Anti-HER2 Polypeptides and Methods of Their Use

Abstract

To provide novel therapeutic agents that can cross the BBB and target HER2 in the brain parenchyma.SOLUTION: In some embodiments, the anti-HER2 constructs (e.g., Fc polypeptide dimer-antibody variable region fusion proteins) retain effector function upon binding to HER2, but do not substantially deplete reticulocytes in vivo. The present disclosure also relates to methods for transcytosing an anti-HER2 antibody variable region across the BBB and treating HER2-positive cancers and metastatic lesions thereof.SELECTED DRAWING: None

Description

[Background technology]

[0001] background Treatment of brain metastases from cancers such as breast cancer currently poses an overwhelming clinical challenge. The incidence of brain metastases among breast cancer patients is as high as 50%. Clinical data indicate that HER2-positive breast cancers tend to metastasize to the brain. In particular, anti-HER2 therapies have proven useful in controlling extracranial tumors, but not intracranial lesions. The failure of these therapies to control metastatic lesions, such as brain metastases from HER2-positive breast cancer, is primarily due to the inability of the therapeutic agents to cross the blood-brain barrier (BBB) ​​and access the brain parenchyma. Therefore, there is a need for new therapeutic agents that can cross the BBB and target HER2 in the brain parenchyma. Summary of the Invention

[0002] overview In some aspects, provided herein is an Fc polypeptide dimer-antibody variable region fusion protein comprising: (a) an antibody variable region capable of binding to human epidermal growth factor receptor 2 (HER2), or an antigen-binding fragment thereof; and (b) a modified Fc polypeptide dimer comprising a first Fc polypeptide comprising amino acid modification(s) that create a TfR-binding site. The antibody variable region may comprise a heavy chain variable region and a light chain variable region.

[0003] In some embodiments, the protein comprises (i) two copies of an antibody light chain, (ii) a first heavy chain variable region fused to a first Fc polypeptide, and (iii) a second heavy chain variable region fused to a second Fc polypeptide. Each light chain can be paired with one of the heavy chain variable regions, and the first and second Fc polypeptides may together form an Fc dimer. The first heavy chain variable region can be fused (N- to C-terminus) to a CH1 domain, which is then fused to a hinge region, which is then fused to the first Fc polypeptide, and / or the second heavy chain variable region can be fused (N- to C-terminus) to a CH1 domain, which is then fused to a hinge region, which is then fused to the second Fc polypeptide.

[0004] In some embodiments, the antibody variable region comprises: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 69, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 70, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 70; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 71, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 71; (d) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 72 or having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 72; (e) a light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 73; and (f) a light chain CDR3 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 74.

[0005] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 70; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 71; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 72; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 73; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 74.

[0006] In some embodiments, the antibody variable regions comprise two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:59 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:60.

[0007] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 75 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 76 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 76; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 77 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 77; (d) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 78 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 78; (e) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 79; and (f) a light chain CDR3 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 80.

[0008] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 76; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 77; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 78; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 79; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 80.

[0009] In some embodiments, the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:61 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:62.

[0010] In some embodiments, the antibody variable region comprises: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 250, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 251, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 251; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 252, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 252; (d) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 253 or having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 253; (e) a light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 254; and (f) a light chain CDR3 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 255.

[0011] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 251; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 252; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 253; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 254; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 255.

[0012] In some embodiments, the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:256 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:257.

[0013] In some embodiments, the TfR-binding site is located within an engineered CH3 domain. In some embodiments, the engineered CH3 domain is derived from a human IgG1, IgG2, IgG3, or IgG4 CH3 domain. In some embodiments, the engineered CH3 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions at the set of amino acid positions including 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 according to EU numbering. In some embodiments, the modified CH3 domain contains, according to EU numbering, 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, Gly, Ser, Thr, or at position 390; Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr at position 391; an acidic amino acid, 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 an acidic amino acid at position 416; and / or an aromatic amino acid, His, or Lys at position 421.

[0014] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein binds to the apical domain of TfR. In some embodiments, one of the Fc polypeptides comprises an amino acid modification that reduces FcγR binding when the Fc polypeptide dimer is bound to TfR (e.g., but has limited or no reduced binding when not bound to TfR). These modifications may include Ala at positions 234 and 235 on the first Fc polypeptide, according to EU numbering. In some embodiments, both Fc polypeptides comprise an amino acid modification that reduces FcγR binding (e.g., both polypeptides comprise Ala at positions 234 and 235).

[0015] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion is fucose-deficient or afucosylated (eg, as described herein).

[0016] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises an amino acid modification that increases serum half-life. In some embodiments, the amino acid modification that increases serum half-life comprises (i) a Leu at position 428 and a Ser at position 434, or (ii) a Ser or an Ala at position 434, according to EU numbering.

[0017] In some embodiments, the first Fc polypeptide further comprises knob mutation T366W, and the second Fc polypeptide comprises hole mutations T366S, L368A, and Y407V, according to EU numbering. In some embodiments, the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 67 and 68.

[0018] In another aspect, provided herein is an Fc polypeptide dimer-antibody variable region fusion protein comprising: (a) an antibody variable region capable of binding to human HER2, or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising a modification that creates a TfR-binding site and knob mutation T366W, according to EU numbering; and (c) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, according to EU numbering, and no TfR-binding site.

[0019] In some embodiments, the antibody variable region comprises: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 69, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 70, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 70; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 71, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 71; (d) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 72 or having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 72; (e) a light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 73; and (f) a light chain CDR3 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 74.

[0020] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 70; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 71; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 72; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 73; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 74.

[0021] In some embodiments, the antibody variable regions comprise two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:59 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:60.

[0022] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 75 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 76 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 76; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 77 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 77; (d) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 78 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 78; (e) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 79; and (f) a light chain CDR3 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 80.

[0023] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 76; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 77; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 78; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 79; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 80.

[0024] In some embodiments, the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:61 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:62.

[0025] In some embodiments, the antibody variable region comprises: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 250, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 251, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 251; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 252, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 252; (d) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 253 or having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 253; (e) a light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 254; and (f) a light chain CDR3 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 255.

[0026] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 251; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 252; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 253; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 254; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 255.

[0027] In some embodiments, the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:256 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:257.

[0028] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, and 81. In some embodiments, the first Fc polypeptide further comprises amino acid modifications L234A and L235A. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 2, 10, 18, and 82. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequences of SEQ ID NO: 57.

[0029] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 29, 37, 45, and 89. In some embodiments, the first Fc polypeptide further comprises amino acid modifications L234A and L235A. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 30, 38, 46, and 90. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0030] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 258, 266, 274, and 282. In some embodiments, the first Fc polypeptide further comprises amino acid modifications L234A and L235A. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 259, 267, 275, and 283. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 290. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0031] In another aspect, provided herein is an Fc polypeptide dimer-antibody variable region fusion protein comprising: (a) an antibody variable region capable of binding to human HER2, or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising the amino acid modifications N434S, with or without knob mutations T366W and M428L, modifications that create a TfR binding site, according to EU numbering; and (c) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, according to EU numbering, and that does not comprise a TfR binding site.

[0032] In some embodiments, the antibody variable region comprises: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 69, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 70, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 70; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 71, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 71; (d) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 72 or having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 72; (e) a light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 73; and (f) a light chain CDR3 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 74.

[0033] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 70; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 71; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 72; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 73; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 74.

[0034] In some embodiments, the antibody variable regions comprise two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:59 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:60.

[0035] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 75 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 76 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 76; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 77 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 77; (d) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 78 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 78; (e) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 79; and (f) a light chain CDR3 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 80.

[0036] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 76; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 77; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 78; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 79; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 80.

[0037] In some embodiments, the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:61 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:62.

[0038] In some embodiments, the antibody variable region comprises: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 250, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 251, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 251; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 252, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 252; (d) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 253 or having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 253; (e) a light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 254; and (f) a light chain CDR3 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 255.

[0039] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 251; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 252; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 253; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 254; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 255.

[0040] In some embodiments, the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:256 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:257.

[0041] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 3, 11, 19, and 83. In some embodiments, the first Fc polypeptide further comprises amino acid modifications L234A and L235A. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 4, 12, 20, and 84. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequences of SEQ ID NO: 57.

[0042] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 31, 39, 47, and 91. In some embodiments, the first Fc polypeptide further comprises amino acid modifications L234A and L235A. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 32, 40, 48, and 92. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0043] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 260, 268, 276, and 284. In some embodiments, the first Fc polypeptide further comprises amino acid modifications L234A and L235A. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 261, 269, 277, and 285. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 290. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0044] In another aspect, provided herein is an Fc polypeptide dimer-antibody variable region fusion protein comprising: (a) an antibody variable region capable of binding to human HER2, or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising knob mutation T366W and modifications that create a TfR-binding site, according to EU numbering; and (c) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, and amino acid modification N434S, with or without M428L, according to EU numbering, and that does not comprise a TfR-binding site.

[0045] In some embodiments, the antibody variable region comprises: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 69, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 70, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 70; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 71, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 71; (d) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 72 or having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 72; (e) a light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 73; and (f) a light chain CDR3 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 74.

[0046] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 70; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 71; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 72; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 73; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 74.

[0047] In some embodiments, the antibody variable regions comprise two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:59 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:60.

[0048] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 75 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 76 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 76; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 77 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 77; (d) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 78 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 78; (e) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 79; and (f) a light chain CDR3 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 80.

[0049] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 76; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 77; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 78; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 79; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 80.

[0050] In some embodiments, the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:61 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:62.

[0051] In some embodiments, the antibody variable region comprises: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 250, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 251, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 251; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 252, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 252; (d) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 253 or having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 253; (e) a light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 254; and (f) a light chain CDR3 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 255.

[0052] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 251; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 252; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 253; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 254; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 255.

[0053] In some embodiments, the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:256 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:257.

[0054] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, and 81. In some embodiments, the first Fc polypeptide further comprises amino acid modifications L234A and L235A. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 2, 10, 18, and 82. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequences of SEQ ID NO: 57.

[0055] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 29, 37, 45, and 89. In some embodiments, the first Fc polypeptide further comprises amino acid modifications L234A and L235A. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 30, 38, 46, and 90. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 56. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0056] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 258, 266, 274, and 282. In some embodiments, the first Fc polypeptide further comprises amino acid modifications L234A and L235A. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 259, 267, 275, and 283. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 291. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0057] In another aspect, provided herein is an Fc polypeptide dimer-antibody variable region fusion protein comprising: (a) an antibody variable region capable of binding to human HER2, or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising modifications that create a TfR-binding site, knob mutation T366W, and amino acid modification N434S, with or without M428L, according to EU numbering; and (c) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, and amino acid modification N434S, with or without M428L, according to EU numbering, and that does not comprise a TfR-binding site.

[0058] In some embodiments, the antibody variable region comprises: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 69, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 70, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 70; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 71, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 71; (d) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 72 or having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 72; (e) a light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 73; and (f) a light chain CDR3 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 74.

[0059] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 70; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 71; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 72; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 73; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 74.

[0060] In some embodiments, the antibody variable regions comprise two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:59 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:60.

[0061] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 75 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 76 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 76; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 77 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 77; (d) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 78 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 78; (e) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 79; and (f) a light chain CDR3 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 80.

[0062] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 76; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 77; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 78; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 79; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 80.

[0063] In some embodiments, the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:61 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:62.

[0064] In some embodiments, the antibody variable region comprises: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 250, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 251, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 251; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 252, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 252; (d) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 253 or having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 253; (e) a light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 254; and (f) a light chain CDR3 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 255.

[0065] In some embodiments, the antibody variable region comprises one or more (e.g., all six) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 251; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 252; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 253; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 254; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 255.

[0066] In some embodiments, the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:256 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:257.

[0067] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 3, 11, 19, and 83. In some embodiments, the first Fc polypeptide further comprises amino acid modifications L234A and L235A. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 4, 12, 20, and 84. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequences of SEQ ID NO: 57.

[0068] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 31, 39, 47, and 91. In some embodiments, the first Fc polypeptide further comprises amino acid modifications L234A and L235A. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 32, 40, 48, and 92. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 56. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0069] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 260, 268, 276, and 284. In some embodiments, the first Fc polypeptide further comprises amino acid modifications L234A and L235A. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 261, 269, 277, and 285. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 291. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0070] In some embodiments, the modified Fc polypeptide dimer does not substantially reduce reticulocytes. In some embodiments, the amount of reticulocytes reduced following administration of the Fc polypeptide dimer-antibody variable region fusion protein is less than the amount of reticulocytes reduced following administration of a control. In some embodiments, the control is a corresponding TfR-binding polypeptide dimer-antibody variable region fusion protein that has full effector function and / or does not contain a mutation that reduces FcγR binding.

[0071] In another aspect, provided herein is an antibody heavy chain comprising: (a) an anti-human HER2 antibody heavy chain variable region, or a fragment thereof; and (b) an altered Fc polypeptide comprising modifications that create a TfR-binding site. In some embodiments, the altered Fc polypeptide comprises one or more amino acid modifications that reduce FcγR binding upon binding to TfR.

[0072] In some embodiments, the antibody heavy chain variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 69, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 70, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 70; and (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 71, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 71. In some embodiments, the antibody heavy chain variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59.

[0073] In some embodiments, the antibody variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 75, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 76, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 76; and (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 76; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61.

[0074] In some embodiments, the antibody variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 250, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 251, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 251; and (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 252, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the antibody variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 251; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 252. In some embodiments, the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 256.

[0075] In some embodiments, the TfR-binding site is located within an engineered CH3 domain. In some embodiments, the engineered CH3 domain is derived from a human IgG1, IgG2, IgG3, or IgG4 CH3 domain. In some embodiments, the engineered CH3 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions at the set of amino acid positions including 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 according to EU numbering. In some embodiments, the modified CH3 domain contains, according to EU numbering, 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, Gly, Ser, Thr, or at position 390; Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr at position 391; an acidic amino acid, 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 an acidic amino acid at position 416; and / or an aromatic amino acid, His, or Lys at position 421.

