conformation can reduce reticulocytes in the blood and bone marrow (see, e.g., Figures 2A-2D).

[0150] In one embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds to TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme, and (b) a second Fc polypeptide that does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0151] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme and the amino acid modification N434S, with or without M428L; and (b) a second Fc polypeptide that does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0152] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme and the amino acid modification N434S, with or without M428L; and (b) a second Fc polypeptide comprising the amino acid modification N434S, with or without M428L, and which does not comprise a TfR-binding site or any modification that reduces FcγR binding.

[0153] In one embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme and the knob mutation T366W; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0154] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234, L235A, and P329G according to the EU numbering scheme and the knob mutation T366W; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0155] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A, knob mutation T366W, and amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0156] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme, the knob mutation T366W, and the amino acid modification N434S with or without M428L; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0157] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A, L235A, and P329G, knob mutation T366W, and amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0158] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, the knob mutation T366W, and the amino acid modification N434S with or without M428L; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0159] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme and the knob mutation T366W; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V and the amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0160] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme and the knob mutation T366W; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme and the amino acid modification N434S with or without M428L, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0161] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme and the knob mutation T366W; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V and the amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0162] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme and the knob mutation T366W; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme and the amino acid modification N434S with or without M428L, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0163] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme, a knob mutation T366W, and the amino acid modifications M252Y, S254T, and T256E; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and the amino acid modifications M252Y, S254T, and T256E, and the TfR-binding site does not comprise any modifications that reduce FcγR binding.

[0164] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme, the knob mutation T366W, and the amino acid modification N434S with or without M428L; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and the amino acid modification N434S with or without M428L, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0165] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, knob mutation T366W, and amino acid modifications M252Y, S254T, and T256E; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and amino acid modifications M252Y, S254T, and T256E, and does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0166] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, the knob mutation T366W, and the amino acid modification N434S, with or without M428L; and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme, and the amino acid modification N434S, with or without M428L, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0167] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme and the hole mutations T366S, L368A, and Y407V; and (b) a second Fc polypeptide comprising the knob mutation T366W according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0168] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme and the hole mutations T366S, L368A, and Y407V; and (b) a second Fc polypeptide comprising the knob mutation T366W according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0169] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and amino acid modifications M252Y, S254T, and T256E; and (b) a second Fc polypeptide comprising a knob mutation T366W according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0170] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and the amino acid modification N434S with or without M428L; and (b) a second Fc polypeptide comprising a knob mutation T366W according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0171] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and amino acid modifications M252Y, S254T, and T256E; and (b) a second Fc polypeptide comprising a knob mutation T366W according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0172] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and the amino acid modification N434S with or without M428L; and (b) a second Fc polypeptide comprising a knob mutation T366W according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0173] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme, and hole mutations T366S, L368A, and Y407V; and (b) a second Fc polypeptide comprising a knob mutation T366W and the amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0174] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme and the hole mutations T366S, L368A, and Y407V; and (b) a second Fc polypeptide comprising the knob mutation T366W according to the EU numbering scheme and the amino acid modification N434S with or without M428L, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0175] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, and hole mutations T366S, L368A, and Y407V; and (b) a second Fc polypeptide comprising a knob mutation T366W and the amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0176] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, and the hole mutations T366S, L368A, and Y407V; and (b) a second Fc polypeptide comprising the knob mutation T366W according to the EU numbering scheme and the amino acid modification N434S, with or without M428L, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0177] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and amino acid modifications M252Y, S254T, and T256E; and (b) a second Fc polypeptide comprising a knob mutation T366W and the amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0178] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A and L235A according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and the amino acid modification N434S, with or without M428L; and (b) a second Fc polypeptide comprising a knob mutation T366W according to the EU numbering scheme and the amino acid modification N434S, with or without M428L, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0179] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and amino acid modifications M252Y, S254T, and T256E; and (b) a second Fc polypeptide comprising a knob mutation T366W and the amino acid modifications M252Y, S254T, and T256E according to the EU numbering scheme, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0180] In another embodiment, the effector function-positive TfR-binding Fc polypeptide dimer comprises: (a) a first Fc polypeptide comprising a TfR-binding site that specifically binds TfR and the amino acid modifications L234A, L235A, and P329G according to the EU numbering scheme, hole mutations T366S, L368A, and Y407V, and the amino acid modification N434S, with or without M428L; and (b) a second Fc polypeptide comprising a knob mutation T366W according to the EU numbering scheme and the amino acid modification N434S, with or without M428L, and which does not comprise a TfR-binding site or any modifications that reduce FcγR binding.

[0181] VI. Measuring Effector Function or FcγR Binding Methods for analyzing binding affinity, binding kinetics, and cross-reactivity between Fc polypeptide dimers and FcγRs are well known in the art. These methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore™ (GE Healthcare, Piscataway, NJ)), kinetic exclusion assays (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), biolayer interferometry (e.g., Octet® (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to determine binding affinity and / or cross-reactivity. Methods for performing ELISA assays are well 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 cross-reactivity. In some embodiments, biolayer interferometry is used to determine binding affinity, binding kinetics, and / or cross-reactivity.

[0182] ADCC is a type of immune response in which antibodies bind to antigens on the surface of pathogenic or tumorigenic target cells and identify them for destruction by effector cells (e.g., natural killer (NK) cells, T cells, and B cells), such as peripheral blood mononuclear cells. Effector cells bearing FcγR recognize and bind the Fc region of antibodies bound to target cells. Thus, antibodies confer specificity to target cell killing. CDC is initiated by the binding of C1q, the initiating component of the classical complement pathway, to the Fc region of target-bound antibodies. ADCC and CDC activity can be measured by standard in vivo or in vitro cell killing assays. Methods for measuring ADCC and CDC activity are available in the art. In some embodiments, such methods involve killing target cells.51 This may involve labeling with a radioactive substance such as Cr or a fluorescent dye such as calcein-AM. The labeled cells are incubated with antibodies and effector cells, and target cell killing by ADCC and CDC can be detected by the emission of radioactivity or fluorescence.

[0183] Other assays for measuring ADCC and CDC activity include, for example, lactate dehydrogenase (LDH) release assays. When the cell membrane is disrupted or damaged in some way, LDH, a soluble but stable enzyme in the cytoplasm, is released into the surrounding extracellular space. The presence of this enzyme in the culture medium can be used as a cell death marker. The relative amount of live and dead cells in the culture medium can then be quantified by measuring the amount of released LDH using a colorimetric or fluorometric LDH cytotoxicity assay.

[0184] VII. Further Mutations in the Fc Region Containing Modified CH3 Domain Polypeptides The Fc polypeptides provided herein that are modified to bind to TfR and initiate transport across the BBB may contain additional mutations, e.g., to increase serum stability or serum half-life, modulate effector function, affect glycosylation, reduce immunogenicity in humans, and / or enable knob-and-hole heterodimerization of the Fc polypeptide.

[0185] In some embodiments, the modified Fc polypeptides described herein have at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a corresponding wild-type Fc polypeptide (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide).

[0186] The modified Fc polypeptides described herein may have other mutations introduced outside of the designated set of amino acids, e.g., to affect glycosylation, increase serum half-life, or, in the case of the CH3 domain, to enable knob-and-hole heterodimerization of the polypeptides comprising the modified CH3 domain. Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding hole ("hole") at the interface of a second polypeptide, such that the protrusion can locate within the hole, promoting heterodimer formation and preventing homodimer formation. The protrusion is formed by replacing a small amino acid side chain at the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary hole of the same or similar size as the protrusion is formed at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine). Such additional mutations are introduced at positions within the polypeptide that do not negatively affect binding of the modified CH3 domain to TfR.

[0187] In one exemplary embodiment of a knobs-and-holes approach to dimerization, a first Fc polypeptide subunit to be dimerized has a tryptophan instead of the native threonine at a position corresponding to position 136 of SEQ ID NO:1, and a second Fc polypeptide subunit of the dimer has a valine instead of the native tyrosine at a position corresponding to position 177 of SEQ ID NO:1. The second subunit of the Fc polypeptide may further comprise a substitution of serine for the native threonine at a position corresponding to position 136 of SEQ ID NO:1, and an alanine for the native leucine at a position corresponding to position 138 of SEQ ID NO:1.

[0188] The modified Fc polypeptides described herein can also be engineered to have other modifications for heterodimerization (e.g., electrostatic manipulation of contact residues within the naturally charged CH3-CH3 interface, or hydrophobic patch modifications, etc.).

[0189] In some embodiments, modifications can be introduced to increase serum half-life. For example, in some embodiments, the modified Fc polypeptides described herein comprise a CH2 domain comprising a Tyr at position corresponding to position 22 of SEQ ID NO:1, a Thr at position corresponding to position 24 of SEQ ID NO:1, and a Glu at position corresponding to position 26 of SEQ ID NO:1. Alternatively, the modified Fc polypeptides described herein may comprise the substitutions M198L and N204S when numbered relative to SEQ ID NO:1. Alternatively, the modified Fc polypeptides described herein may comprise the substitutions N204S or N204A when numbered relative to SEQ ID NO:1.

[0190] Exemplary Fc Polypeptides Containing Additional Mutations The modified Fc polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.35.23.2.1, and CH3C.35.23.1.1) may include knob mutations (e.g., T136W when numbered relative to SEQ ID NO:1), hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), effector function-modulating mutations (e.g., T136S ... The antibody may include additional mutations including L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1, and / or mutations that increase serum stability or serum half-life (e.g., (i) M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1, or (ii) N204S with or without M198L when numbered relative to SEQ ID NO:1).

[0191] In some embodiments, the modified Fc polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.35.23.2.1, and CH3C.35.23.1.1) comprise a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), as well as a variant of ... In some embodiments, the modified Fc polypeptide may have at least 85% identity, at least at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270). In some embodiments, the modified Fc polypeptide may have the sequence of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270).

[0192] In some embodiments, the modified Fc polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.35.23.2.1, and CH3C.35.23.1.1) comprise a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1). The modified Fc polypeptide may have at least 85% identity, at least at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270). In some embodiments, a modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270) can be modified to have knob mutations and mutations that modulate effector function.

[0193] In some embodiments, the modified Fc polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.35.23.2.1, and CH3C.35.23.1.1) comprise a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., (i) M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1, or (ii) a mutation that increases ... The modified Fc polypeptide may have at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270), including a knob mutation and a mutation that increases serum stability or serum half-life.

[0194] In some embodiments, the modified Fc polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.35.23.2.1, and CH3C.35.23.1.1) include a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., (i) SEQ ID NO: or (ii) N204S with or without M198L when numbered based on SEQ ID NO:1), and any one of SEQ ID NOs:4-29, 64-127, and 268-274 (e.g., SEQ ID NOs:66, 68, 94, 107-109, 119, and 268-270). In some embodiments, modified Fc polypeptides having the sequence of any one of SEQ ID NOs:4-29, 64-127, and 268-274 (e.g., SEQ ID NOs:66, 68, 94, 107-109, 119, and 268-270) can be modified to have knob mutations, mutations that modulate effector function, and mutations that increase serum stability or serum half-life.