[0076] In some embodiments, the amino acid modification that reduces FcγR binding upon binding to TfR comprises Ala at position 234 and at position 235, according to EU numbering. In some embodiments, the modified Fc polypeptide further comprises an amino acid modification that increases serum half-life. In some embodiments, the amino acid modification that increases serum half-life comprises (i) Leu at position 428 and Ser at position 434, or (ii) Ser or Ala at position 434, according to EU numbering. In some embodiments, the modified Fc polypeptide further comprises knob mutation T366W, according to EU numbering. In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 63.

[0077] In another aspect, provided herein is an antibody heavy chain comprising: (a) an anti-human HER2 antibody heavy chain variable region, or a fragment thereof; and (b) an altered Fc polypeptide comprising a modification that creates a TfR binding site and the knob mutation T366W, according to EU numbering.

[0078] In some embodiments, the antibody heavy chain variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 69, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 70, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 70; and (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 71, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 71. In some embodiments, the antibody heavy chain variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59.

[0079] In some embodiments, the antibody variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 75, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 76, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 76; and (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 76; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61.

[0080] In some embodiments, the antibody variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 250, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 251, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 251; and (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 252, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the antibody variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 251; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 252. In some embodiments, the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 256.

[0081] In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, and 81. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 29, 37, 45, and 89. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 258, 266, 274, and 282. In some embodiments, the modified Fc polypeptide further comprises the amino acid modifications L234A and L235A. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 2, 10, 18, and 82. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 30, 38, 46, and 90. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 259, 267, 275, and 283.

[0082] In another aspect, provided herein is an antibody heavy chain comprising: (a) an anti-human HER2 antibody heavy chain variable region, or a fragment thereof; and (b) a modified Fc polypeptide comprising the amino acid modification N434S with or without knob mutation T366W and M428L, modifications that create a TfR binding site, according to EU numbering.

[0083] In some embodiments, the antibody heavy chain variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 69, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 70, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 70; and (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 71, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 71. In some embodiments, the antibody heavy chain variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59.

[0084] In some embodiments, the antibody variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 75, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 76, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 76; and (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 76; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61.

[0085] In some embodiments, the antibody variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 250, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 251, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 251; and (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 252, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the antibody variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 251; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 252. In some embodiments, the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 256.

[0086] In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 3, 11, 19, and 83. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 31, 39, 47, and 91. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 260, 268, 276, and 284. In some embodiments, the modified Fc polypeptide further comprises the amino acid modifications L234A and L235A. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 4, 12, 20, and 84. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 32, 40, 48, and 92. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 261, 269, 277, and 285.

[0087] In another aspect, provided herein is a pharmaceutical composition comprising an Fc polypeptide dimer-antibody variable region fusion protein described herein and a pharmaceutically acceptable carrier.

[0088] In another aspect, provided herein are methods for transcytosis across an endothelium of an antibody variable region, or antigen-binding fragment thereof, capable of binding to human HER2, the method comprising contacting the endothelium with a composition comprising an Fc polypeptide dimer-antibody variable region fusion protein described herein. In some embodiments, the endothelium is the BBB.

[0089] In another aspect, provided herein are methods for treating cancer in a subject, the methods comprising administering to the subject a therapeutically effective amount of a composition comprising an Fc polypeptide dimer-antibody variable region fusion protein described herein. In some embodiments, the cancer is a HER2-positive cancer. In some embodiments, the HER2-positive cancer is a HER2-positive breast cancer. In some embodiments, the HER2-positive cancer is a HER2-positive gastric adenocarcinoma and / or a HER2-positive gastroesophageal junction adenocarcinoma. The HER2-positive cancer is a metastatic cancer.

[0090] In another aspect, provided herein are methods for treating brain metastasis of a HER2-positive cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an Fc polypeptide dimer-antibody variable region fusion protein described herein. In some embodiments, the HER2-positive cancer is HER2-positive breast cancer. In some embodiments, the HER2-positive cancer is HER2-positive gastric adenocarcinoma and / or HER2-positive gastroesophageal junction adenocarcinoma.

[0091] In some embodiments, a combination of different Fc polypeptide dimer-antibody variable region fusion proteins (e.g., a combination of Fc polypeptide dimer-antibody variable region fusion proteins binding to subdomains IV and II of HER2) are administered. In some embodiments, a first Fc polypeptide dimer-antibody variable region fusion protein and a second Fc polypeptide dimer-antibody variable region fusion protein are administered to a subject, wherein the antibody variable region of the first Fc polypeptide dimer-antibody variable region fusion protein comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 59 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO: 60 (i.e., an anti-HER2 subdomain IV Fc polypeptide dimer-antibody variable region fusion protein), and the antibody variable region of the second Fc polypeptide dimer-antibody variable region fusion protein comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 61 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO: 62 (i.e., an anti-HER2 subdomain II Fc polypeptide dimer-antibody variable region fusion protein). In some embodiments, an anti-HER2 subdomain IV Fc polypeptide dimer-antibody variable region fusion protein may be administered alone or in combination with an anti-HER2 subdomain II Fc polypeptide dimer-antibody variable region fusion protein. In some embodiments, an anti-HER2 subdomain II Fc polypeptide dimer-antibody variable region fusion protein may be administered alone or in combination with an anti-HER2 subdomain IV Fc polypeptide dimer-antibody variable region fusion protein. In certain embodiments, an anti-HER2 subdomain IV Fc polypeptide dimer-antibody variable region fusion protein may be administered alone. In certain embodiments, an anti-HER2 subdomain II Fc polypeptide dimer-antibody variable region fusion protein may be administered alone.

[0092] In some embodiments, a combination of different Fc polypeptide dimer-antibody variable region fusion proteins (e.g., a combination of Fc polypeptide dimer-antibody variable region fusion proteins that bind subdomains II and I of HER2) are administered. In some embodiments, a first Fc polypeptide dimer-antibody variable region fusion protein and a second Fc polypeptide dimer-antibody variable region fusion protein are administered to a subject, wherein the antibody variable region of the first Fc polypeptide dimer-antibody variable region fusion protein comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 61 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO: 62 (i.e., an anti-HER2 subdomain II Fc polypeptide dimer-antibody variable region fusion protein), and the antibody variable region of the second Fc polypeptide dimer-antibody variable region fusion protein comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 256 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO: 257 (i.e., an anti-HER2 subdomain I Fc polypeptide dimer-antibody variable region fusion protein). In some embodiments, an anti-HER2 subdomain II Fc polypeptide dimer-antibody variable region fusion protein may be administered alone or in combination with an anti-HER2 subdomain I Fc polypeptide dimer-antibody variable region fusion protein. In some embodiments, an anti-HER2 subdomain I Fc polypeptide dimer-antibody variable region fusion protein may be administered alone or in combination with an anti-HER2 subdomain II Fc polypeptide dimer-antibody variable region fusion protein. In certain embodiments, an anti-HER2 subdomain II Fc polypeptide dimer-antibody variable region fusion protein may be administered alone. In certain embodiments, an anti-HER2 subdomain I Fc polypeptide dimer-antibody variable region fusion protein may be administered alone.

[0093] In some embodiments, a combination of different Fc polypeptide dimer-antibody variable region fusion proteins (e.g., a combination of Fc polypeptide dimer-antibody variable region fusion proteins that bind to subdomains IV and I of HER2) are administered. In some embodiments, a first Fc polypeptide dimer-antibody variable region fusion protein and a second Fc polypeptide dimer-antibody variable region fusion protein are administered to a subject, wherein the antibody variable region of the first Fc polypeptide dimer-antibody variable region fusion protein comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 59 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO: 60 (i.e., an anti-HER2 subdomain IV Fc polypeptide dimer-antibody variable region fusion protein), and the antibody variable region of the second Fc polypeptide dimer-antibody variable region fusion protein comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 256 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO: 257 (i.e., an anti-HER2 subdomain I Fc polypeptide dimer-antibody variable region fusion protein). In some embodiments, an anti-HER2 subdomain IV Fc polypeptide dimer-antibody variable region fusion protein may be administered alone or in combination with an anti-HER2 subdomain I Fc polypeptide dimer-antibody variable region fusion protein. In some embodiments, an anti-HER2 subdomain I Fc polypeptide dimer-antibody variable region fusion protein may be administered alone or in combination with an anti-HER2 subdomain IV Fc polypeptide dimer-antibody variable region fusion protein. In certain embodiments, an anti-HER2 subdomain IV Fc polypeptide dimer-antibody variable region fusion protein may be administered alone. In certain embodiments, an anti-HER2 subdomain I Fc polypeptide dimer-antibody variable region fusion protein may be administered alone.

[0094] In some embodiments, the composition comprising the Fc polypeptide dimer-antibody variable region fusion protein antagonizes HER2 activity, hi some embodiments, the subject has not been previously treated with anti-HER2 therapy and / or chemotherapy for metastatic disease.

[0095] In yet another aspect, the disclosure features a method for treating cancer or treating brain metastasis of cancer in a subject, the method includes administering to the subject a therapeutically effective amount of an anti-HER2 construct that binds to (a) subdomain I or II of human HER2 and (b) transferrin receptor (TfR), wherein the anti-HER2 construct alone is therapeutically effective to treat the cancer.

[0096] In some embodiments of this aspect, the anti-HER2 construct comprises an antibody variable region that binds to subdomain I or II of human HER2. The anti-HER2 construct can comprise a modified Fc polypeptide dimer comprising a first Fc polypeptide that includes a modification that creates a TfR-binding site. For example, the anti-HER2 construct is an Fc polypeptide dimer-antibody variable region fusion protein.

[0097] In other embodiments of this aspect, the anti-HER2 construct comprises an antibody variable region that binds to TfR. For example, the anti-HER2 construct may be a bispecific construct comprising an antibody variable region that binds to subdomain I or II of human HER2 and an antibody variable region that binds to TfR.

[0098] In some embodiments, the anti-HER2 construct is administered to the subject as a monotherapy. In some embodiments, the anti-HER2 construct is administered in combination with chemotherapy or radiation therapy.

[0099] In some embodiments, the anti-HER2 construct specifically binds to HER2 and TfR on the same cell.

[0100] In another aspect, the disclosure features a method for treating cancer or treating brain metastasis of cancer in a subject, the method includes administering a therapeutically effective amount of: (a) a first anti-HER2 construct that binds to subdomain II of human HER2; and (b) a second anti-HER2 construct that binds to subdomain IV of human HER2; or (a) a first anti-HER2 construct that binds to subdomain I of human HER2; and (b) a second anti-HER2 construct that binds to subdomain IV of human HER2; or (a) a first anti-HER2 construct that binds to subdomain I of human HER2; and (b) A second anti-HER2 construct that binds to subdomain II of human HER2. administering to a subject The first and / or second anti-HER2 constructs also bind to TfR.

[0101] In some embodiments of this aspect, the first anti-HER2 construct binds to TfR, but not the second anti-HER2 construct. The first anti-HER2 construct can specifically bind to TfR and HER2 on the same cell.

[0102] In some embodiments, the second anti-HER2 construct binds to TfR, but not the first anti-HER2 construct. The second anti-HER2 construct can specifically bind to TfR and HER2 on the same cell.

[0103] In some embodiments of this aspect, the first and / or second anti-HER2 construct comprises an modified Fc polypeptide dimer comprising an antibody variable region that binds to subdomain I, II, or IV of human HER2 and a first Fc polypeptide comprising a modification that creates a TfR-binding site. In certain embodiments, the first anti-HER2 construct is an Fc polypeptide dimer-antibody variable region fusion protein. In certain embodiments, the second anti-HER2 construct is an Fc polypeptide dimer-antibody variable region fusion protein. In certain embodiments, the first and second anti-HER2 constructs are Fc polypeptide dimer-antibody variable region fusion proteins.

[0104] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two heavy chain variable regions and two light chain variable regions that bind to subdomain II of HER2, wherein each of the two heavy chain variable regions comprises a heavy chain CDR1 (CDR-H1), CDR H2, and CDR H3, and each of the two light chain variable regions comprises a light chain CDR1 (CDR-L1), CDR L2, and CDR L3; (1) CDR-H1 comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 75 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 75; (2) CDR-H2 comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 76 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 76; (3) CDR-H3 comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 77 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 77; (4) CDR-L1 comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 78 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 78; (5) CDR-L2 comprises a sequence having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 79; (6) CDR-L3 comprises a sequence having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 80.

[0105] In some embodiments of an Fc polypeptide dimer-antibody variable region fusion protein that binds to subdomain II of HER2, each of the two heavy chain variable regions comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 61, and each of the two light chain variable regions comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 62.

[0106] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two heavy chain variable regions and two light chain variable regions that bind to subdomain I of HER2, wherein each of the two heavy chain variable regions comprises a heavy chain CDR1 (CDR-H1), CDR H2, and CDR H3, and each of the two light chain variable regions comprises a light chain CDR1 (CDR-L1), CDR L2, and CDR L3; (1) CDR-H1 comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 250 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 250; (2) CDR-H2 comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 251 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 251; (3) CDR-H3 comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 252 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 252; (4) CDR-L1 comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 253 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 253; (5) CDR-L2 comprises a sequence having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 254; (6) CDR-L3 comprises a sequence having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 255.

[0107] In some embodiments of an Fc polypeptide dimer-antibody variable region fusion protein that binds to subdomain I of HER2, each of the two heavy chain variable regions comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:256, and each of the two light chain variable regions comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:257.

[0108] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two heavy chain variable regions and two light chain variable regions that bind to subdomain IV of HER2; each of the two heavy chain variable regions comprises a heavy chain CDR1 (CDR-H1), CDR H2, and CDR H3; and each of the two light chain variable regions comprises a light chain CDR1 (CDR-L1), CDR L2, and CDR L3; (1) CDR-H1 comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 69 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 69; (2) CDR-H2 comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 70 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 70; (3) CDR-H3 comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 71 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 71; (4) CDR-L1 comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 72 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 72; (5) CDR-L2 comprises a sequence having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 73; (6) CDR-L3 comprises a sequence having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 74.

[0109] In some embodiments of an Fc polypeptide dimer-antibody variable region fusion protein that binds to subdomain IV of HER2, each of the two heavy chain variable regions comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:59, and each of the two light chain variable regions comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:60.

[0110] In some embodiments of this aspect, the TfR-binding site in the Fc polypeptide of the Fc polypeptide dimer-antibody variable region fusion protein comprises a modified CH3 domain. The modified CH3 domain can be derived from a human IgG1, IgG2, IgG3, or IgG4 CH3 domain. The modified CH3 domain can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions at the set of amino acid positions including 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 according to EU numbering.

[0111] In some embodiments, the modified CH3 domain contains, according to EU numbering, 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, Gly, Ser, Thr, or at position 390; Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr at position 391; an acidic amino acid, 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 an acidic amino acid at position 416; and / or an aromatic amino acid, His, or Lys at position 421.

[0112] In some embodiments of this aspect of the disclosure, the anti-HER2 construct binds to the apical domain of TfR. In some embodiments, the modified Fc polypeptide dimer comprises a first Fc polypeptide comprising an amino acid modification that reduces FcγR binding upon binding to TfR. In certain embodiments, the amino acid modification comprises Ala at positions 234 and 235, according to EU numbering. In some embodiments, one or both Fc polypeptides present in the Fc polypeptide dimer comprise an amino acid modification that increases serum half-life. In certain embodiments, the amino acid modification that increases serum half-life comprises (i) Leu at position 428 and Ser at position 434, or (ii) Ser or Ala at position 434, according to EU numbering.

[0113] In some embodiments of this aspect of the disclosure, the first Fc polypeptide further comprises knob mutation T366W, and the second Fc polypeptide in the Fc polypeptide dimer comprises hole mutations T366S, L368A, and Y407V, according to EU numbering. For example, in some embodiments, the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 67 and 68.

[0114] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein that binds to subdomain II of human HER2 comprises: (a) a first heavy chain having the sequence of SEQ ID NO: 38, a second heavy chain having the sequence of SEQ ID NO: 55; or (b) a first heavy chain having the sequence of SEQ ID NO: 46, a second heavy chain having the sequence of SEQ ID NO: 55; or (c) a first heavy chain having the sequence of SEQ ID NO: 30, a second heavy chain having the sequence of SEQ ID NO: 55 Includes.

[0115] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein that binds to subdomain I of human HER2 comprises: (a) a first heavy chain having the sequence of SEQ ID NO: 267, a second heavy chain having the sequence of SEQ ID NO: 290; or (b) a first heavy chain having the sequence of SEQ ID NO: 275, a second heavy chain having the sequence of SEQ ID NO: 290; or (c) a first heavy chain having the sequence of SEQ ID NO: 259, a second heavy chain having the sequence of SEQ ID NO: 290 Includes.

[0116] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein that binds to subdomain IV of human HER2 is (a) a first heavy chain having the sequence of SEQ ID NO: 10, a second heavy chain having the sequence of SEQ ID NO: 27; or (b) a first heavy chain having the sequence of SEQ ID NO: 18, a second heavy chain having the sequence of SEQ ID NO: 27; or (c) a first heavy chain having the sequence of SEQ ID NO: 2, a second heavy chain having the sequence of SEQ ID NO: 27 Includes.

[0117] In other embodiments of this aspect, the first and / or second anti-HER2 constructs comprise an antibody variable region that binds to TfR. For example, the first anti-HER2 construct can be a bispecific construct comprising an antibody variable region that binds to human HER2 and an antibody variable region that binds to TfR. The second anti-HER2 construct can be a bispecific construct comprising an antibody variable region that binds to human HER2 and an antibody variable region that binds to TfR. Both the first and second anti-HER2 constructs can be bispecific constructs comprising an antibody variable region that binds to human HER2 and an antibody variable region that binds to TfR.

[0118] In some embodiments of this aspect of the disclosure, the cancer is HER2-positive breast cancer. The HER2-positive cancer may be HER2-positive gastric adenocarcinoma and / or HER2-positive gastroesophageal junction adenocarcinoma.

[0119] In another aspect, the disclosure features a method of reducing transferrin receptor (TfR) expression levels on the surface of a cell by contacting the cell with an anti-HER2 construct that binds to (a) subdomain I, II, or IV of human HER2 and (b) TfR, wherein the anti-HER2 construct alone is effective to reduce TfR expression levels on the cell surface of the cell, and the anti-HER2 construct binds to both TfR and HER2 on the same cell. In this aspect, the anti-HER2 construct can be any of the constructs described herein, such as an Fc polypeptide dimer-antibody variable region fusion protein (comprising a human HER2 and an antibody variable region capable of binding to the modified Fc polypeptide dimer, where the first Fc polypeptide comprises a modification that creates a TfR-binding site) or an anti-HER2 bispecific construct (comprising an antibody variable region capable of binding to human HER2 and an antibody variable region that binds to TfR).

[0120] In yet another aspect, the disclosure features a method of binding a construct to transferrin receptor (TfR) and human HER2 expressed on a cell (e.g., TfR and human HER2 expressed on the same cell), comprising contacting the cell with an anti-HER2 construct that binds (a) subdomain I, II, or IV of human HER2 and (b) TfR, wherein the anti-HER2 construct reduces the level of TfR expression on the cell surface of the cell when the cell is contacted with the anti-HER2 construct. In this aspect, the construct that binds to TfR and human HER2 can be any of the constructs described herein, such as an Fc polypeptide dimer-antibody variable region fusion protein (comprising an Fc polypeptide comprising a first Fc polypeptide that includes a modification that creates a TfR-binding site and an antibody variable region capable of binding to the modified Fc polypeptide dimer) or an anti-HER2 bispecific construct (comprising an antibody variable region capable of binding human HER2 and an antibody variable region that binds to TfR).

[0121] In some embodiments, the anti-HER2 construct reduces the level of TfR expression on the cell surface of a cell by at least 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) when the cell is contacted with the fusion protein.

[0122] In some embodiments, the anti-HER2 construct comprises an antibody variable region that binds to subdomain I, II, or IV of human HER2. In certain embodiments, the anti-HER2 construct comprises an antibody variable region that binds to subdomain I, II, or IV of human HER2 and a modified Fc polypeptide dimer comprising a first Fc polypeptide that includes a modification that creates a TfR-binding site. For example, the anti-HER2 construct can be an Fc polypeptide dimer-antibody variable region fusion protein. In yet other embodiments, the anti-HER2 construct can be a bispecific construct comprising an antibody variable region that binds to human HER2 and an antibody variable region that binds to TfR.

[0123] In some embodiments, in any of the aspects described herein, the cell is a cancer cell (e.g., a metastatic cancer cell). In certain embodiments, the cancer cell expresses both HER2 and TfR. In some embodiments, the cell may be a breast cancer cell (e.g., a HER2-positive cancer cell). In some embodiments of any of the aspects described herein, the cell is a mammalian cell, such as a human cell (e.g., a human cancer cell).