[0195] In some embodiments, the modified Fc polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.35.23.2.1, and CH3C.35.23.1.1) contain hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), as well as those of SEQ ID NOs:4-29, 64-127, and 268-274 (e.g., SEQ ID NOs:4-29, 64-127, and 268-274). In some embodiments, the modified Fc polypeptide may have at least 85% identity, at least at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270). In some embodiments, the modified Fc polypeptide may have the sequence of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270) may be modified to have a hole mutation.

[0196] In some embodiments, the modified Fc polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.35.23.2.1, and CH3C.35.23.1.1) comprise a modified Fc polypeptide comprising a hole mutation (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1). The modified Fc polypeptide may have at least 85% identity, at least at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270). In some embodiments, a modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270) can be modified to have hole mutations and mutations that modulate effector function.

[0197] In some embodiments, the modified Fc polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.35.23.2.1, and CH3C.35.23.1.1) comprise a hole mutation (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., (i) M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1, or (ii) a mutation that increases ... The modified Fc polypeptide may have at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270), including any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270). In some embodiments, a modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270) can be modified to have a whole mutation and a mutation that increases serum stability or serum half-life.

[0198] In some embodiments, the modified Fc polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.35.23.2.1, and CH3C.35.23.1.1) comprise mutations that modulate effector function (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), or mutations that modulate effector function (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1). L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., (i) M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1, or (ii) N204S with or without M198L when numbered relative to SEQ ID NO:1), and can have at least 85% identity, at least at least 90% identity, or at least 95% identity to any one of SEQ ID NOs:4-29, 64-127, and 268-274 (e.g., SEQ ID NOs:66, 68, 94, 107-109, 119, and 268-270). In some embodiments, modified Fc polypeptides having the sequence of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270) can be modified to have a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life.

[0199] Clone CH3C.35.20.1 In some embodiments, clone CH3C.35.20.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:177. In some embodiments, clone CH3C.35.20.1 with the knob mutation has the sequence of SEQ ID NO:177.

[0200] In some embodiments, clone CH3C.35.20.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:178 or 179. In some embodiments, clone CH3C.35.20.1 having a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:178 or 179.

[0201] In some embodiments, clone CH3C.35.20.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 180. In some embodiments, clone CH3C.35.20.1 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO: 180.

[0202] In some embodiments, clone CH3C.35.20.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:322. In some embodiments, clone CH3C.35.20.1 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:322.

[0203] In some embodiments, clone CH3C.35.20.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:181 or 182. In some embodiments, clone CH3C.35.20.1 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:181 or 182.

[0204] In some embodiments, clone CH3C.35.20.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:323 or 324. In some embodiments, clone CH3C.35.20.1 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:323 or 324.

[0205] In some embodiments, clone CH3C.35.20.1 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 183. In some embodiments, clone CH3C.35.20.1 with hole mutations has the sequence of SEQ ID NO: 183.

[0206] In some embodiments, clone CH3C.35.20.1 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:184 or 185. In some embodiments, clone CH3C.35.20.1 having hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:184 or 185.

[0207] In some embodiments, clone CH3C.35.20.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:186. In some embodiments, clone CH3C.35.20.1 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:186.

[0208] In some embodiments, clone CH3C.35.20.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:325. In some embodiments, clone CH3C.35.20.1 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:325.

[0209] In some embodiments, clone CH3C.35.20.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:187 or 188. In some embodiments, clone CH3C.35.20.1, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:187 or 188.

[0210] In some embodiments, clone CH3C.35.20.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:326 or 327. In some embodiments, clone CH3C.35.20.1, which has the whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:326 or 327.

[0211] Clone CH3C.35.23.2 In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:189. In some embodiments, clone CH3C.35.23.2 with the knob mutation has the sequence of SEQ ID NO:189.

[0212] In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:190 or 191. In some embodiments, clone CH3C.35.23.2 having a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:190 or 191.

[0213] In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 192. In some embodiments, clone CH3C.35.23.2 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO: 192.

[0214] In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:329. In some embodiments, clone CH3C.35.23.2 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:329.

[0215] In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:193 or 194. In some embodiments, clone CH3C.35.23.2 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:193 or 194.

[0216] In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T136W when numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered with reference to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:330 or 331. In some embodiments, clone CH3C.35.23.2 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:330 or 331.

[0217] In some embodiments, clone CH3C.35.23.2 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:195. In some embodiments, clone CH3C.35.23.2 with hole mutations has the sequence of SEQ ID NO:195.

[0218] In some embodiments, clone CH3C.35.23.2 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:196 or 197. In some embodiments, clone CH3C.35.23.2 with hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:196 or 197.

[0219] In some embodiments, clone CH3C.35.23.2 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:198. In some embodiments, clone CH3C.35.23.2 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:198.

[0220] In some embodiments, clone CH3C.35.23.2 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:332. In some embodiments, clone CH3C.35.23.2 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:332.

[0221] In some embodiments, clone CH3C.35.23.2 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:199 or 200. In some embodiments, clone CH3C.35.23.2, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:199 or 200.

[0222] In some embodiments, clone CH3C.35.23.2 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:333 or 334. In some embodiments, clone CH3C.35.23.2, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:333 or 334.

[0223] Clone CH3C.35.23.3 In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:201. In some embodiments, clone CH3C.35.23.3 with the knob mutation has the sequence of SEQ ID NO:201.

[0224] In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:202 or 203. In some embodiments, clone CH3C.35.23.3 with a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:202 or 203.

[0225] In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:204. In some embodiments, clone CH3C.35.23.3 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:204.

[0226] In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:336. In some embodiments, clone CH3C.35.23.3 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:336.

[0227] In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:205 or 206. In some embodiments, clone CH3C.35.23.3 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:205 or 206.

[0228] In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T136W when numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered with reference to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:337 or 338. In some embodiments, clone CH3C.35.23.3 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:337 or 338.

[0229] In some embodiments, clone CH3C.35.23.3 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:207. In some embodiments, clone CH3C.35.23.3 with hole mutations has the sequence of SEQ ID NO:207.

[0230] In some embodiments, clone CH3C.35.23.3 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:208 or 209. In some embodiments, clone CH3C.35.23.3 with hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:208 or 209.

[0231] In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:210. In some embodiments, clone CH3C.35.23.3 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:210.

[0232] In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:339. In some embodiments, clone CH3C.35.23.3 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:339.

[0233] In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:211 or 212. In some embodiments, clone CH3C.35.23.3, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:211 or 212.

[0234] In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:340 or 341. In some embodiments, clone CH3C.35.23.3, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:340 or 341.

[0235] Clone CH3C.35.23.4 In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:213. In some embodiments, clone CH3C.35.23.4 with the knob mutation has the sequence of SEQ ID NO:213.

[0236] In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:214 or 215. In some embodiments, clone CH3C.35.23.4 with a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:214 or 215.

[0237] In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:216. In some embodiments, clone CH3C.35.23.4 with knob mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:216.

[0238] In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:343. In some embodiments, clone CH3C.35.23.4 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:343.

[0239] In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:217 or 218. In some embodiments, clone CH3C.35.23.4 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:217 or 218.

[0240] In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:344 or 345. In some embodiments, clone CH3C.35.23.4 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:344 or 345.

[0241] In some embodiments, clone CH3C.35.23.4 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:219. In some embodiments, clone CH3C.35.23.4 with hole mutations has the sequence of SEQ ID NO:219.

[0242] In some embodiments, clone CH3C.35.23.4 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:220 or 221. In some embodiments, clone CH3C.35.23.4 with hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:220 or 221.

[0243] In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:222. In some embodiments, clone CH3C.35.23.4 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:222.

[0244] In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:346. In some embodiments, clone CH3C.35.23.4 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:346.

[0245] In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:223 or 224. In some embodiments, clone CH3C.35.23.4, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:223 or 224.

[0246] In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:347 or 348. In some embodiments, clone CH3C.35.23.4, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:347 or 348.

[0247] Clone CH3C.35.21.17.2 In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:225. In some embodiments, clone CH3C.35.21.17.2 with the knob mutation has the sequence of SEQ ID NO:225.

[0248] In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:226 or 227. In some embodiments, clone CH3C.35.21.17.2 having a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:226 or 227.

[0249] In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:228. In some embodiments, clone CH3C.35.21.17.2 with knob mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:228.

[0250] In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:350. In some embodiments, clone CH3C.35.21.17.2 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:350.

[0251] In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:229 or 230. In some embodiments, clone CH3C.35.21.17.2 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:229 or 230.

[0252] In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T136W when numbering relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbering relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbering relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:351 or 352. In some embodiments, clone CH3C.35.21.17.2, which has knob mutations, mutations that modulate effector function, and mutations that increase serum stability or serum half-life, has the sequence of SEQ ID NO:351 or 352.

[0253] In some embodiments, clone CH3C.35.21.17.2 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:231. In some embodiments, clone CH3C.35.21.17.2 with hole mutations has the sequence of SEQ ID NO:231.

[0254] In some embodiments, clone CH3C.35.21.17.2 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:232 or 233. In some embodiments, clone CH3C.35.21.17.2 with hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:232 or 233.

[0255] In some embodiments, clone CH3C.35.21.17.2 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:234. In some embodiments, clone CH3C.35.21.17.2 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:234.

[0256] In some embodiments, clone CH3C.35.21.17.2 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:353. In some embodiments, clone CH3C.35.21.17.2 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:353.

[0257] In some embodiments, clone CH3C.35.21.17.2 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:235 or 236. In some embodiments, clone CH3C.35.21.17.2, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:235 or 236.

[0258] In some embodiments, clone CH3C.35.21.17.2 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:354 or 355. In some embodiments, clone CH3C.35.21.17.2, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:354 or 355.

[0259] Clone CH3C.35.23 In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:237. In some embodiments, clone CH3C.35.23 with the knob mutation has the sequence of SEQ ID NO:237.

[0260] In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:238 or 239. In some embodiments, clone CH3C.35.23 with a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:238 or 239.

[0261] In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:240. In some embodiments, clone CH3C.35.23 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:240.

[0262] In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:357. In some embodiments, clone CH3C.35.23 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:357.

[0263] In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:241 or 242. In some embodiments, clone CH3C.35.23 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:241 or 242.

[0264] In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T136W when numbering with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbering with reference to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbering with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:358 or 359. In some embodiments, clone CH3C.35.23 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:358 or 359.

[0265] In some embodiments, clone CH3C.35.23 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:243. In some embodiments, clone CH3C.35.23 with hole mutations has the sequence of SEQ ID NO:243.

[0266] In some embodiments, clone CH3C.35.23 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:244 or 245. In some embodiments, clone CH3C.35.23 with hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:244 or 245.

[0267] In some embodiments, clone CH3C.35.23 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:246. In some embodiments, clone CH3C.35.23 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:246.

[0268] In some embodiments, clone CH3C.35.23 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:360. In some embodiments, clone CH3C.35.23 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:360.

[0269] In some embodiments, clone CH3C.35.23 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:247 or 248. In some embodiments, clone CH3C.35.23, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:247 or 248.