[0124] In some embodiments, in any of the aspects described herein, the cell is in a mammal, such as a human. In some embodiments, the human has HER2-positive cancer or has been diagnosed with HER2-positive cancer. In certain embodiments, the HER2-positive cancer is metastatic cancer. In certain embodiments, the HER2-positive metastatic cancer has metastasized to the brain. [The present invention 1001] (a) an antibody variable region, or an antigen-binding fragment thereof, capable of binding to human epidermal growth factor receptor 2 (HER2); (b) an altered Fc polypeptide dimer comprising a first Fc polypeptide comprising an alteration that creates a TfR binding site; and An Fc polypeptide dimer-antibody variable region fusion protein comprising: [The present invention 1002] 1001. The Fc polypeptide dimer-antibody variable region fusion protein of the present invention, wherein said antibody variable region forms part of a Fab domain. [The present invention 1003] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1001 or 1002, wherein the antibody variable region binds to subdomain IV, II, or I of human HER2. [The present invention 1004] 1003. An Fc polypeptide dimer-antibody variable region fusion protein of the present invention, wherein the antibody variable region binds to subdomain IV of human HER2. [The present invention 1005] the antibody variable region (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 69 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 70 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 70; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 71 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 71; (d) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 72 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 72; (e) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 73; and (f) a light chain CDR3 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 74; 1004. An Fc polypeptide dimer-antibody variable region fusion protein of the present invention, comprising one or more complementarity determining regions (CDRs) selected from the group consisting of: [The present invention 1006] the antibody variable region (a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 69; (b) a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 70; (c) a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 71; (d) a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 72; (e) a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 73, and (f) a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 74 1005. An Fc polypeptide dimer-antibody variable region fusion protein of the present invention, comprising one or more CDRs selected from the group consisting of: [The present invention 1007] An Fc polypeptide dimer-antibody variable region fusion protein of any of claims 1004 to 1006, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 59 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 60. [The present invention 1008] 1003. An Fc polypeptide dimer-antibody variable region fusion protein of the present invention, wherein the antibody variable region binds to subdomain II of human HER2. [The present invention 1009] the antibody variable region (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 75 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 76 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 76; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 77 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 77; (d) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 78 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 78; (e) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 79; and (f) a light chain CDR3 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 80; 1008. An Fc polypeptide dimer-antibody variable region fusion protein of the present invention, comprising one or more CDRs selected from the group consisting of: [The present invention 1010] the antibody variable region (a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 75; (b) a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 76; (c) a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77; (d) a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78; (e) a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79; and (f) a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80 1009. An Fc polypeptide dimer-antibody variable region fusion protein of the present invention, comprising one or more CDRs selected from the group consisting of: [The present invention 1011] An Fc polypeptide dimer-antibody variable region fusion protein of any of claims 1008 to 1010 of the present invention, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 61 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 62. [The present invention 1012] 1003. An Fc polypeptide dimer-antibody variable region fusion protein of the present invention, wherein the antibody variable region binds to subdomain I of human HER2. [The present invention 1013] the antibody variable region (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 250 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 251 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 251; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 252 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 252; (d) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 253 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 253; (e) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 254; and (f) a light chain CDR3 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 255; 1012. An Fc polypeptide dimer-antibody variable region fusion protein of the present invention, comprising one or more CDRs selected from the group consisting of: [The present invention 1014] the antibody variable region (a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 250; (b) a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 251; (c) a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 252; (d) a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 253; (e) a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 254, and (f) a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 255 1013. An Fc polypeptide dimer-antibody variable region fusion protein of the present invention, comprising one or more CDRs selected from the group consisting of: [The present invention 1015] An Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 1012 to 1014, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 256 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 257. [The present invention 1016] The Fc polypeptide dimer-antibody variable region fusion protein of any of claims 1001 to 1015, wherein the TfR binding site is within the CH3 domain. [The present invention 1017] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention, wherein the modified CH3 domain is derived from a human IgG1, IgG2, IgG3, or IgG4 CH3 domain. [The present invention 1018] 1016 or 1017. An Fc polypeptide dimer-antibody variable region fusion protein of the invention, wherein the modified CH3 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions at a set of amino acid positions including 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 according to EU numbering. [The present invention 1019] The modified CH3 domain may contain, according to EU numbering, 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; an aliphatic amino acid, Gly, Ser, Thr, or Asn at position 389; Gly, His, Gln, Leu, or An Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 1016 to 1018, comprising Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr, an acidic amino acid, 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 an acidic amino acid at position 416, and / or an aromatic amino acid, His, or Lys at position 421. [The present invention 1020] The Fc polypeptide dimer-antibody variable region fusion protein of any of claims 1001 to 1019, wherein the Fc polypeptide dimer-antibody variable region fusion protein binds to the apical domain of TfR. [The present invention 1021] The Fc polypeptide dimer-antibody variable region fusion protein of any of claims 1001 to 1020, wherein the first Fc polypeptide comprises an amino acid modification that reduces FcγR binding when bound to TfR. [The present invention 1022] 1021. The Fc polypeptide dimer-antibody variable region fusion protein of the present invention, wherein the amino acid modifications include Ala at position 234 and at position 235 according to EU numbering. [The present invention 1023] An Fc polypeptide dimer-antibody variable region fusion protein of any of claims 1001 to 1022, wherein the first Fc polypeptide and / or the second Fc polypeptide present in the Fc polypeptide dimer contains an amino acid modification that increases serum half-life. [The present invention 1024] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1023, wherein the amino acid modification that increases serum half-life includes, according to EU numbering, (i) Leu at position 428 and Ser at position 434, or (ii) Ser or Ala at position 434. [The present invention 1025] An Fc polypeptide dimer-antibody variable region fusion protein of any of claims 1001 to 1024, wherein the first Fc polypeptide further comprises a knob mutation T366W, and the second Fc polypeptide present in the Fc polypeptide dimer comprises hole mutations T366S, L368A, and Y407V, according to EU numbering. [The present invention 1026] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1025, wherein the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 63. [The present invention 1027] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1025 or 1026, wherein the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 67 and 68. [The present invention 1028] (a) an antibody variable region, or an antigen-binding fragment thereof, capable of binding to human HER2; (b) a first Fc polypeptide comprising a modification that creates a TfR binding site and the knob mutation T366W, according to EU numbering; (c) a second Fc polypeptide comprising the hole mutations T366S, L368A, and Y407V according to EU numbering and not comprising a TfR binding site; An Fc polypeptide dimer-antibody variable region fusion protein comprising: [The present invention 1029] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1028, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 59 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 60. [The present invention 1030] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1028, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 61 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 62. [The present invention 1031] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1028, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 256 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 257. [The present invention 1032] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1028 or 1029, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, and 81. [The present invention 1033] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1028 or 1029, wherein said first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering. [The present invention 1034] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1033, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising any one of the amino acid sequences of SEQ ID NOs: 2, 10, 18, and 82. [This invention 1035] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1028, 1029, or 1032 to 1034 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 27. [The present invention 1036] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1028, 1029, or 1032 to 1035 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57. [This invention 1037] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1028 or 1030, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 29, 37, 45, and 89. [The present invention 1038] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1028 or 1030, wherein said first Fc polypeptide further comprises amino acid modifications L234A and L235A. [This invention 1039] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1038, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 30, 38, 46, and 90. [The present invention 1040] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1028, 1030, or 1037 to 1039, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 55. [The present invention 1041] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1028, 1030, or 1037 to 1040 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58. [The present invention 1042] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1028 or 1031, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 258, 266, 274, and 282. [This invention 1043] 1028 or 1031, wherein the first Fc polypeptide further comprises amino acid modifications L234A and L235A. [This invention 1044] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1043, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 259, 267, 275, and 283. [This invention 1045] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1028, 1031, and 1042 to 1044 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 291. [The present invention 1046] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1028, 1031, and 1042 to 1045 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293. [This invention 1047] (a) an antibody variable region, or an antigen-binding fragment thereof, capable of binding to human HER2; (b) a first Fc polypeptide comprising the amino acid modification N434S with or without the knob mutation T366W and M428L, modifications that create a TfR binding site, according to EU numbering; (c) a second Fc polypeptide comprising the hole mutations T366S, L368A, and Y407V according to EU numbering and not comprising a TfR binding site; An Fc polypeptide dimer-antibody variable region fusion protein comprising: [This invention 1048] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1047, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 59 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 60. [This invention 1049] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1047, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 61 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 62. [The present invention 1050] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1047, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 256 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 257. [This invention 1051] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1047 or 1048, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 3, 11, 19, and 83. [This invention 1052] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1047 or 1048, wherein the first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering. [This invention 1053] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1052, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 4, 12, 20, and 84. [This invention 1054] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1047, 1048, and 1051 to 1053 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 27. [This invention 1055] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1047, 1048, and 1051 to 1054 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57. [The present invention 1056] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1047 or 1049, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 31, 39, 47, and 91. [This invention 1057] The Fc polypeptide dimer-antibody variable region fusion protein of the invention 1047 or 1049, wherein said first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering. [This invention 1058] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1057, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 32, 40, 48, and 92. [This invention 1059] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1047, 1049, or 1056 to 1058, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 55. [The present invention 1060] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1047, 1049, or 1056 to 1059, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58. [This invention 1061] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1047 or 1050, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 260, 268, 276, and 284. [This invention 1062] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1047 or 1050, wherein said first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering. [This invention 1063] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1062, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 261, 269, 277, and 285. [This invention 1064] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1047, 1050, or 1061 to 1063, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 290. [This invention 1065] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1047, 1050, or 1061 to 1064, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293. [The present invention 1066] (a) an antibody variable region, or an antigen-binding fragment thereof, capable of binding to human HER2; (b) a first Fc polypeptide comprising a modification that creates a TfR binding site and the knob mutation T366W, according to EU numbering; (c) a second Fc polypeptide comprising the hole mutations T366S, L368A, and Y407V, and the amino acid modification N434S with or without M428L, according to EU numbering, and which does not comprise a TfR binding site; An Fc polypeptide dimer-antibody variable region fusion protein comprising: [This invention 1067] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1066, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 59 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 60. [The present invention 1068] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1066, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 61 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 62. [The present invention 1069] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1066, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 256 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 257. [The present invention 1070] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1066 or 1067, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, and 81. [This invention 1071] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1066 or 1067, wherein the first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering. [This invention 1072] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1071, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 2, 10, 18, and 82. [This invention 1073] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1066, 1067, or 1070 to 1072 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 28. [This invention 1074] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1066, 1067, or 1070 to 1073 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57. [This invention 1075] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1066 or 1068, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 29, 37, 45, and 89. [This invention 1076] The Fc polypeptide dimer-antibody variable region fusion protein of the invention 1066 or 1068, wherein said first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering. [This invention 1077] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1076, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 30, 38, 46, and 90. [This invention 1078] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1066, 1068, or 1075 to 1077 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 56. [This invention 1079] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1066, 1068, or 1075 to 1078 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58. [The present invention 1080] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1066 or 1069, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 258, 266, 274, and 282. [This invention 1081] The Fc polypeptide dimer-antibody variable region fusion protein of the invention 1066 or 1069, wherein said first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering. [This invention 1082] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1080, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 259, 267, 275, and 283. [This invention 1083] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1066, 1069, or 1080 to 1082 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 291. [This invention 1084] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1066, 1069, or 1080 to 1083 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293. [This invention 1085] (a) an antibody variable region, or an antigen-binding fragment thereof, capable of binding to human HER2; (b) a first Fc polypeptide comprising the amino acid modification N434S with or without the knob mutation T366W and M428L, modifications that create a TfR binding site, according to EU numbering; (c) a second Fc polypeptide comprising the hole mutations T366S, L368A, and Y407V, and the amino acid modification N434S with or without M428L, according to EU numbering, and which does not comprise a TfR binding site; An Fc polypeptide dimer-antibody variable region fusion protein comprising: [The present invention 1086] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1085, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 59 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 60. [This invention 1087] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1085, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 61 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 62. [This invention 1088] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1085, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 256 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 257. [This invention 1089] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1085 or 1086, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 3, 11, 19, and 83. [The present invention 1090] The Fc polypeptide dimer-antibody variable region fusion protein of the invention 1085 or 1086, wherein said first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering. [This invention 1091] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1090, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 4, 12, 20, and 84. [This invention 1092] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1085, 1086, or 1089 to 1091 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 28. [This invention 1093] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1085, 1086, or 1089 to 1092 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57. [This invention 1094] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1085 or 1087, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 31, 39, 47, and 91. [This invention 1095] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1085 or 1087, wherein the first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering. [This invention 1096] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1095, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising any one of the amino acid sequences of SEQ ID NOs: 32, 40, 48, and 92. [This invention 1097] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1085, 1087, or 1094 to 1096 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 56. [This invention 1098] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1085, 1087, or 1094 to 1097 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58. [This invention 1099] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1085 or 1088, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 260, 268, 276, and 284. [The present invention 1100] The Fc polypeptide dimer-antibody variable region fusion protein of the invention 1085 or 1088, wherein said first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering. [The present invention 1101] An Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1100, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 261, 269, 277, and 285. [The present invention 1102] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1085, 1088, and 1099 to 1101 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 291. [The present invention 1103] An Fc polypeptide dimer-antibody variable region fusion protein of any of 1085, 1088, and 1099 to 1101 of the present invention, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293. [The present invention 1104] The Fc polypeptide dimer-antibody variable region fusion protein of any of claims 1001 to 1103, wherein the modified Fc polypeptide dimer does not substantially reduce reticulocytes. [This invention 1105] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1104, wherein the amount of reticulocytes reduced after administration of the Fc polypeptide dimer-antibody variable region fusion protein is less than the amount of reticulocytes reduced after administration of a control. [The present invention 1106] The Fc polypeptide dimer-antibody variable region fusion protein of the present invention 1105, wherein the control is a corresponding TfR-binding polypeptide dimer-antibody variable region fusion protein that has full effector function and / or does not contain a mutation that reduces FcγR binding. [This invention 1107] (a) an anti-human HER2 antibody heavy chain variable region, or a fragment thereof; (b) a modified Fc polypeptide comprising a modification that creates a TfR binding site; and an antibody heavy chain comprising: [This invention 1108] The antibody heavy chain of the invention 1107, wherein said modified Fc polypeptide comprises one or more amino acid modifications that reduce FcγR binding when bound to TfR. [This invention 1109] the antibody heavy chain variable region (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 69 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 70 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 70; and (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 71 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 71 The antibody heavy chain of the present invention 1107 or 1108, comprising one or more CDRs selected from the group consisting of: [The present invention 1110] the antibody heavy chain variable region (a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 69; (b) a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 70; and (c) a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 71 The antibody heavy chain of the present invention 1107 or 1108, comprising one or more CDRs selected from the group consisting of: [The present invention 1111] The antibody heavy chain of any of claims 1107 to 1110, wherein the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO:59. [The present invention 1112] the antibody variable region (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 75 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 76 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 76; and (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 77 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 77 The antibody heavy chain of the present invention 1107 or 1108, comprising one or more CDRs selected from the group consisting of: [The present invention 1113] the antibody variable region (a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 75; (b) a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 76; and (c) a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77 The antibody heavy chain of the present invention 1107 or 1108, comprising one or more CDRs selected from the group consisting of: [This invention 1114] The antibody heavy chain of any one of claims 1107, 1108, 1112, or 1113, wherein the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO:61. [This invention 1115] the antibody variable region (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 250 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 251 or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 251; and (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 252 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 252. The antibody heavy chain of the present invention 1107 or 1108, comprising one or more CDRs selected from the group consisting of: [The present invention 1116] the antibody variable region (a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 250; (b) a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 251; and (c) a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 252 The antibody heavy chain of the present invention 1107 or 1108, comprising one or more CDRs selected from the group consisting of: [This invention 1117] The antibody heavy chain of any one of claims 1107, 1108, 1115, or 1116, wherein the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 256. [This invention 1118] The antibody heavy chain of any of claims 1107 to 1117, wherein the TfR-binding site comprises a modified CH3 domain. [This invention 1119] The antibody heavy chain of the invention 1118, wherein said modified CH3 domain is derived from a human IgG1, IgG2, IgG3, or IgG4 CH3 domain. [The present invention 1120] 1118 or 1119. The antibody heavy chain of the invention, wherein said modified CH3 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions at a set of amino acid positions including 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 according to EU numbering. [This invention 1121] The modified CH3 domain may contain, according to EU numbering, 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; an aliphatic amino acid, Gly, Ser, Thr, or Asn at position 389; and Gly, Hi at position 390. 413 an acidic amino acid, Ala, Ser, Leu, Thr, Pro, Ile, or His, at position 414; Glu, Ser, Asp, Gly, Thr, Pro, Gln, or Arg at position 415; Thr, Arg, Asn, or an acidic amino acid at position 416; and / or an aromatic amino acid, His, or Lys at position 421. [This invention 1122] The antibody heavy chain of any of claims 1108 to 1121, wherein the amino acid modifications that reduce FcγR binding upon binding to TfR include Ala at positions 234 and 235 according to EU numbering. [This invention 1123] The antibody heavy chain of any of claims 1107 to 1122, wherein the modified Fc polypeptide further comprises an amino acid modification that increases serum half-life. [This invention 1124] The antibody heavy chain of the present invention 1123, 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 EU numbering. [Invention 1125] The antibody heavy chain of any of claims 1107 to 1124, wherein the modified Fc polypeptide further comprises the knob mutation T366W according to EU numbering. [The present invention 1126] The antibody heavy chain of the present invention 1125, wherein said modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 63. [This invention 1127] (a) an anti-human HER2 antibody heavy chain variable region, or a fragment thereof; (b) a modified Fc polypeptide comprising modifications that create a TfR binding site and the knob mutation T366W, according to EU numbering; an antibody heavy chain comprising: [This invention 1128] The antibody heavy chain of the present invention 1127, wherein the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59. [This invention 1129] The antibody heavy chain of the present invention 1127, wherein the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61. [The present invention 1130] The antibody heavy chain of the present invention 1127, wherein the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 256. [This invention 1131] The antibody heavy chain of the present invention 1128, wherein the antibody heavy chain comprises any one of the amino acid sequences of SEQ ID NOs: 1, 9, 17, and 81. [This invention 1132] The antibody heavy chain of the present invention 1129, wherein the antibody heavy chain comprises any one of the amino acid sequences of SEQ ID NOs: 29, 37, 45, and 89. [This invention 1133] The antibody heavy chain of the present invention 1130, wherein the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 258, 266, 274, and 282. [This invention 1134] The antibody heavy chain of any of claims 1127 to 1133, wherein said modified Fc polypeptide further comprises the amino acid modifications L234A and L235A according to EU numbering. [This invention 1135] The antibody heavy chain of the present invention 1134, wherein the antibody heavy chain comprises any one of the amino acid sequences of SEQ ID NOs: 2, 10, 18, and 82. [This invention 1136] The antibody heavy chain of the present invention 1134, wherein the antibody heavy chain comprises any one of the amino acid sequences of SEQ ID NOs: 30, 38, 46, and 90. [This invention 1137] The antibody heavy chain of the present invention 1134, wherein the antibody heavy chain comprises any one of the amino acid sequences of SEQ ID NOs: 259, 267, 275, and 283. [This invention 1138] (a) an anti-human HER2 antibody heavy chain variable region, or a fragment thereof; (b) a modified Fc polypeptide comprising the modifications that create a TfR binding site, the knob mutation T366W, and the amino acid modification N434S with or without M428L, according to EU numbering; an antibody heavy chain comprising: [This invention 1139] The antibody heavy chain of the present invention 1138, wherein the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59. [The present invention 1140] The antibody heavy chain of the present invention 1138, wherein the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61. [This invention 1141] The antibody heavy chain of the present invention 1138, wherein the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 256. [This invention 1142] The antibody heavy chain of the present invention 1139, wherein the antibody heavy chain comprises any one of the amino acid sequences of SEQ ID NOs: 3, 11, 19, and 83. [This invention 1143] The antibody heavy chain of the present invention 1140, wherein the antibody heavy chain comprises any one of the amino acid sequences of SEQ ID NOs: 31, 39, 47, and 91. [This invention 1144] The antibody heavy chain of the present invention 1140, wherein the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 260, 268, 276, and 284. [Invention 1145] The antibody heavy chain of any of claims 1138 to 1144, wherein said modified Fc polypeptide further comprises the amino acid modifications L234A and L235A according to EU numbering. [Invention 1146] The antibody heavy chain of the present invention 1145, wherein the antibody heavy chain comprises any one of the amino acid sequences of SEQ ID NOs: 4, 12, 20, and 84. [This invention 1147] The antibody heavy chain of the present invention 1145, wherein the antibody heavy chain comprises any one of the amino acid sequences of SEQ ID NOs: 32, 40, 48, and 92. [This invention 1148] The antibody heavy chain of the present invention 1145, wherein the antibody heavy chain comprises any one of the amino acid sequences of SEQ ID NOs: 261, 269, 277, and 285. [This invention 1149] A pharmaceutical composition comprising any one of the Fc polypeptide dimer-antibody variable region fusion proteins of the present inventions 1001 to 1106 and a pharmaceutically acceptable carrier. [This invention 1150] A method for transcytosis across endothelium of an antibody variable region, or an antigen-binding fragment thereof, capable of binding to human HER2, comprising contacting the endothelium with a composition comprising an Fc polypeptide dimer-antibody variable region fusion protein of any one of claims 1001 to 1106 of the present invention. [This invention 1151] 1150. The method of claim 1150, wherein said endothelium is the BBB. [This invention 1152] A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising any one of the Fc polypeptide dimer-antibody variable region fusion proteins of the present inventions 1001 to 1106. [This invention 1153] The method of claim 1152, wherein said cancer is a HER2-positive cancer. [This invention 1154] The method of claim 1153, wherein said HER2-positive cancer is HER2-positive breast cancer. [This invention 1155] The method of claim 1153, wherein said HER2-positive cancer is HER2-positive gastric adenocarcinoma and / or HER2-positive gastroesophageal junction adenocarcinoma. [Invention 1156] The method of any one of claims 1153 to 1155, wherein the HER2-positive cancer is a metastatic cancer. [This invention 1157] A method for treating brain metastasis of HER2-positive cancer in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising any one of the Fc polypeptide dimer-antibody variable region fusion proteins of the present inventions 1001 to 1106. [This invention 1158] 1157. The method of claim 1157, wherein said HER2-positive cancer is HER2-positive breast cancer. [This invention 1159] 1157. The method of claim 1157, wherein said HER2-positive cancer is HER2-positive gastric adenocarcinoma and / or HER2-positive gastroesophageal junction adenocarcinoma. [The present invention 1160] The method of any of claims 1152 to 1159, wherein the composition comprising the Fc polypeptide dimer-antibody variable region fusion protein antagonizes HER2 activity. [This invention 1161] a first Fc polypeptide dimer-antibody variable region fusion protein and a second Fc polypeptide dimer-antibody variable region fusion protein are administered to the subject; the antibody variable region of the first Fc polypeptide dimer-antibody variable region fusion protein comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 59 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 60; the antibody variable region of the second Fc polypeptide dimer-antibody variable region fusion protein comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 61 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 62; Any of the methods of the present invention 1152 to 1160. [This invention 1162] a first Fc polypeptide dimer-antibody variable region fusion protein and a second Fc polypeptide dimer-antibody variable region fusion protein are administered to the subject; the antibody variable region of the first Fc polypeptide dimer-antibody variable region fusion protein comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 59 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 60; the antibody variable region of the second Fc polypeptide dimer-antibody variable region fusion protein comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 256 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 257; Any of the methods of the present invention 1152 to 1160. [This invention 1163] a first Fc polypeptide dimer-antibody variable region fusion protein and a second Fc polypeptide dimer-antibody variable region fusion protein are administered to the subject; the antibody variable region of the first Fc polypeptide dimer-antibody variable region fusion protein comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 61 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 62; the antibody variable region of the second Fc polypeptide dimer-antibody variable region fusion protein comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 256 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO: 257; Any of the methods of the present invention 1152 to 1160. [Invention 1164] The method of any of claims 1152 to 1163, wherein said subject has not been previously treated with anti-HER2 therapy and / or chemotherapy for metastatic disease. [Brief explanation of the drawings]

[0125] [Figure 1A]

[0023] Figure 1 shows Biacore™ data showing binding affinity to the HER2 extracellular domain using anti-HER2_DIV, HER2_DIV-35.23.1.1cisLALA, anti-HER2_DII, and HER2_DII-35.23.1.1cisLALA. [Figure 1B]

[0023] Figure 1 shows Biacore™ data showing binding affinity to human apical hTfR using HER2_DIV-35.23.1.1cisLALA and HER2_DII-35.23.1.1cisLALA. [Figure 2] 1 shows a growth inhibition assay of BT474 cells using WST1 reagent showing inhibition of cancer cell proliferation at day 6 by anti-HER2_DIV, HER2_DIV-35.23.1.1cisLALA, a combination of anti-HER2_DIV and anti-HER2_DII, and a combination of HER2_DIV-35.23.1.1cisLALA and HER2_DII-35.23.1.1cisLALA. [Figure 3] 1 shows Western blot data demonstrating a decrease in phosphorylated AKT (p-AKT) in BT474 cells treated with anti-HER2_DIV and HER2_DIV-35.23.1.1cisLALA. [Figure 4] 1 shows a growth inhibition assay of BT474 cells using WST1 reagent showing the response of cancer cell proliferation at day 6 to anti-HER2_DIV, HER2_DIV-35.23.1.1cisLALA, a combination of anti-HER2_DIV and anti-HER2_DII, and a combination of HER2_DIV-35.23.1.1cisLALA and HER2_DII-35.23.1.1cisLALA in the presence of neuregulin-1. [Figure 5]1 shows Western blot data of phosphorylated AKT (p-AKT) protein levels in BT474 cells treated with anti-HER2_DIV and HER2_DIV-35.23.1.1cisLALA, a combination of anti-HER2_DIV and anti-HER2_DII, and a combination of HER2_DIV-35.23.1.1cisLALA and HER2_DII-35.23.1.1cisLALA in the presence of neuregulin-1. [Figure 6] 1 shows ADCC activity in SK-BR-3 cells using HER2_DIV-35.23.1.1cisLALA. [Figure 7A] Figure 1 shows the in vivo antitumor efficacy of ATV:HER2-DIV and ATV:HER2-DII in a BT474 xenograft tumor model in SCID mice, demonstrating significantly greater tumor growth inhibition in animals treated with ATV:HER2-DIV + ATV:HER2-DII compared to anti-HER2-DIV + anti-HER2-DII. [Figure 7B] Figure 1 shows the in vivo antitumor efficacy of ATV:HER2-DIV and ATV:HER2-DII in a BT474 xenograft tumor model in SCID mice. The dose-response relationship using doses of 3, 10, and 20 mg / kg of each test article is shown, demonstrating that ATV:HER2-DIV + ATV:HER2-DII is more potent than anti-HER2-DIV + anti-HER2-DII. [Figure 7C] This figure shows the in vivo antitumor efficacy of ATV:HER2-DIV and ATV:HER2-DII in a BT474 xenograft tumor model in SCID mice. Both anti-HER2-DIV + anti-HER2-DII and ATV:HER2-DIV + ATV:HER2-DII treatments significantly reduced pAKT levels, consistent with a mechanism by which targeting HER2 may suppress the PI3K / Akt signaling pathway activated in HER2+ tumors. "ATV" refers to the TfR-binding Fc polypeptide. [Figure 8A] Plasma exposure, brain uptake, and brain-to-plasma ratios of TfRmu / hu KI mice treated with ATV:HER2-DIV and ATV:HER2-DII are shown. [Figure 8B] Plasma exposure, brain uptake, and brain-to-plasma ratios of TfRmu / hu KI mice treated with ATV:HER2-DIV and ATV:HER2-DII are shown. [Figure 8C] Plasma exposure, brain uptake, and brain-to-plasma ratios of TfRmu / hu KI mice treated with ATV:HER2-DIV and ATV:HER2-DII are shown. [Figure 9A] 1 shows a growth inhibition assay of BT474 and OE19 cells using ATV:HER2-DIV, demonstrating that ATV:HER2-DIV has an increased anti-proliferative effect compared to anti-HER2_DIV in anti-HER2_DIV-resistant HER2+ cancer cell lines. [Figure 9B] 1 shows a growth inhibition assay of BT474 and OE19 cells using ATV:HER2-DIV, demonstrating that ATV:HER2-DIV has an increased anti-proliferative effect compared to anti-HER2_DIV in anti-HER2_DIV-resistant HER2+ cancer cell lines. [Figure 10A] Figure 1 shows growth inhibition assays of BT474 and OE19 cells using ATV:HER2-DII, showing that ATV:HER2-DII has superior growth inhibition to anti-HER2-DII, while ATV:ctrl and anti-TfR with or without anti-HER2-DII have minimal effect in HER2+ cancer cell lines. [Figure 10B] Figure 1 shows growth inhibition assays of BT474 and OE19 cells using ATV:HER2-DII, showing that ATV:HER2-DII has superior growth inhibition to anti-HER2-DII, while ATV:ctrl and anti-TfR with or without anti-HER2-DII have minimal effect in HER2+ cancer cell lines. [Figure 11] 1 shows a growth inhibition assay of OE19 cells using ATV:HER2-DI, demonstrating that ATV:HER2-DI has superior growth inhibition to anti-HER2-DI in HER2+ cancer cell lines. [Figure 12]1 shows a growth inhibition assay of BT474 cells without NRG1 using ATV:HER2-DIV+ATV:HER2-DII, showing that the combination of ATV:HER2-DIV+ATV:HER2-DII is more potent in growth inhibition than the combination of anti-HER2-DIV+anti-HER2-DII with or without NRG1. [Figure 13A] Figure 1 shows cell surface TfR expression in BT474 cells treated with ATV:HER2-DIV, ATV:HER2-DII, and a combination of ATV:HER2-DIV and ATV:HER2-DII. Treatment with ATV:HER2-DIV and ATV:HER2-DII enhanced downregulation of cell surface TfR expression under internalization conditions (30 min at 37°C). [Figure 13B] Figure 1 shows cell surface TfR expression in BT474 cells treated with ATV:HER2-DIV, ATV:HER2-DII, and a combination of ATV:HER2-DIV and ATV:HER2-DII. Treatment with ATV:HER2-DIV and ATV:HER2-DII enhanced downregulation of cell surface TfR expression under internalization conditions (30 min at 37°C). [Figure 13C] Figure 1 shows cell surface TfR expression in BT474 cells treated with ATV:HER2-DIV, ATV:HER2-DII, and a combination of ATV:HER2-DIV and ATV:HER2-DII. Treatment with ATV:HER2-DIV and ATV:HER2-DII enhanced downregulation of cell surface TfR expression under internalization conditions (30 min at 37°C). DETAILED DESCRIPTION OF THE INVENTION

[0126] Detailed Description I. Introduction Anti-HER2 constructs capable of crossing the BBB have been developed. Generally, these anti-HER2 constructs comprise an antibody variable region capable of binding to human HER2 (e.g., subdomain I, II, or IV of human HER2). In some embodiments, the anti-HER2 construct comprises an antibody variable region capable of binding to HER2 fused to a modified Fc polypeptide that has been engineered to contain a non-native TfR binding site, also referred to herein as an "Fc polypeptide dimer-antibody variable region fusion protein." For example, the Fc polypeptide dimer-antibody variable region fusion protein can comprise an anti-HER2 Fab fused to a modified Fc polypeptide dimer containing a TfR binding site.

[0127] In other embodiments, the anti-HER2 construct comprises an antibody variable region capable of binding to HER2 (e.g., subdomain I, II, or IV of human HER2) and an antibody variable region capable of binding to TfR.

[0128] Thus, the present disclosure relates, in part, to Fc polypeptide dimer-antibody variable region fusion proteins, and fragments thereof, that bind to subdomain IV, subdomain II, or subdomain I of HER2, bind to TfR, and have reduced effector function (e.g., ADCC or CDC) upon binding to TfR, yet retain and exhibit a level of effector function (e.g., ADCC or CDC) upon binding to HER2. The present disclosure also relates, in part, to methods for delivering anti-HER2 therapeutic constructs across the BBB to treat HER2-positive cancers, as well as metastases of HER2-positive cancers. Surprisingly, as described herein, it has been found that using a combination of Fc polypeptide dimer-antibody variable region fusion proteins targeting HER2 subdomains IV and II is more effective in inhibiting breast cancer cell growth than using a combination of anti-HER2 subdomain IV and anti-HER2 subdomain II antibodies. Furthermore, in some embodiments, using a combination of Fc polypeptide dimer-antibody variable region fusion proteins targeting HER2 subdomains IV and I is more effective at inhibiting breast cancer cell growth than using a combination of anti-HER2 subdomain IV and anti-HER2 subdomain I antibodies. Furthermore, in some embodiments, using a combination of Fc polypeptide dimer-antibody variable region fusion proteins targeting HER2 subdomains II and I is more effective at inhibiting breast cancer cell growth than using a combination of anti-HER2 subdomain II and anti-HER2 subdomain I antibodies.

[0129] Furthermore, the use of Fc polypeptide dimer-antibody variable region fusion proteins targeting HER2 subdomain IV, subdomain II, or subdomain I alone is more effective at inhibiting breast cancer cell growth than the use of anti-HER2 subdomain IV, anti-HER2 subdomain II, or anti-HER2 subdomain I antibodies, respectively. Furthermore, the use of Fc polypeptide dimer-antibody variable region fusion proteins targeting HER2 subdomain IV, subdomain II, or subdomain I alone is more effective at inhibiting breast cancer cell growth than the use of a combination of an anti-HER2 subdomain IV antibody and an anti-TfR antibody, a combination of an anti-HER2 subdomain II antibody and an anti-TfR antibody, or a combination of an anti-HER2 subdomain I antibody and an anti-TfR antibody, respectively.

[0130] In other embodiments, the present disclosure also relates, in part, to using anti-HER2 constructs comprising an antibody variable region capable of binding to HER2 (e.g., subdomain I, II, or IV of human HER2) and an antibody variable region capable of binding to TfR to inhibit breast cancer cell growth. The use of such anti-HER2 constructs with dual specificity for HER2 (e.g., subdomain I, II, or IV of human HER2) and TfR is more effective in inhibiting breast cancer cell growth than using an anti-HER2 antibody (e.g., an anti-HER2 subdomain I antibody, an anti-HER2 subdomain II antibody, or an anti-HER2 subdomain IV antibody) alone.

[0131] 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" can include two or more such molecules, and the like.

[0132] As used herein, the terms "about" and "approximately," when used to modify a quantity specified in a numerical value or range, indicate a reasonable deviation from the numerical value, similar to values ​​known to one of ordinary skill in the art, e.g., ±20%, ±10%, or ±5%, within the intended meaning of the recited value.

[0133] "Human epidermal growth factor receptor 2," "HER2," "HER2 / neu," and "ERBB2" (also known as CD340, receptor tyrosine-protein kinase erbB-2, proto-oncogene, and Neu) refer to the tyrosine receptor kinase protein encoded by the ERBB2 gene in humans, a member of the human epidermal growth factor receptor (HER / EGFR / ERBB) family. Amplification or overexpression of HER2 plays a prominent role in the development and progression of certain aggressive types of cancer, including breast cancer. Non-limiting examples of human HER2 nucleotide sequences are set forth in GenBank Reference Nos. NP_001005862, NP_001289936, NP_001289937, NP_001289938, and NP_004448. Non-limiting examples of human HER2 peptide sequences are set forth in GenBank reference numbers NP_001005862, NP_001276865, NP_001276866, NP_001276867, and NP_004439.