[0270] In some embodiments, clone CH3C.35.23 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:361 or 362. In some embodiments, clone CH3C.35.23, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:361 or 362.

[0271] Clone CH3C.35.21 In some embodiments, clone CH3C.35.21 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:250. In some embodiments, clone CH3C.35.21 with the knob mutation has the sequence of SEQ ID NO:250.

[0272] In some embodiments, clone CH3C.35.21 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:252 or 275. In some embodiments, clone CH3C.35.21 with a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:252 or 275.

[0273] In some embodiments, clone CH3C.35.21 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:276. In some embodiments, clone CH3C.35.21 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:276.

[0274] In some embodiments, clone CH3C.35.21 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:364. In some embodiments, clone CH3C.35.21 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:364.

[0275] In some embodiments, clone CH3C.35.21 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:277 or 278. In some embodiments, clone CH3C.35.21 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:277 or 278.

[0276] In some embodiments, clone CH3C.35.21 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:365 or 366. In some embodiments, clone CH3C.35.21 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:365 or 366.

[0277] In some embodiments, clone CH3C.35.21 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:279. In some embodiments, clone CH3C.35.21 with hole mutations has the sequence of SEQ ID NO:279.

[0278] In some embodiments, clone CH3C.35.21 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:280 or 281. In some embodiments, clone CH3C.35.21 with hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:280 or 281.

[0279] In some embodiments, clone CH3C.35.21 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:282. In some embodiments, clone CH3C.35.21 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:282.

[0280] In some embodiments, clone CH3C.35.21 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:367. In some embodiments, clone CH3C.35.21 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:367.

[0281] In some embodiments, clone CH3C.35.21 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:283 or 284. In some embodiments, clone CH3C.35.21, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:283 or 284.

[0282] In some embodiments, clone CH3C.35.21 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:368 or 369. In some embodiments, clone CH3C.35.21, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:368 or 369.

[0283] Clone CH3C.35.20.1.1 In some embodiments, clone CH3C.35.20.1.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:285. In some embodiments, clone CH3C.35.20.1.1 with the knob mutation has the sequence of SEQ ID NO:285.

[0284] In some embodiments, clone CH3C.35.20.1.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:286 or 287. In some embodiments, clone CH3C.35.20.1.1 having a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:286 or 287.

[0285] In some embodiments, clone CH3C.35.20.1.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:288. In some embodiments, clone CH3C.35.20.1.1 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:288.

[0286] In some embodiments, clone CH3C.35.20.1.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:371. In some embodiments, clone CH3C.35.20.1.1 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:371.

[0287] In some embodiments, clone CH3C.35.20.1.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:289 or 290. In some embodiments, clone CH3C.35.20.1.1 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:289 or 290.

[0288] In some embodiments, clone CH3C.35.20.1.1 can have a knob mutation (e.g., T136W when numbered with reference to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered with reference to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered with reference to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:372 or 373. In some embodiments, clone CH3C.35.20.1.1 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:372 or 373.

[0289] In some embodiments, clone CH3C.35.20.1.1 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:291. In some embodiments, clone CH3C.35.20.1.1 with hole mutations has the sequence of SEQ ID NO:291.

[0290] In some embodiments, clone CH3C.35.20.1.1 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:292 or 293. In some embodiments, clone CH3C.35.20.1.1 having hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:292 or 293.

[0291] In some embodiments, clone CH3C.35.20.1.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:294. In some embodiments, clone CH3C.35.20.1.1 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:294.

[0292] In some embodiments, clone CH3C.35.20.1.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:374. In some embodiments, clone CH3C.35.20.1.1 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:374.

[0293] In some embodiments, clone CH3C.35.20.1.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:295 or 296. In some embodiments, clone CH3C.35.20.1.1, which has the whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:295 or 296.

[0294] In some embodiments, clone CH3C.35.20.1.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:375 or 376. In some embodiments, clone CH3C.35.20.1.1, which has the whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:375 or 376.

[0295] Clone CH3C.35.23.2.1 In some embodiments, clone CH3C.35.23.2.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:297. In some embodiments, clone CH3C.35.23.2.1 with the knob mutation has the sequence of SEQ ID NO:297.

[0296] In some embodiments, clone CH3C.35.23.2.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:298 or 299. In some embodiments, clone CH3C.35.23.2.1 having a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:298 or 299.

[0297] In some embodiments, clone CH3C.35.23.2.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:300. In some embodiments, clone CH3C.35.23.2.1 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:300.

[0298] In some embodiments, clone CH3C.35.23.2.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:378. In some embodiments, clone CH3C.35.23.2.1 having a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:378.

[0299] In some embodiments, clone CH3C.35.23.2.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:301 or 302. In some embodiments, clone CH3C.35.23.2.1 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:301 or 302.

[0300] In some embodiments, clone CH3C.35.23.2.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:379 or 380. In some embodiments, clone CH3C.35.23.2.1 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:379 or 380.

[0301] In some embodiments, clone CH3C.35.23.2.1 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:303. In some embodiments, clone CH3C.35.23.2.1 with hole mutations has the sequence of SEQ ID NO:303.

[0302] In some embodiments, clone CH3C.35.23.2.1 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:304 or 305. In some embodiments, clone CH3C.35.23.2.1 with hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:304 or 305.

[0303] In some embodiments, clone CH3C.35.23.2.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:306. In some embodiments, clone CH3C.35.23.2.1 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:306.

[0304] In some embodiments, clone CH3C.35.23.2.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:381. In some embodiments, clone CH3C.35.23.2.1 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:381.

[0305] In some embodiments, clone CH3C.35.23.2.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:307 or 308. In some embodiments, clone CH3C.35.23.2.1, which has a whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:307 or 308.

[0306] In some embodiments, clone CH3C.35.23.2.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:382 or 383. In some embodiments, clone CH3C.35.23.2.1, which has the whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:382 or 383.

[0307] Clone CH3C.35.23.1.1 In some embodiments, clone CH3C.35.23.1.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:309. In some embodiments, clone CH3C.35.23.1.1 with the knob mutation has the sequence of SEQ ID NO:309.

[0308] In some embodiments, clone CH3C.35.23.1.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:310 or 311. In some embodiments, clone CH3C.35.23.1.1 having a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:310 or 311.

[0309] In some embodiments, clone CH3C.35.23.1.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:312. In some embodiments, clone CH3C.35.23.1.1 with a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:312.

[0310] In some embodiments, clone CH3C.35.23.1.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:385. In some embodiments, clone CH3C.35.23.1.1 having a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:385.

[0311] In some embodiments, clone CH3C.35.23.1.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:313 or 314. In some embodiments, clone CH3C.35.23.1.1 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:313 or 314.

[0312] In some embodiments, clone CH3C.35.23.1.1 can have a knob mutation (e.g., T136W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:386 or 387. In some embodiments, clone CH3C.35.23.1.1 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO:386 or 387.

[0313] In some embodiments, clone CH3C.35.23.1.1 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:315. In some embodiments, clone CH3C.35.23.1.1 with hole mutations has the sequence of SEQ ID NO:315.

[0314] In some embodiments, clone CH3C.35.23.1.1 can have hole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:316 or 317. In some embodiments, clone CH3C.35.23.1.1 having hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:316 or 317.

[0315] In some embodiments, clone CH3C.35.23.1.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:318. In some embodiments, clone CH3C.35.23.1.1 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:318.

[0316] In some embodiments, clone CH3C.35.23.1.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:388. In some embodiments, clone CH3C.35.23.1.1 with whole mutations and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:388.

[0317] In some embodiments, clone CH3C.35.23.1.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:319 or 320. In some embodiments, clone CH3C.35.23.1.1, which has the whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:319 or 320.

[0318] In some embodiments, clone CH3C.35.23.1.1 can have whole mutations (e.g., T136S, L138A, and Y177V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:389 or 390. In some embodiments, clone CH3C.35.23.1.1, which has the whole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:389 or 390.

[0319] VIII. TfR-binding protein formats In some embodiments, the modified TfR-binding polypeptides described herein are subunits of a protein dimer. In some embodiments, the dimer is a heterodimer. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer comprises one Fc polypeptide that binds to the TfR receptor (i.e., is monovalent for binding to the TfR receptor). In some embodiments, the dimer comprises a second polypeptide that binds to the TfR receptor. The second polypeptide may comprise the same modified Fc polypeptide to provide a bivalent homodimeric protein, or a second modified Fc polypeptide described herein may provide a second TfR receptor-binding site.

[0320] The TfR-binding polypeptides and dimeric or multimeric proteins comprising the polypeptides described herein can have a wide range of binding affinities, depending, for example, on the format of the polypeptide. For example, in some embodiments, polypeptides comprising the modified Fc polypeptides described herein have affinities for TfR ranging from 1 pM to 10 μM. In some embodiments, affinity can be measured in a monovalent format. In other embodiments, affinity can be measured in a bivalent format, for example, as a protein dimer comprising the modified Fc polypeptide.

[0321] Methods for analyzing binding affinity, binding kinetics, and cross-reactivity to analyze binding to TfR are well known in the art. These methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore™ (GE Healthcare, Piscataway, NJ)), kinetic exclusion assays (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), biolayer interferometry (e.g., Octet® (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to examine binding affinity and / or cross-reactivity. Methods for performing ELISA assays are well known in the art and are also described in the Examples section below. In some embodiments, surface plasmon resonance (SPR) is used to examine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, kinetic exclusion assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, biolayer interferometry is used to determine binding affinity, binding kinetics, and / or cross-reactivity. FcRn binding of TfR-binding polypeptides can also be assessed using these types of assays. FcRn binding is typically assayed under acidic conditions, e.g., at a pH of about 5 to about 6.

[0322] IX. TfR-binding protein conjugates In some embodiments, the modified polypeptide that binds to TfR and initiates transport across the BBB comprises a modified Fc polypeptide described herein and further comprises a partial or complete hinge region. The hinge region may be from any immunoglobulin subclass or isotype. An exemplary immunoglobulin hinge is an IgG hinge region, such as the IgG1 hinge region (e.g., the amino acid sequence of the human IgG1 hinge, EPKSCDKTHTCPPCP (SEQ ID NO:62)). In further embodiments, the polypeptide, which may comprise the hinge or partial hinge region, is further fused to another moiety, such as an immunoglobulin variable region, to create a TfR-binding polypeptide-variable region fusion polypeptide. The variable region can bind to any antigen of interest, such as a therapeutic or diagnostic neurological target.

[0323] In some embodiments, the TfR-binding polypeptide (e.g., a modified Fc polypeptide) is fused to the variable region via a linker. As noted in the preceding paragraph, the TfR-binding polypeptide (e.g., a modified Fc polypeptide) can be fused to the variable region via a hinge region. In some embodiments, the TfR-binding polypeptide (e.g., a modified Fc polypeptide) can be fused to the variable region via a peptide linker. The peptide linker can be configured to allow rotation of the variable region and the TfR-binding polypeptide relative to each other and / or be resistant to protease digestion. In some embodiments, the linker can be a flexible linker comprising amino acids such as Gly, Asn, Ser, Thr, and Ala. Such linkers can be designed using well-known parameters. For example, the linker can have a repeating sequence such as a Gly-Ser repeat.