[0134] The extracellular domain of HER2, which contains approximately 600 amino acids, contains four subdomains (subdomains I, II, III, and IV). Subdomains I and III form the ligand-binding site. Cysteine-rich subdomains II and IV are involved in receptor homodimerization and heterodimerization. Anti-HER2 therapeutic constructs can bind to specific subdomains (e.g., subdomain II or subdomain IV).

[0135] When HER2 is amplified or overexpressed in a cell, the cell is said to be "HER2 positive" or "HER2+." The level of HER2 amplification or overexpression in a HER2-positive cell is generally expressed as a score ranging from 0 to 3 (i.e., HER2 0, HER2 1+, HER2 2+, or HER2 3+), with higher scores corresponding to greater degrees of expression.

[0136] HER2 testing methods include immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), ELISA, and RNA quantification (e.g., of HER2 expression), such as RT-PCR and microarray analysis. HER2 testing can be performed on subjects (e.g., patients) being considered for anti-HER2 therapy.

[0137] As used herein, the term "anti-HER2 construct" refers to a molecular (e.g., protein) construct that binds to (a) subdomain I, II, or IV of human HER2 and (b) transferrin receptor (TfR). The anti-HER2 construct can comprise an antibody variable region capable of binding to human HER2. In some embodiments, the anti-HER2 construct is an Fc polypeptide dimer-antibody variable region fusion protein, comprising an antibody variable region capable of binding to human HER2 and a modified Fc polypeptide dimer comprising a first Fc polypeptide containing a modification that creates a TfR-binding site. In other embodiments, the anti-HER2 construct is a bispecific construct comprising an antibody variable region capable of binding to human HER2 and an antibody variable region that binds to TfR. The anti-HER2 constructs described herein can bind to subdomain I, II, or IV of human HER2.

[0138] As used herein, the terms "anti-HER2-DI," "anti-HER2-DII," or "anti-HER2-DIV" refer to antibodies that bind to subdomains I, II, or IV of human HER2, respectively.

[0139] 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 the hinge region. Generally, an Fc polypeptide does not comprise a variable region.

[0140] "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.

[0141] As used herein, the term "Fc polypeptide dimer" refers to a dimer of two Fc polypeptides. In some embodiments, an Fc polypeptide dimer can bind to an Fc receptor (e.g., FcγR). In an Fc polypeptide dimer, two Fc polypeptides dimerize through interaction between two CH3 antibody constant domains. In some embodiments, two Fc polypeptides may also dimerize through one or more disulfide bonds formed between the hinge domains of two dimerized Fc domain monomers. An Fc polypeptide dimer can 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. An Fc polypeptide dimer can be a heterodimer or a homodimer.

[0142] 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 can 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).

[0143] As used herein, "transferrin receptor" or "TfR" refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is set forth in SEQ ID NO: 102. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee, accession numbers XP_003310238.1; rhesus monkey, NP_001244232.1; dog, NP_001003111.1; cow, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP_990587.1). The term "transferrin receptor" also encompasses allelic variants of exemplary reference sequences, e.g., human sequences encoded by genes at the transferrin receptor protein 1 chromosomal locus. The full-length TfR protein includes a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain. The apical domain sequence of human transferrin receptor 1 is set forth in SEQ ID NO: 103.

[0144] As used herein, the term "Fcγ receptor" or "FcγR" refers to a type of Fc receptor, classified based on the type of antibody they recognize. FcγRs include several members, FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b), whose antibody affinities differ due to their different molecular structures. FcγRs bind to the Fc portion of IgG class antibodies and are important for inducing phagocytosis of opsonized microorganisms. FcγRs are found on the cell surface of cells of the immune system. FcγRs are involved in triggering immune system effector functions and are activated upon binding of the Fc portion of an antibody to the receptor. FcγRs mediate immune functions, for example, by binding to antibodies attached to infected cells or invading pathogens and stimulating phagocytic or cytotoxic cells to destroy microorganisms or infected cells by antibody-mediated phagocytosis or ADCC.

[0145] As used herein, the term "reduced FcγR binding" refers to an altered Fc polypeptide or altered Fc polypeptide dimer comprising a mutation in the CH3 domain of the altered Fc polypeptide, which mutation reduces the affinity of the altered 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 contain the FcγR binding-reducing mutation. FcγR binding can be measured, for example, using surface plasmon resonance (SPR) methods (e.g., a Biacore™ system). Alternatively, FcγR binding can be measured using a functional assay, e.g., an ADCC assay (e.g., an in vivo or in vitro cell killing assay) such as those described herein. Reduction in FcγR binding can be measured when the modified Fc polypeptide dimer binds to TfR. In some embodiments, the modified Fc polypeptide or Fc polypeptide dimer has reduced FcγR binding when bound to TfR, but may have limited reduction (e.g., less than a 25%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1% reduction) or no reduction when not bound to TfR.

[0146] 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 having neither a TfR-binding site nor a mutation that reduces FcγR binding. Thus, upon TfR association, the resulting asymmetric Fc polypeptide dimer having the first and second Fc polypeptides can have an overall reduced affinity for FcγR, whereas when not bound to TfR, there can be a limited reduction in FcγR binding (e.g., as described above) or none.

[0147] The term "FcRn" refers to the fetal 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 is similar to major histocompatibility (MHC) class I proteins, and β2-microglobulin, approximately 15 kDa in size.

[0148] As used herein, "FcRn-binding site" refers to the region of an Fc polypeptide that binds to FcRn. In human IgG, when numbered using the EU numbering scheme, the FcRn-binding site comprises L251, M252, I253, S254, R255, T256, M428, H433, N434, H435, and Y436. These positions correspond to positions 21-26, 198, and 203-206 of SEQ ID NO:99.

[0149] 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.

[0150] As used herein, the term "does not substantially reduce reticulocytes" or "does not substantially reduce reticulocytes in vivo" refers to the extent to which 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-antibody variable region fusion protein described herein comprising an effector function-positive TfR-binding Fc polypeptide dimer, is greater than that caused by a control, e.g., a mutant having intact effector function and / or reduced FcγR binding. "FcγR binding reduction" means that the reduction in reticulocytes (e.g., bone marrow reticulocytes or circulating reticulocytes) caused by a corresponding TfR-binding Fc dimer or Fc polypeptide dimer-antibody variable region fusion protein that does not contain a mutation that reduces FcγR binding, or an antibody that has full effector function and / or does not contain a mutation that reduces FcγR binding.

[0151] The term "does not substantially reduce reticulocytes" or "does not substantially reduce reticulocytes in vivo" refers to the level or percentage of remaining reticulocytes (e.g., remaining reticulocytes 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-antibody variable region fusion protein described herein comprising an effector function-positive TfR-binding Fc polypeptide dimer, compared to a control (e.g., an antibody variable region fusion protein having full effector function and / or FcγR binding). It can also mean exceeding (e.g., at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of) the amount or percentage of remaining reticulocytes (e.g., remaining reticulocytes in the bone marrow or circulation) following administration of a corresponding TfR-binding Fc dimer or Fc polypeptide dimer-antibody variable region fusion protein that does not contain a mutation that reduces FcγR binding, or an antibody comprising a corresponding TfR-binding Fc dimer that has full effector function and / or does not contain a mutation that reduces FcγR binding.

[0152] The amount or percentage of reticulocyte reduction (e.g., reticulocyte reduction in bone marrow or circulation) or the amount or percentage of remaining reticulocytes (e.g., remaining reticulocytes in bone marrow or circulation) may be measured using human TfR knock-in (TfR) mice, which have been 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 intravenously administering the modified Fc dimer or a control, e.g., 25-50 mg / kg (e.g., TfR ms / hu In KI mice, circulating reticulocytes can be measured at 24 hours post-administration by cytochemistry using the Advia 120 hematology system, as described herein. Bone marrow reticulocytes can be measured by Ter119 as described herein. + , hCD71 hi , and FSClow The abundance of the population can be determined using FACS sorting.

[0153] 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 position 341 to about position 447, numbered according to the EU numbering scheme, and a CH2 domain polypeptide refers to the segment of amino acids from about position 231 to about position 340, 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 1-110 and the CH3 domain is numbered 1-107, according to the IMGT Scientific Chart numbering (IMGT website). The CH2 and CH3 domains are part of the Fc region of an immunoglobulin. In the context of an IgG antibody, the Fc region refers to the segment of amino acids from about position 231 to about position 447, 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: 104.

[0154] The term "variable region" refers to the domain in 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. Typically, antibody variable regions contain four conserved "framework" regions interspersed with three hypervariable "complementarity-determining regions."

[0155] The terms "wild-type," "native," and "naturally occurring" with respect to a CH3 or CH2 domain are used herein to refer to a domain having a sequence that occurs in nature.

[0156] As used herein, the term "mutant" with respect to a mutant polypeptide or mutant polynucleotide is used interchangeably with "variant." Variants with respect to a given wild-type CH3 or CH2 domain reference sequence 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 produced by genetic modification, e.g., using genetic engineering or mutagenesis techniques of a native CH3 or CH2 domain polynucleotide or polypeptide. A "variant" includes any domain that contains at least one amino acid mutation with respect to the wild-type. Mutations can include substitutions, insertions, and deletions.

[0157] 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.

[0158] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that have been later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analog" refers to a compound that has 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 as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetic" refers to a chemical compound that has 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.

[0159] Naturally occurring α-amino acids include, without limitation, 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, without limitation, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0160] 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.

[0161] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, 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.

[0162] As used herein, the term "protein" refers to either a single-chain polypeptide or a dimer (i.e., two) or multimer (i.e., three or more) of polypeptides. The single-chain polypeptides of a protein can be linked by covalent bonds, e.g., disulfide bonds, or non-covalent interactions.

[0163] The terms "conservative substitution," "conservative mutation," or "conservatively modified variant" refer to changes that result in the substitution of an amino acid with another amino acid that can be classified as having similar characteristics. Examples of conservative amino acid group classifications defined in this manner include the "charged / polar group" which includes 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); the "aromatic group" which includes Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and the "aliphatic group" which includes 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). Within each group, subgroups may also be identified. For example, the group of charged or polar amino acids can be subdivided 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, the aromatic or cyclic group can be subdivided into subgroups including a "nitrogen ring subgroup" containing Pro, His, and Trp, and a "phenyl subgroup" containing Phe and Tyr. In another further example, the aliphatic group can be subdivided into subgroups, for example, an "aliphatic nonpolar subgroup" containing Val, Leu, Gly, and Ala, and an "aliphatic slightly polar subgroup" containing Met, Ser, Thr, and Cys. Examples of conservative mutation categories include, but are not limited to, amino acid substitutions of amino acids within the above subgroups, such as Arg for Lys or vice versa, such that a positive charge may be maintained; Asp for Glu or vice versa, such that a negative charge may be maintained; Thr for Ser or vice versa, such that a free -OH may be maintained; and Asn for Gln or vice versa, such that a free -NH2 may be maintained.In some embodiments, a hydrophobic amino acid is substituted for a naturally occurring hydrophobic amino acid, for example, in the active site, to preserve hydrophobicity.

[0164] The terms "identical" or percent "identity" in the context of two or more polypeptide sequences refer to two or more sequences or subsequences that are identical, are the same, or have a specified percentage of amino acid residues, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more, over a particular region when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0165] For polypeptide sequence comparison, typically, one amino acid sequence 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, such as visual alignment, or using publicly available software that uses known algorithms to achieve maximum alignment. Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters used for alignment to achieve maximum alignment can be determined by those skilled in the art. For the purposes of polypeptide sequence comparison for the purposes of this application, the BLASTP algorithm, standard protein BLAST, is used to align two protein sequences using default parameters.

[0166] The terms "corresponding to," "determined with reference to," or "numbered with reference to," when used in the particular context of a given amino acid residue in a polypeptide sequence, refer to the position of the residue in the specified reference sequence when the given amino acid sequence is aligned and compared to the reference sequence to the fullest extent possible. Thus, for example, when optimally aligned with SEQ ID NO: 99, an amino acid residue in a polypeptide "corresponds to" an amino acid in the region of SEQ ID NO: 99 when the residue aligns with the amino acid of SEQ ID NO: 99. A polypeptide that is aligned to a reference sequence need not be the same length as the reference sequence.

[0167] As used herein, 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 "is specific for" a particular target (e.g., TfR or FcγR) refer to, for example, a binding affinity of at least 10-fold, ... -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 Equilibrium dissociation constant K DIn some embodiments, the modified CH3 domain polypeptide specifically binds to an epitope on the TfR that is conserved across species (e.g., structurally conserved across species), e.g., conserved between non-human primate and human species (e.g., structurally conserved between non-human primate and human species). In some embodiments, the polypeptide may bind exclusively to the human TfR.

[0168] As used herein, the term "binding affinity" refers to the strength of a non-covalent interaction between two molecules, e.g., between a single binding site on a polypeptide and the target to which it binds, e.g., TfR. Thus, for example, unless otherwise indicated or clear from the context, the term may refer to a 1:1 interaction between a polypeptide and its target. Binding affinity is measured by the equilibrium dissociation constant (K D ) and the binding rate constant (k a ,time -1 M -1 ) divided by the dissociation rate constant (k d ,time -1 ) refers to K D can be determined by measuring the kinetics of complex formation and dissociation, for example, using 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" refers not only to formal binding affinities, such as those reflecting a 1:1 interaction between a polypeptide and its target, but also to K values, which may reflect strong binding. D It also includes the apparent affinity, from which the

[0169] The terms "antigen-binding portion" and "antigen-binding fragment" are used interchangeably herein and refer to one or more fragments of an antibody variable region that retain the ability to specifically bind to an antigen (e.g., HER2). Examples of antigen-binding fragments include, but are not limited to, Fab fragments (monovalent fragments consisting of the VL, VH, CL, and CH1 domains), F(ab')2 fragments (bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region), single-chain Fvs (scFvs), disulfide-linked Fvs (dsFvs), complementarity-determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), nanobodies, diabodies, and other formats described in Spiess et al., Mol. Immun. 67 (2015) 95-106, which is incorporated herein by reference.

[0170] The term "complementarity-determining region" or "CDR" refers to the three hypervariable regions in each chain that interrupt the four framework regions established by the light and heavy chain variable regions. The CDRs are primarily responsible for antibody binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, are numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which a particular CDR is located. Thus, a VH CDR3 or CDR-H3 is located in the variable region of the heavy chain of the antibody in which it is found, while a VL CDR1 or CDR-L1 is the CDR1 from the variable region of the light chain of the antibody in which it is found.

[0171] The "framework regions" or "FRs" of different light or heavy chains are relatively conserved within a species. The framework regions of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serve to position and align the CDRs in three-dimensional space. Framework sequences can be obtained from public DNA databases or published references containing germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBASE2" germline variable gene sequence database of human and mouse sequences.

[0172] The amino acid sequences of the CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, the International ImMunoGeneTics database (IMGT), AbM, and observed antigen contact ("Contact"). In some embodiments, CDRs are determined according to the Contact definition. See MacCallum et al., J. Mol. Biol., 262:732-745 (1996). In some embodiments, CDRs are determined by a combination of the Kabat, Chothia, and Contact CDR definitions.

[0173] As used interchangeably herein, the terms "subject," "individual," and "patient" refer to mammals, including but not limited to humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In one embodiment, the patient is a human.

[0174] The terms "treatment," "treating," and the like are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. "Treating" or "treatment" can refer to any indication of successful treatment or improvement of cancer (e.g., HER2-positive and / or metastatic cancer), including any objective or subjective parameter, such as relief, remission, improved patient survival, increased survival time or rate, attenuation of symptoms or making the disease more tolerable for the patient, slowing in the rate of degeneration or decline, or improving the patient's physical or mental health. The treatment or improvement of symptoms can be based on objective or subjective parameters. The effect of treatment can be compared to an individual or pool of individuals not receiving treatment, or to the same patient at different time points before or during treatment.

[0175] The term "pharmaceutically acceptable excipient" refers to inactive pharmaceutical ingredients that are biologically or pharmacologically compatible for use in humans or animals, such as, but not limited to, buffers, carriers, or preservatives.

[0176] As used herein, a "therapeutic amount" or "therapeutically effective amount" of a construct (e.g., an antibody described herein) is an amount of the construct that treats, alleviates, relieves, or reduces the severity of symptoms of a disease in a subject. A "therapeutic amount" or "therapeutically effective amount" of a construct (e.g., an Fc polypeptide dimer-antibody variable region fusion protein or an antibody heavy chain) may improve patient survival, increase survival time or rate, attenuate symptoms, make an injury, disease, or condition (e.g., cancer, such as HER2-positive and / or metastatic cancer) more tolerable, slow the rate of degeneration or decline, or improve the physical or mental well-being of a patient.

[0177] The term "administering" refers to a method of delivering a construct, compound, or composition to a desired site of biological effect. These methods include, but are not limited to, topical, parenteral, intravenous, intradermal, intramuscular, intrathecal, colonic, rectal, or intraperitoneal delivery. In one embodiment, the antibodies described herein are administered intravenously.

[0178] III. FC Polypeptide Dimer-Antibody Variable Region Fusion Proteins In some aspects, the present disclosure provides Fc polypeptide dimer-antibody variable region fusion proteins capable of binding to human epidermal growth factor 2 (HER2) and modified to bind to transferrin receptor (TfR), thus enabling the Fc polypeptide dimer-antibody variable region fusion protein to cross the blood-brain barrier (BBB). In some embodiments, the Fc polypeptide dimer-antibody variable region fusion proteins provided herein retain effector function upon binding to HER2 but have reduced effector function upon TfR binding. In this manner, the Fc polypeptide dimer-antibody variable region fusion protein can transport an anti-HER2 antibody variable region (e.g., forming part of the Fab domain) across the BBB without substantial reduction in reticulocytes (which also contain TfR on the cell surface), yet still exhibit effector function capable of targeting cancer cells (e.g., HER2-positive cancer cells or their metastases).

[0179] In some embodiments, provided herein are effector function-positive Fc polypeptide dimer-antibody variable region fusion proteins having a cis configuration, meaning that only one (but not both) of the Fc polypeptides in the Fc polypeptide dimer is modified to have a TfR-binding site and a modification that reduces FcγR binding upon binding to TfR. In these embodiments, the other Fc polypeptide in the Fc polypeptide dimer does not contain either a TfR-binding site or a modification that substantially reduces FcγR binding. A trans configuration 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, while the other Fc polypeptide contains a modification that, for example, reduces effector function upon binding to TfR. Modified Fc polypeptide dimers having a cis configuration but not a trans configuration can reduce reticulocytopenia in the blood and bone marrow.

[0180] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion proteins provided herein do not substantially reduce reticulocytes (e.g., in the bone marrow and / or circulation). In some embodiments, the Fc polypeptide dimer-antibody variable region fusion proteins do not substantially reduce reticulocytes in vivo. In some embodiments, the amount of reticulocytes reduced following administration of the Fc polypeptide dimer-antibody variable region fusion protein is less than the amount of reticulocytes reduced following administration of a control. In some embodiments, the control is a corresponding TfR-binding polypeptide dimer-antibody variable region fusion protein that has full effector function and / or does not contain a mutation that reduces FcγR binding. In some cases, the control is an Fc polypeptide dimer-antibody variable region fusion protein in which the first Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 81, 83, 85, 87, 89, 91, 93, 95, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, or 280 (i.e., a first Fc polypeptide that specifically binds to TfR comprising a TfR binding site but does not comprise a LALA substitution or other modification that reduces FcγR binding), and the second Fc polypeptide does not comprise a TfR binding site or any modification that reduces FcγR binding.

[0181] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises (a) an antibody variable region capable of binding to HER2 (e.g., subdomain II or IV of HER2), or an antigen-binding fragment thereof, and (b) an altered Fc polypeptide dimer comprising a first Fc polypeptide comprising a modification that creates a TfR-binding site. In some embodiments, the altered Fc polypeptide dimer comprises a second Fc polypeptide that does not comprise a TfR-binding site. In some embodiments, the first Fc polypeptide comprises an amino acid modification that reduces FcγR binding upon binding to TfR. In some embodiments, the second Fc polypeptide comprises an amino acid modification that reduces FcγR binding upon binding to TfR. In some embodiments, the first and second Fc polypeptides comprise amino acid modifications that reduce FcγR binding upon binding to TfR. In some embodiments, the first and / or second Fc polypeptides comprise amino acid modifications that reduce FcγR binding upon binding to TfR. In some embodiments, the amino acid modifications that reduce FcγR binding upon binding to TfR include Ala at position 234 and at position 235 according to EU numbering.

[0182] In some embodiments, the first and / or second Fc polypeptide comprises an amino acid modification that increases serum half-life. In some embodiments, the first Fc polypeptide comprises an amino acid modification that increases serum half-life. In some embodiments, the second Fc polypeptide comprises an amino acid modification that increases serum half-life. In some embodiments, the first and second Fc polypeptides comprise amino acid modifications that increase serum half-life. In some embodiments, the amino acid modifications that increase serum half-life comprise (i) Leu at position 428 and Ser at position 434, or (ii) Ser or Ala at position 434, according to EU numbering.

[0183] In some embodiments, the antibody variable region forms part of a Fab domain.

[0184] Anti-HER2 variable region anti-HER2_DIV In some embodiments, the antibody variable region comprises one or more complementarity determining regions (CDRs) selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 69, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 70, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 70; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 71, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 71; (d) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 72, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 72; (e) a light chain CDR2 having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 73; and (f) a light chain CDR3 having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 74.

[0185] In some embodiments, the antibody variable region comprises two, three, four, five, or all six of (a) through (f). In some embodiments, the antibody variable region comprises a heavy chain CDR1 of (a), a heavy chain CDR2 of (b), and a heavy chain CDR3 of (c). In some embodiments, the antibody variable region comprises a light chain CDR1 of (d), a light chain CDR2 of (e), and a light chain CDR3 of (f). In some embodiments, a CDR with a maximum of two amino acid substitutions has one amino acid substitution relative to the reference sequence. In some embodiments, a CDR with a maximum of two amino acid substitutions has two amino acid substitutions relative to the reference sequence. In some embodiments, the maximum two amino acid substitutions are conservative substitutions.

[0186] In some embodiments, the antibody variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 69; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 70; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 71; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 72; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 73; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 74.

[0187] In some embodiments, the antibody variable region comprises two, three, four, five, or all six of (a) through (f). In some embodiments, the antibody variable region comprises a heavy chain CDR1 of (a), a heavy chain CDR2 of (b), and a heavy chain CDR3 of (c). In some embodiments, the antibody variable region comprises a light chain CDR1 of (d), a light chain CDR2 of (e), and a light chain CDR3 of (f).

[0188] In some embodiments, the antibody variable region comprises (a) a CDR-H1, CDR-H2, and CDR-H3 having (i) at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 59, and (ii) CDR-H1, CDR-H2, and CDR-H3 identical to SEQ ID NOs: 69, 70, and 71, respectively. and / or (b) a light chain variable region comprising (i) at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 60, and (ii) CDR-L1, CDR-L2, and CDR-L3 identical to SEQ ID NOs: 72, 73, and 74, respectively.

[0189] In some embodiments, the antibody variable region comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 59. In some embodiments, the antibody variable region comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:59.

[0190] In some embodiments, the antibody variable region comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 60. In some embodiments, the antibody variable region comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:60.

[0191] In some embodiments, the antibody variable regions comprise two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:59 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:60.

[0192] Anti-HER2_DII In some embodiments, the antibody variable region comprises one or more complementarity determining regions (CDRs) selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 75 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 76 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 76; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 77 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 77; (d) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 78 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 78; (e) a light chain CDR2 having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 79; and (f) a light chain CDR3 having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 80.

[0193] In some embodiments, the antibody variable region comprises two, three, four, five, or all six of (a) through (f). In some embodiments, the antibody variable region comprises a heavy chain CDR1 of (a), a heavy chain CDR2 of (b), and a heavy chain CDR3 of (c). In some embodiments, the antibody variable region comprises a light chain CDR1 of (d), a light chain CDR2 of (e), and a light chain CDR3 of (f). In some embodiments, a CDR with a maximum of two amino acid substitutions has one amino acid substitution relative to the reference sequence. In some embodiments, a CDR with a maximum of two amino acid substitutions has two amino acid substitutions relative to the reference sequence. In some embodiments, the maximum two amino acid substitutions are conservative substitutions.

[0194] In some embodiments, the antibody variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 75; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 76; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 77; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 78; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 79; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 80.

[0195] In some embodiments, the antibody variable region comprises two, three, four, five, or all six of (a) through (f). In some embodiments, the antibody variable region comprises a heavy chain CDR1 of (a), a heavy chain CDR2 of (b), and a heavy chain CDR3 of (c). In some embodiments, the antibody variable region comprises a light chain CDR1 of (d), a light chain CDR2 of (e), and a light chain CDR3 of (f).

[0196] In some embodiments, the antibody variable region comprises (a) a CDR-H1, CDR-H2, and CDR-H3 having (i) at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 61, and (ii) CDR-H1, CDR-H2, and CDR-H3 identical to SEQ ID NOs: 75, 76, and 77, respectively. and / or (b) a light chain variable region comprising (i) at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 62, and (ii) CDR-L1, CDR-L2, and CDR-L3 identical to SEQ ID NOs: 78, 79, and 80, respectively.

[0197] In some embodiments, the antibody variable region comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 61. In some embodiments, the antibody variable region comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 61.

[0198] In some embodiments, the antibody variable region comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 62. In some embodiments, the antibody variable region comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62.

[0199] In some embodiments, the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:61 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:62.

[0200] anti-HER2_DI In some embodiments, the antibody variable region comprises: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 250, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 251, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 251; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 252, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 252; (d) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 253 or having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 253; (e) a light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 254; and (f) a light chain CDR3 having at most two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 255.

[0201] In some embodiments, the antibody variable region comprises two, three, four, five, or all six of (a) through (f). In some embodiments, the antibody variable region comprises a heavy chain CDR1 of (a), a heavy chain CDR2 of (b), and a heavy chain CDR3 of (c). In some embodiments, the antibody variable region comprises a light chain CDR1 of (d), a light chain CDR2 of (e), and a light chain CDR3 of (f). In some embodiments, a CDR with a maximum of two amino acid substitutions has one amino acid substitution relative to the reference sequence. In some embodiments, a CDR with a maximum of two amino acid substitutions has two amino acid substitutions relative to the reference sequence. In some embodiments, the maximum two amino acid substitutions are conservative substitutions.

[0202] In some embodiments, the antibody variable region comprises one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 250; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 251; (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 252; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 253; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 254; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 255.

[0203] In some embodiments, the antibody variable region comprises two, three, four, five, or all six of (a) through (f). In some embodiments, the antibody variable region comprises a heavy chain CDR1 of (a), a heavy chain CDR2 of (b), and a heavy chain CDR3 of (c). In some embodiments, the antibody variable region comprises a light chain CDR1 of (d), a light chain CDR2 of (e), and a light chain CDR3 of (f).

[0204] In some embodiments, the antibody variable region comprises (a) a CDR-H1, CDR-H2, and CDR-H3 sequence having (i) at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 256, and (ii) a CDR-H1, CDR-H2, and CDR-H3 sequence identical to SEQ ID NOs: 250, 251, and 252, respectively. and / or (b) a light chain variable region comprising (i) at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 257, and (ii) CDR-L1, CDR-L2, and CDR-L3 identical to SEQ ID NOs: 253, 254, and 255, respectively.

[0205] In some embodiments, the antibody variable region comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 256. In some embodiments, the antibody variable region comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:256.

[0206] In some embodiments, the antibody variable region comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 257. In some embodiments, the antibody variable region comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:257.

[0207] In some embodiments, the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:256 and two light chain variable regions comprising the amino acid sequence of SEQ ID NO:257.

[0208] Exemplary Fc Polypeptide Dimer-Antibody Variable Region Fusion Proteins The Fc polypeptide dimer-antibody variable region fusion proteins described herein can include any combination of the anti-HER2 variable regions described above. In some embodiments, the first Fc polypeptide comprises a TfR-binding site comprising a modified CH3 domain. Non-limiting examples of modified CH3 domains that can be used in the compositions and methods are described herein in the section titled "TfR-Binding Fc Polypeptides." In some embodiments, the first Fc polypeptide further comprises the knob mutation T366W, and the second Fc polypeptide comprises the hole mutations T366S, L368A, and Y407V, according to EU numbering. In some embodiments, the first Fc polypeptide comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 63. In some cases, the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the second Fc polypeptide comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 67 or 68. In some cases, the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 67 and 68.