[0324] The variable regions can be in any antibody format, such as Fab or scFv format, hi some embodiments, the antibody variable region sequences comprise two antibody variable region heavy chains and two antibody variable region light chains, or fragments of each thereof.

[0325] TfR-binding polypeptides (e.g., modified Fc polypeptides) can also be fused to polypeptides other than immunoglobulin variable regions that target an antigen of interest. In some embodiments, such polypeptides are fused to the TfR-binding polypeptide using a peptide linker, such as the flexible linkers described above.

[0326] In some embodiments, the TfR-binding polypeptide can be fused to a polypeptide, e.g., a therapeutic polypeptide, that is desirable for targeting cells expressing the TfR-binding polypeptide. In some embodiments, the TfR-binding polypeptide is fused to a polypeptide having biological activity for transport across the BBB, e.g., a soluble protein, e.g., the extracellular domain of a receptor or a growth factor, cytokine, or enzyme.

[0327] In still other embodiments, the TfR-binding polypeptide can be fused to a peptide or protein useful for protein purification, e.g., polyhistidine, an epitope tag such as FLAG, c-Myc, a hemagglutinin tag, glutathione S-transferase (GST), thioredoxin, protein A, protein G, or maltose-binding protein (MBP). Optionally, the peptide or protein fused to the TfR-binding polypeptide can include a protease cleavage site, such as a factor Xa or thrombin cleavage site. In certain embodiments, the linkage is cleavable by an enzyme present in the central nervous system.

[0328] Substances other than polypeptides can also be bound to TfR-binding polypeptides.Such substances include cytotoxic agents, contrast agents, DNA or RNA molecules, or compounds.In some embodiments, the substance is a therapeutic or imaging compound.In some embodiments, the substance is a small molecule, for example, less than 1000 Da, less than 750 Da, or less than 500 Da.

[0329] The substance, whether polypeptide or non-polypeptide, may bind to the N-terminal or C-terminal region of the TfR-binding polypeptide, or may bind to any region of the polypeptide, so long as the substance does not interfere with binding of the TfR-binding polypeptide to the TfR.

[0330] In various embodiments, conjugates can be prepared using well-known chemical cross-linking reagents and protocols. For example, many chemical cross-linking agents known to those skilled in the art are useful for cross-linking polypeptides with target substances. For example, the cross-linking agent is a heterobifunctional cross-linking agent that can be used to link molecules in a stepwise manner. Heterobifunctional cross-linking agents allow for the design of more specific linking methods for protein binding, thereby reducing the occurrence of unwanted side reactions such as homoprotein polymers.

[0331] The agent of interest may be a therapeutic agent, such as a cytotoxic agent, or including a chemical moiety, hi some embodiments, the agent may be a peptide or small molecule therapeutic or imaging agent.

[0332] X. How to enhance effector function For certain applications, it may be desirable to introduce modifications into the modified Fc polypeptides or modified Fc polypeptide dimers described herein that enhance effector function (e.g., ADCC). One method for enhancing effector function involves generating modified Fc polypeptides or modified Fc polypeptide dimers that are hypofucosylated or fucose-deficient.

[0333] One approach to generating fucose-deficient modified Fc polypeptides or modified Fc polypeptide dimers is to use fucose analogs, such as 2-fluorofucose (2-FF), which can reduce or decrease the availability of GDP-fucose, the substrate required by fucosyltransferases to incorporate fucose into proteins.

[0334] An alternative approach for producing fucose-deficient modified Fc polypeptides or modified Fc polypeptide dimers, which is widely used in commercial production, is to use an α-1,6 fucosyltransferase (FUT8) knockout cell line to express the modified Fc polypeptide or modified Fc polypeptide dimer. A non-limiting example of a suitable FUT8 knockout cell line is the Chinese hamster ovary (CHO) FUT8 knockout cell line sold by Lonza Biologics. Furthermore, as described by Mori et al. (Biotechnol. Bioeng. (2004) 88:901-908; the entire contents of which are incorporated herein by reference), FUT8 small interfering RNA (siRNA) can be used to convert CHO cell lines to produce fucose-deficient proteins (e.g., by constitutive expression of FUT8 siRNA).

[0335] XI. Nucleic Acids, Vectors, and Host Cells The modified TfR-binding polypeptides described herein are typically prepared using recombinant methods. Accordingly, the invention provides isolated nucleic acids comprising nucleic acid sequences encoding any of the polypeptides, including the modified Fc polypeptides described herein, and host cells into which nucleic acids have been introduced that are used to replicate the nucleic acids encoding the polypeptides and / or express the polypeptides. In some embodiments, the host cells are eukaryotic cells, e.g., human cells.

[0336] In another aspect, a polynucleotide is provided that comprises a nucleotide sequence that encodes the polypeptide described herein.The polynucleotide can be single-stranded or double-stranded.In some embodiments, the polynucleotide is DNA.In certain embodiments, the polynucleotide is cDNA.In some embodiments, the polynucleotide is RNA.

[0337] In some embodiments, the polynucleotide is comprised within a nucleic acid construct. In some embodiments, the construct is a replicable vector. In some embodiments, the vector is selected from a plasmid, a viral vector, a phagemid, a yeast chromosomal vector, and a non-episomal mammalian vector.

[0338] In some embodiments, the polynucleotide is operably linked to one or more regulatory nucleotide sequences in an expression construct. In one set of embodiments, the nucleic acid expression construct is adapted for use as a surface expression library. In some embodiments, the library is adapted for surface expression in yeast. In some embodiments, the library is adapted for surface expression in phage. In another set of embodiments, the nucleic acid expression construct is adapted for expression of polypeptides in a system that allows for isolation of polypeptides in milligram or gram quantities. In some embodiments, the system is a mammalian cell expression system. In some embodiments, the system is a yeast cell expression system.

[0339] Expression vehicles for producing recombinant polypeptides include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotes such as E. coli. pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotes. Alternatively, derivatives of viruses such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205) can be used for transient expression of polypeptides in eukaryotes. In some embodiments, it may be desirable to express recombinant polypeptides using a baculovirus expression system. Such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors. Additional expression systems include adenovirus, adeno-associated virus, and other viral expression systems.

[0340] The vector can be transformed into any suitable host cell. In some embodiments, the host cell, such as a bacterial or yeast cell, can be adapted for use as a surface expression library. In some cells, the vector is expressed within the host cell to express relatively large amounts of the polypeptide. Such host cells include mammalian cells, yeast cells, insect cells, and prokaryotic cells. In some embodiments, the cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a baby hamster kidney (BHK) cell, an NS0 cell, a YO cell, an HEK293 cell, a COS cell, a Vero cell, or a HeLa cell.

[0341] Host cells transfected with an expression vector encoding a TfR-binding polypeptide can be cultured under appropriate conditions to result in expression of the polypeptide. The polypeptide can be secreted and isolated from the mixture of cells and the medium containing the polypeptide. Alternatively, the polypeptide can be retained in the cytoplasm or a membrane fraction, and the cells can be harvested, lysed, and the polypeptide isolated by any desired method. [Example]

[0342] XII. Examples The following examples are included to demonstrate specific embodiments of the present disclosure. Those skilled in the art will recognize that the techniques disclosed in the examples below represent techniques that may function effectively in practicing the present disclosure and, therefore, may be considered to constitute specific aspects of its implementation. However, those skilled in the art will recognize, in light of the present disclosure, that many changes can be made to the specific embodiments disclosed and still obtain similar or equivalent results without departing from the spirit and scope of the present disclosure.

[0343] Example 1: Preparation of TfR-binding polypeptides Fc polypeptides that bind to TfR were generated by using combinatorial libraries as specific positions within the CH3 region and selecting these libraries for binding to human TfR. Affinity maturation of the initial TfR-binding sequences identified specific TfR-binding Fc polypeptides, such as the Fc polypeptide having the sequence of SEQ ID NO:66. An Fc polypeptide dimer-Fab fusion containing a heterodimeric Fc polypeptide dimer was constructed by coexpressing the following three polypeptides in a 1:1:2 ratio, respectively: the resulting tetrameric Fc polypeptide dimer-Fab fusion protein is designated BACE1-3C.35.21.

[0344] (1) A heavy chain in which a Fab region, a hinge region, and a modified Fc polypeptide are fused to each other in tandem in that order. The Fab region contains the heavy chain variable region of a BACE1-binding antibody. The hinge region has the sequence of SEQ ID NO:62. The Fc polypeptide has the sequence of SEQ ID NO:250 and contains a "knob" mutation (T366W according to the EU numbering scheme) and a TfR-binding mutation.

[0345] (2) A heavy chain comprising a Fab region, a hinge region, and a modified Fc polypeptide fused to each other in tandem in that order. The Fab region comprises the heavy chain variable region of a BACE1-binding antibody. The hinge region has the sequence of SEQ ID NO:62. The Fc polypeptide has the sequence of SEQ ID NO:251 and contains "hole" mutations (T366S, L368A, and Y407V according to the EU numbering scheme).

[0346] (3) a light chain comprising the light chain variable region of a BACE1-binding antibody;

[0347] DNA encoding the genes for expressing the three polypeptides was cloned into an expression vector and transfected into ExpiCHO cells (Thermo Fisher Scientific) at a 1:1:2 ratio. After 5–7 days, the cells were harvested, and the resulting polypeptides were purified with protein A and then subjected to hybridization using methods well known to those skilled in the art. The resulting polypeptides were analyzed by mass spectrometry to confirm the absence of fusion proteins containing homodimeric Fc polypeptide dimers (i.e., Fc polypeptide dimers containing two Fc polypeptides, both of which have "knob" mutations, or Fc polypeptide dimers containing two Fc polypeptides, both of which have "hole" mutations). Additional tetrameric polypeptides were produced in a similar manner.

[0348] Example 2: Generation of human apical domain knock-in mice (human TfR knock-in mice (TfR ms / hu KI) Mouse) Methods for generating knock-in / knock-out mice have been published in the literature and are well known to those skilled in the art. In summary, TfR knock-in / knock-out mice expressing the human Tfrc apical domain within the mouse Tfrc gene using CRISPR / Cas9 technology have been developed. ms / hu KI mice were generated, and the resulting chimeric TfR was expressed in vivo under the control of the endogenous promoter. As described in International Application No. PCT / US2018 / 018302, the entire contents of which are incorporated herein by reference, a knock-in human apical TfR mouse line was generated by pronuclear microinjection of single-cell embryos using C57B16 mice followed by embryo transfer into pseudopregnant females. Specifically, Cas9, single guide RNAs having the sequences of SEQ ID NOs: 264 and 265, and donor DNA having SEQ ID NO: 267 were introduced into the embryos. The donor DNA contained the coding sequence for the human apical domain (SEQ ID NO: 266, codon-optimized for mouse expression). The coding sequence for the apical domain was flanked by a left (nucleotides 1-817 of SEQ ID NO:267) and a right (nucleotides 1523-2329 of SEQ ID NO:267) homology arm. The donor sequence was designed so that the apical domain was inserted after the fourth mouse exon and immediately adjacent to the ninth exon at the 3' end. Founder males from the offspring of the implanted females were mated with wild-type females to generate F1 heterozygous mice. Homozygous mice were then generated by mating the F1 heterozygous mice.

[0349] Example 3 TfR-binding Fc polypeptides with LALA mutations in both Fc polypeptides prevent reticulocyte loss in mice Antibodies that bind to TfR have been shown to reduce both circulating and bone marrow reticulocytes when administered to mice (see, e.g., Couch et al., Sci Transl Med, 5:183ra57, 1-12, 2013). An Fc polypeptide dimer-Fab fusion similar to BACE1-3C.35.21 described in Example 1 was generated. This Fc polypeptide dimer-Fab fusion is referred to as "BACE1-3C.35.21". 2XLALA " and include: (1) a heavy chain having a Fab region with a heavy chain variable region of a BACE1-binding antibody, a hinge region, and a modified Fc polypeptide with a "knob" mutation (T366W according to the EU numbering scheme), mutations that reduce effector function (L234A and L235A according to the EU numbering scheme), and a TfR-binding mutation (anti-BACE1 Fab region fused to a hinge region (SEQ ID NO:62) and SEQ ID NO:252 (clone CH3C.35.21 with knob and LALA mutations)); (2) a Fab region with a heavy chain variable region of a BACE1-binding antibody, a hinge region, and a modified Fc polypeptide with a "hole" mutation (T366S, L368A, and Y407V according to the EU numbering scheme) and mutations that reduce effector function (L234A and L235A according to the EU numbering scheme) (hinge region (SEQ ID NO:62) and SEQ ID NO:252 (clone CH3C.35.21 with knob and LALA mutations)). (3) two light chains each containing the light chain variable region of a BACE1-binding antibody; and (4) two light chains each containing the light chain variable region of a BACE1-binding antibody.

[0350] To evaluate the Fc polypeptide dimer-Fab fusion protein in vivo, we used a homozygous human TfR knock-in (TfR ms / hu We generated KI) mice. Briefly, these mice were engineered to replace the mouse TfR with a human apical domain / mouse chimeric TfR protein (see Example 2). These mice were then transfected with a BACE1-Fc dimer. 2XLALAPG(anti-BACE1 Fab fused to an Fc dimer with LALA and P329G mutations (according to the EU numbering scheme) in both Fc polypeptides), or BACE1-3C.35.21 2XLALA Circulating and bone marrow reticulocytes were assessed 24 hours after administration by CBC and FACS analysis, respectively. Bone marrow reticulocytes were assessed by Ter119 + , hCD71 高 , and FSC 低 Consistent with the above experiments, BACE1-3C.35.21 2XLALA The Fc polypeptide dimer-Fab fusion, designated as , did not induce a reduction in reticulocytes in vivo, similar to the non-TfR-binding Fc polypeptide dimer (FIGS. 1A and 1B).

[0351] Example 4 Design of TfR-binding Fc polypeptides with asymmetric LALA mutations TfR is highly expressed on reticulocytes, immature red blood cells present in both the bone marrow and circulation. It has been shown that TfR antibodies with intact effector function can rapidly reduce reticulocytes in both the blood and bone marrow. Therefore, reduction of reticulocytes is a major safety issue in TfR-based antibody therapy. However, complete removal of effector function of TfR-based antibodies is not a suitable solution, as in certain cases, it is desirable for effector function to be induced upon Fab binding but not upon binding to TfR by an engineered TfR-binding Fc region fused to a therapeutic Fab. Because Fc mutations such as L234A and L235A (LALA) according to the EU numbering scheme reduce FcγR binding to Fc polypeptide dimers, engineered TfR-binding Fc polypeptide dimers fused to Fabs bearing such mutations in both Fc polypeptides of the Fc polypeptide dimer cannot induce effector function by either TfR binding or Fab binding to a target.

[0352] An engineered TfR-binding Fc polypeptide dimer fused to a therapeutic Fab is desirable, capable of inducing effector function when the Fab binds to its target but not when the TfR-binding site binds to TfR. To this end, Fc polypeptide dimers have been developed in which one (but not the other) of the two Fc polypeptides has a mutation that reduces FcγR binding upon binding to TfR. A series of TfR-binding Fc polypeptide dimers fused to antigen-binding Fab regions that bind either BACE1, human CD20 (hCD20), or mouse CD20 (mCD20), with LALA mutations in one or both Fc polypeptides or neither Fc polypeptide, were generated as described in Tables 1 and 2 below. Table 1 shows the mutations within the Fc regions of each of the two heavy chains. In all variants shown in Table 1, heavy chain 1 contains the TfR binding site and knob mutation T366W (according to the EU numbering scheme) in the Fc region, and heavy chain 2 contains hole mutations T366S, L368A, and Y407V (according to the EU numbering scheme) in the Fc region. Variant zz-3C.35.21 does not contain any additional mutations in the Fc region; 2xLALA contains the LALA mutation in the Fc region of both heavy chains 1 and 2, and the variant zz-3C.35.21 シスLALA contains the LALA mutation (cisLALA or cis form) within the same Fc region as the one containing the TfR binding site, and the mutant zz-3C.35.21 トランスLALA contains the LALA mutation in the Fc region that does not contain the TfR binding site (trans-LALA or trans-form). 2xLALA , mutant zz-3C.35.23 シスLALA , and mutant zz-3C.35.23 トランスLALA This applies to:

[0353] Table 2 shows the SEQ ID NOs for the heavy and light chains of each Fc polypeptide dimer-Fab fusion, e.g., BACE1-3C.35.21 2xLALA The mutant zz-3C.35.21 shown in Table 12xLALA and a Fab region that targets BACE1.

[0354] Table 1. Effector function mutations within the Fc polypeptide TIFF2026010691000001.tif65160

[0355] Table 2. SEQ ID NOs of Fc polypeptide dimer-Fab fusions TIFF2026010691000002.tif115160TIFF2026010691000003.tif230160TIFF2026010691000004.tif123160

[0356] Example 5 TfR binding in cis-form attenuates reticulocyte loss in mice To determine whether Fc polypeptide dimers with cis or trans configurations can attenuate reticulocyte loss, we used homozygous human TfR knock-in (TfR ms / hu KI) mice were treated with these Fc polypeptides and the corresponding wild-type human IgG (hIgG), and reticulocytes from both the peripheral blood and bone marrow of these animals were analyzed. Circulating reticulocytes were quantified from peripheral blood using the Advia 120 Hematology System. Briefly, cells were stained with the ADVIA autoRETIC reagent, and reticulocytes were identified based on RNA content and differential absorbance. For bone marrow reticulocytes, bone marrow cells were collected from the femur of each animal, blocked with an Fc blocker (anti-mouse CD16 / CD32), stained with anti-mTer119 and anti-hCD71, and analyzed by fluorescence-activated cell sorting (FACS). Reticulocytes were analyzed using FlowJo analysis software to identify mTer119 and hCD71. + , hCD71 高 , and FSC 低The Fc polypeptide dimers were gated as a population. Analysis revealed that the cis-configuration Fc polypeptide dimer (i.e., only one Fc polypeptide contains both the TfR-binding site and the LALA mutation, while the other Fc polypeptide contains neither the TfR-binding site nor the LALA mutation) reduced the reticulocyte reduction in both blood and bone marrow observed with the wild-type hIgG control and the trans-configuration Fc polypeptide dimer (i.e., one Fc polypeptide contains the TfR-binding site, and the other Fc polypeptide contains the LALA mutation). Surprisingly, at 25 mg / kg, the cis-configuration Fc polypeptide did not pose the major safety issues typically observed with TfR-binding polypeptides with effector function (Figures 2A and 2B). Furthermore, this system was challenged with a TfR-binding polypeptide with low TfR affinity at a high dose of 50 mg / kg. The cis-configuration partially attenuated the reduction in blood reticulocytes but did not affect the reduction in bone marrow reticulocytes, which is more representative of clinical safety (Figures 2C and 2D). In contrast, the Fc polypeptide dimer having the trans conformation reduced blood and bone marrow reticulocytes to the same extent as the wild-type IgG control. These results demonstrate that the Fc polypeptide dimer having the cis conformation can reduce reticulocyte loss in vivo.

[0357] Example 6 TfR binding in cis-conformation attenuates TfR-mediated ADCC activity in vitro The lack of in vivo reticulocyte reduction by the cis-conformation Fc polypeptide dimer was demonstrated to be due to its inability to induce TfR-mediated ADCC. Ramos cells, which express high levels of human TfR, were used as target cells in in vitro ADCC assays. Target cells were seeded at 10,000 cells / well and opsonized for 30 minutes with (1) hIgG1 containing a TfR-binding site, (2) hIgG1 containing a TfR-binding site and LALA mutations in both Fc polypeptides, and (3) hIgG1 containing a cis-conformation Fc polypeptide dimer. Effector natural killer (NK) cells were isolated from human peripheral blood, incubated overnight with IL-21 (20 ng / ml), and then incubated with target cells at a 25:1 effector:target cell ratio (250,000 cells / well) for 4 hours. Cytotoxicity was assessed by LDH expression, normalized to a control without polypeptide, and calculated as the % maximum lysis of target cells. Because effector immune cells (in this case, natural killer cells) express FcγR, the Fc portion of wild-type IgG1 binds to FcγR and induces an ADCC response. Indeed, hIgG1 with a TfR-binding site induced a strong ADCC response, whereas hIgG1 with LALA mutations in both Fc polypeptides and an Fc polypeptide dimer with a cis configuration did not. This data was consistent with the in vivo reticulocyte data. Specifically, the Fc polypeptide dimer with a cis configuration and the Fc polypeptide dimer with TfR-binding sites and LALA mutations in both Fc polypeptides (with two different TfR affinities (Figure 3A: CH3C.35.21, Figure 3B: CH3C.35.23)) prevented TfR-mediated ADCC, thereby reducing reticulocyte depletion.

[0358] Example 7 TfR-binding polypeptides prevent TfR-mediated in vitro CDC activity TfR-mediated CDC activity may also contribute to the in vivo reduction of reticulocytes by antibodies binding to TfR. Interestingly, Fc polypeptide dimers bound to TfR were unable to induce CDC, presumably due to their inability to form 3D polypeptide hexamers that trigger the complement response. CHO cells engineered to overexpress TfR (CHO-hTfR) were seeded at 200,000 cells / well in serum-free medium. Cells were opsonized for 30 minutes with (1) control hIgG1, (2) Ab204 (anti-TfR positive control antibody), and (3) hIgG1 containing the TfR-binding site. 50 μL of diluted baby rabbit serum was added to each well, and the cells were incubated for 4 hours. Cytotoxicity was assessed by LDH expression, normalized to the control without polypeptide, and calculated as the % maximum lysis of target cells. Indeed, anti-TfR Ab204 failed to induce CDC in CHO-hTfR cells, whereas hIgG1, which contains the TfR-binding site in its Fc region, had no effect on CDC (Fig. 4 ).

[0359] Example 8 TfR-binding polypeptides with cis conformations stimulate pSyk activity in primary human microglia The engineered Fc polypeptide dimers have been confirmed to have functional Fc as measured by FcγR-induced phosphorylation of spleen tyrosine kinase (pSYK) in primary human microglial cells. FcγR on effector immune cells typically binds the Fc region of antibodies to elicit numerous responses important in innate immunity. One of these responses is the Syk tyrosine kinase signaling pathway, which plays a key role in immune cell activation, including phagocytosis, cytokine release, and ADCC (DeFranco et al., J. of Exp Med., 1997, 186(7):1027-39). Upon immune cell FcγR binding to immune complexes, immunoreceptor tyrosine-based activation motifs (ITAMs) recruit Syk kinase, which is phosphorylated by Src family kinases, leading to downstream signaling pathways that trigger immune cell activation (Hirose et al., J of Biol Chem, 2004, 279:32308-15). Therefore, pSyk is used as a readout of FcγR-induced immune cell activation. Microglial cells obtained from mixed glial cultures derived from human embryonic tissue were harvested and used to assess pSyk activity upon binding to TfR-binding polypeptides. Microglia were then added to 96-well plates coated with TfR-binding polypeptides, incubated at 37°C for 10 minutes, lysed, and pSyk levels were quantified using a pSyk sandwich immunoassay. Although the cis-LALA mutant did not induce TfR-mediated ADCC, it was able to induce pSyk responses in human microglial cells (approximately 3-fold increase from the LALA mutant control, Figure 5), similar to the wild-type IgG peptide. This result indicates that the modified Fc polypeptide dimer with the cis conformation retains its Fc function and can have effector functions.

[0360] Example 9 TfR-binding polypeptides having cis conformations induce Fab-mediated ADCC and CDC in target cells In addition to inducing pSYK activity, the ability of the cis-conformal TfR-binding polypeptides to induce Fab-mediated ADCC and CDC was assessed. Fab-mediated ADCC was assessed using target cells expressing murine CD20 (A20 cell line). As with the TfR-mediated ADCC described in Example 8, target cells were seeded at 10,000 cells / well, opsonized, and incubated with NK cells at an effector:target cell ratio of 25:1, and cytotoxicity was assessed by LDH expression. Target cells were opsonized with (1) control hIgG, (2) anti-mCD20 antibody, (3) hIgG1 containing the TfR-binding site and the Fab binding site of mCD20 (CH3C.35.21 hIgG1:α-mCD20(WT)), and (4) hIgG1 containing a cis-configuration and an Fc polypeptide dimer containing the Fab binding site of mCD20 (CH3C.35.21 hIgG1:α-mCD20(cisLALA)). Because target cells do not express human TfR, the assay was designed to assess only Fab binding. Consistent with the induction of pSYK activity, hIgG1 containing a cis-configuration and an Fc polypeptide dimer containing the Fab binding site of mCD20 induced ADCC similarly to anti-mCD20 antibody and hIgG1 containing the TfR-binding site and the Fab binding site of mCD20 (Figure 6A).

[0361] In addition to ADCC, CDC was also evaluated. Raji cells have previously been shown to be sensitive to anti-hCD20-mediated CDC. Therefore, we used Raji cells as target cells to evaluate hIgG1 carrying the TfR-binding site and the Fab-binding site of hCD20. Raji cells were seeded at 200,000 cells / well in serum-free medium and opsonized for 30 minutes with (1) control hIgG, (2) anti-hCD20 antibody, (3) hIgG1 carrying the TfR-binding site and the Fab-binding site of hCD20 (CH3C.35.21 hIgG1:α-hCD20(WT)), and (4) hIgG1 containing a cis-form Fc polypeptide dimer carrying the Fab-binding site of hCD20 (CH3C.35.21 hIgG1:α-hCD20(cisLALA)). Fifty microliters of diluted baby rabbit serum was added to each well, and the cells were incubated for 4 hours. Cytotoxicity was assessed by LDH expression, normalized to a control without polypeptide, and calculated as the percent maximum lysis of target cells. Similar to Fab-mediated ADCC, hIgG1 containing an Fc polypeptide dimer in cis configuration and carrying the Fab-binding site of hCD20 induced CDC to a similar extent as anti-hCD20 and hIgG1 containing the TfR-binding site and the Fab-binding site of hCD20 (Figure 6B). Taken together, these functional in vitro cytotoxicity assays demonstrate that hIgG1 containing an Fc polypeptide dimer in cis configuration does not interfere with the ability of hIgG1 to induce Fab-mediated effector function.

[0362] Example 10 TfR-binding polypeptides with cis-forms and the Fab-binding site of mCD20 induce effector function in vitro As demonstrated in in vivo safety analyses, hIgG1 containing an Fc polypeptide dimer with a cis conformation reduced reticulocyte depletion upon binding to TfR. Next, we tested whether this conformation induced Fab-mediated effector function in vivo, which is essential for inducing a therapeutic response. Antibodies against mCD20 have previously been shown to significantly deplete peripheral blood and splenic B cells in vivo and have therefore been used to assess Fab-mediated effector function. The ability of hIgG1 containing an Fc polypeptide dimer with a cis conformation and the Fab-binding site of mCD20 to deplete blood and splenic B cells was evaluated in wild-type (WT) mice and compared with the responses observed with an anti-mCD20 antibody and hIgG1 containing both the TfR-binding site and the Fab-binding site of mCD20. WT mice were treated with 25 mg / kg of (1) control IgG, (2) anti-mCD20 antibody, (3) hIgG1 containing the TfR-binding site and the Fab binding site of mCD20 (CH3C.35.21 hIgG1:α-mCD20(WT)), (4) hIgG1 containing the TfR-binding site, LALA mutations in both Fc polypeptides, and the Fab binding site of mCD20 (CH3C.35.21 hIgG1:α-mCD20(LALA)), and (5) hIgG1 containing the cis form and an Fc polypeptide dimer containing the Fab binding site of mCD20 (CH3C.35.21 hIgG1:α-mCD20(cisLALA)). Mature peripheral blood B cells and spleen B cells were assessed on days 1 and 5, respectively. Briefly, peripheral blood and spleens were collected. Cells from peripheral blood and spleen cells were treated with ACK lysis buffer, incubated with Fc blocker, and stained with anti-B220 and anti-IgM. Mature B cells were identified by FACS analysis. 高 IgM 高Consistent with the Fab-mediated in vitro ADCC and CDC assays, hIgG1 containing an Fc polypeptide dimer with a cis configuration and the Fab-binding site of mCD20 induced stable B cell depletion, as did anti-mCD20 antibody and hIgG1 containing a TfR-binding site and the Fab-binding site of mCD20 (Figures 7A and 7B). These results demonstrate that the modified Fc polypeptide dimer with a cis configuration retains its Fc function and has Fab-mediated effector function in vivo.

[0363] Example 11 Modified Fc Polypeptides that Bind to TfR This example describes the modification of an Fc polypeptide to confer binding to TfR and cross the BBB.

[0364] Unless otherwise specified, amino acid residue positions in this section are numbered based on EU numbering for the wild-type Fc region of human IgG1.

[0365] Generation and characterization of Fc polypeptides (CH3C clones) containing modifications at positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 Yeast libraries containing Fc regions with modifications introduced at positions including amino acid positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 were generated as described below. Exemplary clones that bind to TfR are shown in Tables 3 and 4.

[0366] After two further rounds of sorting, single clones were sequenced and four unique sequences were identified, all of which contained a conserved Trp at position 388 and an aromatic residue (i.e., Trp, Tyr, or His) at position 421. High diversity was observed at other positions.

[0367] Four clones selected from the library were expressed as Fc fusions with Fab fragments in CHO or 293 cells, purified by protein A and size-exclusion chromatography, and screened by ELISA for binding to human TfR in the presence or absence of holo-Tf. Each clone bound to human TfR, and binding was unaffected by the addition of excess holo-Tf (5 μM). Each clone was also tested for binding to 293F cells, which endogenously express the human TfR. Although each clone bound to 293F cells, overall binding was significantly weaker than that of the high-affinity positive control.

[0368] Next, we tested whether each clone could be internalized by TfR-expressing cells using clone CH3C.3 as the test clone. Adherent HEK293 cells were grown to approximately 80% confluence in 96-well plates, and the medium was removed. The following samples were added at 1 μM: clone CH3C.3, an anti-TfR benchmark positive control antibody (Ab204), an anti-BACE1 benchmark negative control antibody (Ab107), and a human IgG isotype control (obtained from Jackson Immunoresearch). The cells were incubated for 30 minutes at 37°C and 8% CO2, then washed, permeabilized with 0.1% Triton® X-100, and stained with anti-human IgG-Alexa Fluor® 488 secondary antibody. After further washing, the cells were imaged under a high-content fluorescence microscope (i.e., the Opera Phenix® system), and the number of fluorescent puncta per cell was quantified. At 1 μM, clone CH3C.3 showed a similar tendency for internalization as the anti-TfR positive control, whereas the negative control showed no internalization.

[0369] Further manipulation of the clone Further libraries were generated to enhance the affinity of the initial hits for human TfR using a soft randomization approach, in which DNA oligos were generated and soft mutagenesis was introduced based on each of the first four hits. Additional clones that bound to TfR were identified and selected. The selected clones fell into two major sequence groups. Group 1 clones (i.e., clones CH3C.18, CH3C.21, CH3C.25, and CH3C.34) contained a semi-conservative Leu at position 384, a Leu or His at position 386, a conserved and semi-conserved Val at positions 387 and 389, respectively, and a semi-conservative PTW motif at positions 413, 416, and 421, respectively. Group 2 clones contained a conserved Tyr at position 384, the motif TXWSX at positions 386–390, and the conserved motif S / TEF at positions 413, 416, and 421, respectively. Clones CH3C.18 and CH3C.35 were used in further experiments as representative sequences of their respective sequence groups.

[0370] Epitope mapping To test whether the engineered Fc region binds to the apical domain of TfR, the TfR apical domain was expressed on the surface of phage. Proper folding and display of the apical domain requires truncation of one of the loops and a circular rearrangement of the sequence. Clones CH3C.18 and CH3C.35 were coated onto ELISA plates and assayed using a phage ELISA plate. Briefly, after washing and blocking with 1% PBSA, dilutions of the phage display were added and incubated for 1 hour at room temperature. Each plate was then washed, anti-M13-HRP was added, and after further washing, each plate was developed with TMB substrate and quenched with 2N H2SO4. In this assay, both clones CH3C.18 and CH3C.35 bound to the apical domain.

[0371] Paratope Mapping To understand which residues in the Fc domain are most important for binding to the TfR, we generated a series of mutant clones, clone CH3C.18 and clone CH3C.35, in which one position in the TfR-binding register of each mutant was reverted to wild type. The resulting mutants were recombinantly expressed as Fab-Fc fusions and tested for binding to human or cynomolgus monkey TfR. In clone CH3C.35, positions 388 and 421 were critical for binding, and reverting either of these to wild type completely abolished binding to the human TfR.

[0372] Characterization of binding of mature clones As described above, binding ELISA was performed between the purified Fab-Fc fusion variants and plate-coated human or cynomolgus monkey TfR. Clone CH3C.3.2-1, clone CH3C.3.2-5, and clone CH3C.3.2-19, which are variants from the clone CH3C.18 maturation library, showed roughly equivalent EC2s to human and cynomolgus monkey TfR. 50 Whereas the parental clones CH3C.18 and CH3C.35 showed 10-fold higher binding to the human TfR versus the cynomolgus TfR.

[0373] Next, we tested whether the modified Fc polypeptides could be internalized into human and monkey cells. Using the protocol described above, we tested internalization into human HEK293 cells and rhesus monkey LLC-MK2 cells. Clones CH3C.3.2-5 and CH3C.3.2-19, which bind similarly to human and cynomolgus monkey TfRs, showed significantly improved internalization into LLC-MK2 cells compared to clone CH3C.35.

[0374] Further Clone Operations Further engineering of the affinity-matured clones CH3C.18 and CH3C.35 involved adding additional mutations at positions that improved binding through direct interactions, second hydration shell interactions, or structural stabilization. This was accomplished by creating "NNK walk" or "NNK patch" libraries and selecting from these libraries. In the NNK walk library, NNK mutations were introduced one at a time at residues near the paratope. By examining the structure of Fc bound to FcγRI (PDB ID: 4W4O), 44 residues near the original altered positions were identified as candidates for testing. Specifically, the following residues were targeted for NNK mutagenesis: K248, R255, Q342, R344, E345, Q347, T359, K360, N361, Q362, S364, K370, E380, E382, S383, G385, Y391, K392, T393, D399, S400, D401, S403, K409, L410, T411, V412, K414, S415, Q418, Q419, G420, V422, F423, S424, S426, Q438, S440, S442, L443, S444, P4458, G446, and K447. Kunkel mutagenesis was used to generate these 44 single-point NNK libraries, and the products were pooled and electroporated into yeast as described above for the other yeast libraries.

[0375] These mini-libraries (each containing a single mutation position, resulting in 20 mutants) were combined to generate a smaller library, which was then selected for positions that yielded higher affinity binding using yeast surface display. Selection was performed using the TfR apical domain protein as described above. After three rounds of sorting, clones from the enriched yeast library were sequenced, identifying several "hotspot" positions where specific point mutations significantly improved binding to the apical domain protein. In clone CH3C.35, these mutations included E380 (mutated to Trp, Tyr, Leu, or Gln) and S415 (mutated to Glu). The sequences of single and combined mutants of clone CH3C.35 are shown in SEQ ID NOs: 21-23, 64-69, and 125-127. In clone CH3C.18, these mutations included E380 (mutated to Trp, Tyr, or Leu) and K392 (mutated to Gln, Phe, or His). The sequences of the single mutants of clone CH3C.18 are shown in SEQ ID NOs:70-75.

[0376] Further maturation of the library to improve the affinity of clone CH3C.35 To identify mutation combinations from the NNK walk library while adding several additional positions around them, a further library was generated as described for the yeast library above. In this library, the YxTEWSS (SEQ ID NO: 414) and TxxExxxxF (SEQ ID NO: 415) motifs were kept constant, and the following six positions were fully randomized: E380, K392, K414, S415, S424, and S426. Positions E380 and S415 were included because they were "hot spots" in the NNK walk library. Positions K392, S424, and S426 were included because they constitute part of a core that may determine the location of the binding region, and K414 was selected because it is adjacent to position 415.

[0377] This library was sorted by only the apical domain of the cynomolgus TfR as described above. After five rounds, the enriched pool was sequenced, and the sequences of the modified regions of the identified unique clones are shown in SEQ ID NOS: 76-93.

[0378] The next library was designed to further explore the diversity tolerated within the primary binding paratope. A series of single-position saturation mutagenesis libraries was generated in yeast by individually randomizing each of the original positions (384, 386, 387, 388, 389, 390, 413, 416, and 421) and two hot spots (380 and 415) with NNK codons. Each position was then individually remutated to the wild-type residue, and these individual clones were displayed in yeast. It was noteworthy that positions 380, 389, 390, and 415 were the only positions that maintained appreciable binding to TfR when remutated to the wild-type residue (though remutation of position 413 to the wild-type yielded some residual but significantly reduced binding).

[0379] Single-position NNK libraries were sorted three times against the human TfR apical domain, and the top 5% of binding clones were recovered. At least 16 clones were then sequenced from each library. These results, relative to clone CH3C.35, indicate which amino acids can be tolerated at each position without significantly reducing binding to the human TfR. A summary is provided below. Position 380: Trp, Leu, or Glu; Position 384: Tyr or Phe; 386th place: Thr only; Position 387: Glu only; 388th: Trp only; Position 389: Ser, Ala, or Val (the wild-type Asn residue appeared to maintain some binding, but not in subsequent library sorts); 390th: Ser or Asn; Position 413: Thr or Ser; Position 415: Glu or Ser; Position 416: Glu only; and 421st place: Phe only.

[0380] The above residues represent paratope diversity that, when substituted singly or in combination into clone CH3C.35, maintains binding to the TfR apical domain. Clones with mutations at these positions include those shown in Table 4, and the sequences of the CH3 domains of these clones are shown in SEQ ID NOs:65-69, 92, 94-124, and 268-274.

[0381] Example 12 Method 1. Preparation of Phage Display Libraries Template DNA encoding the wild-type human Fc sequence was synthesized and inserted into a phagemid vector containing the ompA or pelB leader sequence, the Fc insert fused to c-Myc and 6xHis (SEQ ID NO:421) epitope tags, and the M13 coat protein pIII followed by an amber stop codon.

[0382] Primers containing an "NNK" triple codon were generated at the desired positions for modification, where N is any DNA base (i.e., A, C, G, or T) and K is G or T. Alternatively, primers for "soft" randomization were used, with a mix of bases at each randomization position, with 70% matching the wild-type base and 10% matching each of the other three bases. Libraries were generated by PCR amplification of Fc region fragments corresponding to each region of randomization, assembling them with end primers containing SfiI restriction sites, digesting them with SfiI, and ligating them into a phagemid vector. Alternatively, primers for Kunkel mutagenesis were used. The ligated Kunkel products were transformed into electrocompetent E. coli cells of the TG1 strain (obtained from Lucigen®). The recovered E. coli cells were infected with M13K07 helper phage and grown overnight. The library phage were then precipitated with 5% PEG / NaCl, resuspended in PBS containing 15% glycerol, and frozen until use. Typical library sizes were approximately 10 transformants. 9 ~about 10 11 Fc dimers were displayed on the phage by pairing of pIII-fused Fc with soluble Fc that was not bound to pIII (the latter generated due to an amber stop codon before pIII).

[0383] Preparation of yeast display libraries Template DNA encoding wild-type human Fc sequences was synthesized and integrated into yeast display vectors. For the CH2 and CH3 libraries, Fc polypeptides were displayed on the Aga2p cell wall protein. Both vectors contained a pre-pro leader peptide with a Kex2 cleavage sequence and a c-Myc epitope tag fused to the terminus of the Fc polypeptide.

[0384] Yeast display libraries were assembled using methods similar to those described for phage libraries, except that fragment amplification was performed using primers with homologous ends to the vector. The linearized vector and assembled library inserts were electroporated into freshly prepared electrocompetent yeast (i.e., strain EBY100). Electroporation techniques will be familiar to those skilled in the art. After recovery in selective SD-CAA medium, yeast were grown to confluence and split twice before protein expression was induced by transferring to SG-CAA medium. Typical library sizes were approximately 10 transformants. 7 ~about 10 9 Fc dimers were formed by pairing of adjacently displayed Fc monomers.

[0385] General methods for phage selection The phage method was adapted from Phage Display: A Laboratory Manual (Barbas, 2001). Further protocol details are available from this reference.

[0386] Plate sorting method Human TfR targets were coated onto MaxiSorp® microtiter plates (typically 200 μL of 1–10 μg / mL PBS solution) overnight at 4°C. All binding was performed at room temperature unless otherwise noted. Phage libraries were added to each well and incubated overnight for binding. Microtiter wells were thoroughly washed with PBS containing 0.05% Tween® 20 (PBST), and bound phage were eluted by incubating each well with acid (typically 500 mM KCl or 50 mM HCl containing 100 mM glycine, pH 2.7) for 30 minutes. Eluted phage were neutralized with 1 M Tris (pH 8) and amplified using TG1 cells and M13 / KO7 helper phage. The phage were grown overnight at 37°C in 2YT medium containing 50 μg / mL carbenicillin and 50 μg / mL kanamycin. Enrichment was assessed by comparing the titers of phage eluted from wells containing target with those recovered from wells containing no target, and then the stringency of selection was increased by shortening the binding incubation time and increasing the wash time and number.

[0387] Bead sorting method Antigens were biotinylated via free amines using NHS-PEG4-biotin (obtained from Pierce™). A 3- to 5-fold molar excess of biotin reagent was used in the biotinylation reaction in PBS. The reaction was stopped with Tris and then extensively dialyzed into PBS. Biotinylated antigens were immobilized on streptavidin-coated magnetic beads (i.e., M280 streptavidin beads obtained from Thermo Fisher). The phage display library was incubated with the antigen-coated beads for 1 hour at room temperature. After this time, unbound phage were removed, and the beads were washed with PBST. Bound phage were eluted by incubation with 500 mM KCl (or 0.1 M glycine, pH 2.7) in 50 mM HCl for 30 minutes, neutralized, and propagated as described above for plate sorting.

[0388] After three to five rounds of panning, single clones were screened by expressing soluble Fc on phage or in the periplasm of E. coli. Such expression methods will be familiar to those skilled in the art. Individual phage supernatants or periplasmic extracts were exposed to blocked ELISA plates coated with antigen or negative control, and then detected with HRP-conjugated goat anti-Fc (obtained from Jackson Immunoresearch) for periplasmic extracts or anti-M13 (GE Healthcare) for phage, followed by development with TMB reagent (obtained from Thermo Fisher). OD 450 Wells with values ​​greater than approximately 5-fold above background were considered positive clones and sequenced, and some clones were then expressed as soluble Fc fragments or fused to Fab fragments.

[0389] General methods for yeast selection Bead sorting (magnetic cell sorting (MACS)) method MACS and FACS selections were performed as described in Ackerman et al. 2009 Biotechnol. Prog. 25(3), 774. Streptavidin magnetic beads (e.g., M-280 streptavidin beads from Thermo Fisher Scientific) were labeled with biotinylated antigen and incubated with yeast (typically 5–10x library diversity). Unbound yeast were removed, the beads were washed, and bound yeast were grown in selective medium and induced for subsequent rounds of selection.

[0390] Fluorescence-activated cell sorting (FACS) Yeast were labeled with anti-c-Myc antibodies to monitor expression and biotinylated antigen (at varying concentrations depending on the sorting round). In some experiments, the antigen was premixed with streptavidin-Alexa Fluor® 647 to increase the avidity of the interaction. In other experiments, the biotinylated antigen was detected after binding and washed with streptavidin-Alexa Fluor® 647. Bound singlets were sorted using a FACS Aria III cell sorter. Sorted yeast were grown in selective medium and then induced for subsequent rounds of selection.

[0391] After obtaining enriched yeast populations, yeast were plated onto SD-CAA agar plates, and single colonies were grown, induced for expression, and labeled as described above to determine their propensity to bind to targets. Positive single clones were then sequenced for antigen binding, and some clones were expressed as soluble Fc fragments or fused to Fab fragments.

[0392] General screening methods Screening by ELISA Clones were selected from the panning output and grown in individual wells of 96-well deep-well plates. Each clone was either induced for periplasmic expression using autoinduction medium (obtained from EMD Millipore) or infected with helper phage for phage display of individual Fc variants on phage. The cultures were grown overnight and centrifuged to pellet the E. coli. Phage-containing supernatants were used directly for phage ELISA. For periplasmic expression, the pellets were resuspended in 20% sucrose, diluted 4:1 with water, and shaken for 1 hour at 4°C. The plates were centrifuged to pellet the solids, and the supernatants were used for ELISA.

[0393] ELISA plates were coated with antigen (usually overnight at 0.5 mg / ml) and then blocked with 1% BSA before adding phage or periplasmic extract. After a 1-hour incubation and washing away unbound proteins, HRP-conjugated secondary antibodies (i.e., anti-Fc or anti-M13 for soluble Fc or phage-displayed Fc, respectively) were added and incubated for 30 minutes. Each plate was washed again, developed with TMB reagent, and quenched with 2N sulfuric acid. Absorbance at 450 nm was quantified using a plate reader (BioTek®), and binding curves were plotted using Prism software, where applicable. The absorbance signal of the tested clones was compared to a negative control (phage or periplasmic extract lacking Fc). In some assays, soluble transferrin or other competitors are added during the binding step, usually in large molar excess (greater than 10-fold excess).

[0394] Flow cytometry screening Fc variant polypeptides (expressed on phage, in periplasmic extracts, or as soluble fusions with Fab fragments) were added to cells (approximately 100,000 cells per well in PBS + 1% BSA (PBSA)) in 96-well V-bottom plates and incubated for 1 hour at 4°C. After this time, each plate was centrifuged, the medium was removed, and the cells were washed once with PBSA. The cells were resuspended in PBSA containing the secondary antibody (usually goat anti-human IgG-Alexa Fluor® 647 (from Thermo Fisher Scientific)). After 30 minutes, the plates were centrifuged, the medium was removed, and the cells were washed once or twice with PBSA before the plates were read on a flow cytometer (i.e., a FACSCanto™ II flow cytometer). Median fluorescence was calculated for each condition using FlowJo software, and binding curves were plotted using Prism software.

[0395] Example 13 Construction of CH3C.18 Mutants This example describes the construction of a library of CH3C.18 mutants.

[0396] Single clones were isolated and grown overnight in SG-CAA medium supplemented with 0.2% glucose to induce surface expression of CH3C.18 variants. For each clone, 2 million cells were washed three times in PBS + 0.5% BSA (pH 7.4). Cells were stained with 250 nM human TfR, 250 nM cynomolgus TfR, or 250 nM unrelated biotinylated protein as biotinylated targets for 1 hour at 4°C with shaking, followed by two washes in the same buffer. Cells were stained with neutravidin-Alexafluor647 (AF647) for 30 minutes at 4°C and then washed twice again. Expression was measured using an anti-c-myc antibody with an anti-chicken Alexfluor488 (AF488) secondary antibody. Cells were resuspended, and the median fluorescence intensity (MFI) of AF647 and AF488 was measured using a BD FACS Canto II. The MFI was calculated for the TfR-binding population of each population and plotted alongside binding to human TfR, cynomolgus monkey TfR, or control.

[0397] Table 5 shows a library of CH3C.18 variants. Each row represents a variant with the indicated amino acid substitution at each position, with the amino acids at the remaining positions being the same as those in CH3C.18. Each position shown in Table 5 is numbered according to the EU numbering scheme.

[0398] Table 5 CH3C.18 mutants TIFF2026010691000005.tif155160

[0399] Example 14 TfR-binding polypeptides with cis-conformation and Fabs that effectively bind amyloid beta (Aβ), cross the BBB, and induce robust effector function recruit microglia to Aβ plaques and reduce plaques in an Aβ plaque mouse model. To provide further evidence that TfR-binding polypeptides with cis-conformations can be used in therapeutically relevant disease models, we evaluated TfR-binding Fc polypeptides with cis-conformations and Fabs that bind Aβ in a mouse model of Aβ plaque deposition. Specifically, these polypeptides were evaluated for their ability to recruit microglia to Aβ plaques. Briefly, animals (3.5 months old) were treated intraperitoneally with 50 mg / kg TfR-binding polypeptides on days 0, 3, 6, and 9 in the following groups: 1) TfR-binding polypeptides treated with control IgG; ms / hu KI mice (n=6), 2) 5XFAD×TfR treated with control IgG ms / hu KI mice (n = 11), 3) 5XFAD × TfR treated with anti-Aβ (α-Aβ) ms / hu KI mice (n=12), 4) 5XFAD×TfR treated with a TfR-binding Fc polypeptide containing the cis form and Aβ Fab binding site ms / hu KI mice (n=12), and 5) 5XFAD×TfR treated with a TfR-binding Fc polypeptide carrying the LALA mutation in both Fc polypeptides. ms / hu KI mice (n=12). Table 6 shows the SEQ ID NOs for the heavy and light chains of each Fc polypeptide dimer-Fab fusion.

[0400] Table 6. SEQ ID NOs for Fc polypeptide dimer-Fab fusions TIFF2026010691000006.tif124160

[0401] On day 12, mice were perfused, and brains were harvested and sectioned sagitally at 40 μm for immunohistochemistry. Two brain sections per animal (section 1: approximately 3 mm lateral to the midline, section 2: midline) were selected for IHC analysis. Free-floating sections were incubated in blocking solution (5% donkey serum in PBS containing 0.3% Triton X-100) at room temperature for 2 hours, followed by incubation with primary antibodies (anti-CD68, anti-human Aβ) overnight at 4°C. Sections were then washed three times for 15 minutes each, incubated with fluorescently labeled secondary antibodies for 2 hours at room temperature, and in DAPI solution for 20 minutes, followed by three washes in PBS containing 0.3% Triton X-100. Each section was mounted on a slide and coverslipped with Prolong Glass Antifade mounting medium. Slides were imaged using a Zeiss Axioscan.Z1 slide scanner at 20x magnification and processed using custom macros and image processing macros in Zeiss ZEN software. Specifically, images were analyzed to determine expanded plaque area (by size), plaque / CD68 microglia overlap area, and expanded CD68 + Microglia area, count, and pixel intensity summation (by size, 9–14 μm 2 , 14~33μm 2 , 33~75μm 2 , and 75 to 3333 μm 2 ), as well as plaque morphology, area, count, and pixel intensity summation (density <0.7 for irregular and >0.7 for circular to separate round from irregular plaques; size separation (30-125 μm 2 , 125~250μm 2 , 250~500μm 2 , and 500 to 3300 μm 2 )) was measured.

[0402] These experiments demonstrated that 5XFAD×TfR treated with anti-Aβ, which contains a TfR binding site containing a cis-LALA Fc polypeptide dimer, ms / huKI mice induce stable microglial recruitment to Aβ plaques (as measured by colocalization of the microglial marker CD68 with Aβ markers), with sizes ranging from 30 to 125 μm. 2 The anti-Aβ antibody reduced small plaques in a manner similar to that of the anti-Aβ antibody (Figures 8A-8C). Importantly, these effects were not observed with an anti-Aβ antibody bearing a TfR-binding site with LALA mutations in both Fc polypeptides, consistent with the requirement for effector function in this disease paradigm. Overall, these data, along with other in vitro and in vivo data herein, provide strong evidence that a platform scaffold of TfR-binding Fc polypeptides bearing the cis-LALA conformation and associated Fab-binding site can not only mitigate reticulocyte safety but also induce target-mediated effector function in relevant disease models (i.e., microglial involvement in the brain).

[0403] It will be understood that the examples and embodiments set forth herein are for illustrative purposes only, and that various modifications or variations will be suggested to those skilled in the art in light of the same, and that such modifications and variations are intended to be included within the spirit and scope of this application and the appended claims. Sequences with sequence accession numbers cited herein are hereby incorporated by reference.

[0404] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0405] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, or limitation, not specifically disclosed herein. Thus, for example, terms such as "comprise," "include," and "contain" should be read expansively and without limitation. Furthermore, the terms and expressions used herein are used as terms of description and not of limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described, or portions thereof, but rather to recognize that various modifications are possible within the scope of the claimed invention.

[0406] In the event of a discrepancy, the amino acid substitution for each clone listed in a table (e.g., Tables 3 and 4) determines the amino acid substitution at that register position for that clone in preference to the amino acid found in the sequence listed in the Sequence Listing.

[0407] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if individually incorporated. In case of conflict, the present specification, including definitions, will control.

[0408] Table 3: Positions and mutations of the CH3C register TIFF2026010691000007.tif159170

[0409] Table 4: Additional CH3C register positions and mutations TIFF2026010691000008.tif230160

[0410] Unofficial sequence listing TIFF2026010691000009.tif224170TIFF2026010691000010.tif240170TIFF2026010691000011.tif245170TIFF2026010691000012.tif243170TIFF2026010691000013.tif235170TIFF2026010691000014.tif240170TIFF2026010691000015.tif240170TIFF2026010691000016.tif240170TIFF2026010691000017.tif240170TIFF2026010691000018.tif240170TIFF2026010691000019.tif242170TIFF2026010691000020.tif247170TIFF2026010691000021.tif227170TIFF2026010691000022.tif240170TIFF2026010691000023.tif240170TIFF2026010691000024.tif240170TIFF2026010691000025.tif240170TIFF2026010691000026.tif240170TIFF2026010691000027.tif240170TIFF2026010691000028.tif222170TIFF2026010691000029.tif237170TIFF2026010691000030.tif135170TIFF2026010691000031.tif242170TIFF2026010691000032.tif240170TIFF2026010691000033.tif240170TIFF2026010691000034.tif240170TIFF2026010691000035.tif240170TIFF2026010691000036.tif240170TIFF2026010691000037.tif240170TIFF2026010691000038.tif240170TIFF2026010691000039.tif240170TIFF2026010691000040.tif240170TIFF2026010691000041.tif240170TIFF2026010691000042.tif240170TIFF2026010691000043.tif240170TIFF2026010691000044.tif239170TIFF2026010691000045.tif233170TIFF2026010691000046.tif118170.

[0411] Array information SEQUENCE LISTING <110> DENALI THERAPEUTICS INC. <120> TRANSFERRIN RECEPTOR-BINDING POLYPEPTIDES AND USES THEREOF <150> US 62 / 721,275 <151> 2018-08-22 <150> US 62 / 682,639 <151> 2018-06-08 <150> US 62 / 631,281 <151> 2018-02-15 <150> US 62 / 615,914 <151> 2018-01-10 <160> 421 <170> PatentIn version 3.5 <210> 1 <211> 217 <212> PRT <213> Homo sapiens <400> 1 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 2 <211> 110 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 2 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys ...

Claims

[Claim 1] The invention described herein.

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