[0209] In some embodiments, the first Fc polypeptide further comprises hole mutations T366S, L368A, and Y407V, and the second Fc polypeptide comprises knob mutation T366W, according to EU numbering. In some embodiments, the first Fc polypeptide comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 64. In some cases, the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, the second Fc polypeptide comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 65 or 66. In some cases, the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 65 and 66.

[0210] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region capable of binding to HER2 (e.g., human HER2), or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising modifications that create a TfR binding site, amino acid modifications L234A and L235A, and knob mutation T366W, according to EU numbering; and (c) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, according to EU numbering, and does not comprise any modifications that reduce the TfR binding site or FcγR binding.

[0211] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 2, 10, 18, and 82. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0212] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 30, 38, 46, and 90. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0213] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 259, 267, 275, and 283. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 290. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0214] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region capable of binding to HER2 (e.g., human HER2), or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising modifications that create a TfR-binding site, amino acid modifications L234A and L235A, knob mutation T366W, and amino acid modification N434S with or without M428L, according to EU numbering; and (c) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, according to EU numbering, and that does not comprise a TfR-binding site.

[0215] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 4, 12, 20, and 84. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0216] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 32, 40, 48, and 92. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0217] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 261, 269, 277, and 285. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 290. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0218] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region capable of binding to HER2 (e.g., human HER2), or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising modifications that create a TfR binding site, amino acid modifications L234A and L235A, and knob mutation T366W, according to EU numbering; and (c) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, and amino acid modification N434S with or without M428L, according to EU numbering, and that does not comprise a TfR binding site.

[0219] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 2, 10, 18, and 82. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0220] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 30, 38, 46, and 90. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 56. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0221] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 259, 267, 275, and 283. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 291. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0222] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region capable of binding to HER2 (e.g., human HER2), or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising modifications that create a TfR-binding site, amino acid modifications L234A and L235A, knob mutation T366W, and amino acid modification N434S with or without M428L, according to EU numbering; and (c) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, and amino acid modification N434S with or without M428L, according to EU numbering, and that does not comprise a TfR-binding site.

[0223] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 4, 12, 20, and 84. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0224] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 32, 40, 48, and 92. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 56. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0225] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 261, 269, 277, and 285. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 291. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0226] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region capable of binding to HER2 (e.g., human HER2), or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising modifications that create a TfR binding site, amino acid modifications L234A and L235A, and hole mutations T366S, L368A, and Y407V, according to EU numbering; and (c) a second Fc polypeptide comprising knob mutation T366W, according to EU numbering, and no TfR binding site.

[0227] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 6, 14, 22, and 86. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0228] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 34, 42, 50, and 94. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0229] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 263, 271, 279, and 287. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 294. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0230] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region capable of binding to HER2 (e.g., human HER2), or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising modifications that create a TfR binding site, amino acid modifications L234A and L235A, hole mutations T366S, L368A, and Y407V, and amino acid modification N434S with or without M428L, according to EU numbering; and (c) a second Fc polypeptide comprising knob mutation T366W, according to EU numbering, and that does not comprise a TfR binding site.

[0231] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 8, 16, 24, and 88. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0232] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 36, 44, 52, and 96. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0233] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 265, 273, 281, and 289. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 294. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0234] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region capable of binding to HER2 (e.g., human HER2), or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising modifications that create a TfR binding site, amino acid modifications L234A and L235A, and hole mutations T366S, L368A, and Y407V, according to EU numbering; and (c) a second Fc polypeptide comprising knob mutation T366W, and amino acid modification N434S, with or without M428L, according to EU numbering, and that does not comprise a TfR binding site.

[0235] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 6, 14, 22, and 86. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0236] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 34, 42, 50, and 94. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0237] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 263, 271, 279, and 287. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 295. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0238] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region capable of binding to HER2 (e.g., human HER2), or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising modifications that create a TfR-binding site, amino acid modifications L234A and L235A, hole mutations T366S, L368A, and Y407V, and amino acid modification N434S with or without M428L, according to EU numbering; and (c) a second Fc polypeptide comprising knob mutation T366W, and amino acid modification N434S with or without M428L, according to EU numbering, and that does not comprise a TfR-binding site.

[0239] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 8, 16, 24, and 88. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0240] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 36, 44, 52, and 96. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0241] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 265, 273, 281, and 289. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 295. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0242] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region, or antigen-binding fragment thereof, capable of binding to HER2 (e.g., human HER2); (b) a first Fc polypeptide comprising a modification that creates a TfR binding site and knob mutation T366W, according to EU numbering; and (c) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, according to EU numbering, and no TfR binding site.

[0243] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, and 81. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0244] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 29, 37, 45, and 89. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0245] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 258, 266, 274, and 282. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 290. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0246] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region capable of binding to HER2 (e.g., human HER2), or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising the amino acid modifications N434S, with or without knob mutations T366W and M428L, modifications that create a TfR binding site, according to EU numbering; and (c) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, according to EU numbering, and that does not comprise a TfR binding site.

[0247] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 3, 11, 19, and 83. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0248] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 31, 39, 47, and 91. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0249] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 260, 268, 276, and 284. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 290. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0250] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region capable of binding to HER2 (e.g., human HER2), or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising a modification that creates a TfR binding site and knob mutation T366W, according to EU numbering; and (c) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, and amino acid modification N434S with or without M428L, according to EU numbering, and which does not comprise a TfR binding site.

[0251] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, and 81. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0252] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 29, 37, 45, and 89. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 56. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0253] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 258, 266, 274, and 282. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 291. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0254] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region capable of binding to HER2 (e.g., human HER2), or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising modifications that create a TfR binding site, knob mutation T366W, and amino acid modification N434S, with or without M428L, according to EU numbering; and (c) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, and amino acid modification N434S, with or without M428L, according to EU numbering, and that does not comprise a TfR binding site.

[0255] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 3, 11, 19, and 83. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0256] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 31, 39, 47, and 91. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 56. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0257] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 260, 268, 276, and 284. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 291. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0258] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region capable of binding to HER2 (e.g., human HER2), or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising modifications that create a TfR binding site and hole mutations T366S, L368A, and Y407V, according to EU numbering; and (c) a second Fc polypeptide comprising knob mutation T366W, according to EU numbering, and no TfR binding site.

[0259] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 5, 13, 21, and 85. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0260] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 33, 41, 49, and 93. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0261] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 262, 270, 278, and 286. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 294. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0262] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region, or antigen-binding fragment thereof, capable of binding to HER2 (e.g., human HER2); (b) a first Fc polypeptide comprising the amino acid modification N434S, with or without hole mutations T366S, L368A, and Y407V, and M428L, modifications that create a TfR binding site, according to EU numbering; and (c) a second Fc polypeptide comprising the knob mutation T366W, according to EU numbering, and that does not comprise a TfR binding site.

[0263] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 7, 15, 23, and 87. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0264] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 35, 43, 51, and 95. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0265] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 264, 272, 280, and 288. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 294. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0266] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region capable of binding to HER2 (e.g., human HER2), or an antigen-binding fragment thereof; (b) a first Fc polypeptide comprising modifications that create a TfR binding site and hole mutations T366S, L368A, and Y407V, according to EU numbering; and (c) a second Fc polypeptide comprising knob mutation T366W, and amino acid modification N434S, with or without M428L, according to EU numbering, and that does not comprise a TfR binding site.

[0267] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 5, 13, 21, and 85. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0268] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 33, 41, 49, and 93. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0269] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 262, 270, 278, and 286. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 295. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0270] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises: (a) an antibody variable region, or antigen-binding fragment thereof, capable of binding to HER2 (e.g., human HER2); (b) a first Fc polypeptide comprising modifications that create a TfR binding site, hole mutations T366S, L368A, and Y407V, and the amino acid modification N434S, with or without M428L, according to EU numbering; and (c) a second Fc polypeptide comprising knob mutation T366W, and the amino acid modification N434S, with or without M428L, according to EU numbering, and that does not comprise a TfR binding site.

[0271] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 7, 15, 23, and 87. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 57.

[0272] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 35, 43, 51, and 95. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 58.

[0273] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 264, 272, 280, and 288. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 295. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains comprising the amino acid sequence of SEQ ID NO: 293.

[0274] antibody heavy chain In another aspect, provided herein are antibody heavy chains. In some embodiments, the antibody heavy chain comprises (a) an anti-HER2 (e.g., human HER2) antibody heavy chain variable region, or a fragment thereof, and (b) an altered Fc polypeptide comprising a modification that creates a TfR-binding site. The antibody heavy chain can comprise any of the anti-HER2 variable heavy chain CDRs and / or heavy chain variable region sequences in the section entitled "Anti-HER2 Variable Regions" above. The altered Fc polypeptide can comprise any of the TfR-binding sites (e.g., an altered CH3 domain) described herein and / or any of the modifications described herein that increase serum half-life or reduce FcγR binding (e.g., upon binding to TfR).

[0275] In some embodiments, the modified Fc polypeptide further comprises knob mutation T366W, according to EU numbering. In some embodiments, the modified polypeptide comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 63. In some cases, the modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the modified Fc polypeptide further comprises hole mutations T366S, L368A, and Y407V, according to EU numbering. In some embodiments, the modified polypeptide comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 64. In some cases, the modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 64.

[0276] In some embodiments, the antibody heavy chain comprises (a) an anti-HER2 (e.g., human HER2) antibody heavy chain variable region, or a fragment thereof, and (b) a modified Fc polypeptide comprising modifications that create a TfR binding site, amino acid modifications L234A and L235A, and knob mutation T366W, according to EU numbering. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 2, 10, 18, and 82. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 30, 38, 46, and 90. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 259, 267, 275, and 283.

[0277] In some embodiments, the antibody heavy chain comprises (a) an anti-HER2 (e.g., human HER2) antibody heavy chain variable region, or a fragment thereof, and (b) a modified Fc polypeptide comprising modifications that create a TfR binding site, amino acid modifications L234A and L235A, knob mutation T366W, and amino acid modification N434S with or without M428L, according to EU numbering. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 4, 12, 20, and 84. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 32, 40, 48, and 92. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 261, 269, 277, and 285.

[0278] In some embodiments, the antibody heavy chain comprises (a) an anti-HER2 (e.g., human HER2) antibody heavy chain variable region, or a fragment thereof, and (b) a modified Fc polypeptide comprising modifications that create a TfR binding site, amino acid modifications L234A and L235A, and hole mutations T366S, L368A, and Y407V, according to EU numbering. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 6, 14, 22, and 86. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 34, 42, 50, and 94. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 263, 271, 279, and 287.

[0279] In some embodiments, the antibody heavy chain comprises (a) an anti-HER2 (e.g., human HER2) antibody heavy chain variable region, or a fragment thereof, and (b) a modified Fc polypeptide comprising modifications that create a TfR binding site, amino acid modifications L234A and L235A, hole mutations T366S, L368A, and Y407V, and amino acid modification N434S with or without M428L, according to EU numbering. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 8, 16, 24, and 88. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 36, 44, 52, and 96. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 265, 273, 281, and 289.

[0280] In some embodiments, the antibody heavy chain comprises (a) an anti-HER2 (e.g., human HER2) antibody heavy chain variable region, or a fragment thereof, and (b) an engineered Fc polypeptide comprising a modification that creates a TfR binding site and the knob mutation T366W, according to EU numbering. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, and 81. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 29, 37, 45, and 89. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 258, 266, 274, and 282.

[0281] In some embodiments, the antibody heavy chain comprises (a) an anti-HER2 (e.g., human HER2) antibody heavy chain variable region, or a fragment thereof, and (b) a modified Fc polypeptide comprising the amino acid modification N434S, with or without knob mutation T366W and M428L, modifications that create a TfR binding site, according to EU numbering. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 3, 11, 19, and 83. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 31, 39, 47, and 91. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 260, 268, 276, and 284.

[0282] In some embodiments, the antibody heavy chain comprises (a) an anti-HER2 (e.g., human HER2) antibody heavy chain variable region, or a fragment thereof, and (b) a modified Fc polypeptide comprising modifications to create a TfR binding site and hole mutations T366S, L368A, and Y407V, according to EU numbering. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 5, 13, 21, and 85. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 33, 41, 49, and 93. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 262, 270, 278, and 286.

[0283] In some embodiments, the antibody heavy chain comprises (a) an anti-HER2 (e.g., human HER2) antibody heavy chain variable region, or a fragment thereof, and (b) a modified Fc polypeptide comprising the following modifications that create a TfR binding site: hole mutations T366S, L368A, and Y407V, and amino acid modification N434S with or without M428L, according to EU numbering. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 7, 15, 23, and 87. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 35, 43, 51, and 95. In some embodiments, the antibody heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 264, 272, 280, and 288.

[0284] IV. TFR-binding FC polypeptides This section describes modified Fc polypeptides that can bind to TfR and be transported across the blood-brain barrier (BBB).

[0285] 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 can be from 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 position 341 to about position 447, numbered according to the EU numbering scheme. Positions in the CH3 domain for purposes of identifying the corresponding set of amino acid positions for TfR binding are determined with reference to amino acids 111-217 of the EU numbering scheme, SEQ ID NO: 101, or SEQ ID NO: 99, unless otherwise specified. Substitutions are also determined with reference to the EU numbering scheme or SEQ ID NO: 99, i.e., an amino acid is considered to be a substitution for the amino acid at the corresponding position in the EU numbering scheme or SEQ ID NO: 99.

[0286] As noted above, sets of residues in a CH3 domain that may be modified are numbered herein with reference to the EU numbering scheme or SEQ ID NO: 99. Any CH3 domain, e.g., an IgG1, IgG2, IgG3, or IgG4 CH3 domain, may have a modification, e.g., an amino acid substitution, at one or more sets of residues that correspond to residues at recognized positions in the EU numbering scheme or SEQ ID NO: 99. The position in each of the IgG1, IgG2, IgG3, and IgG4 sequences that corresponds to any given position in the EU numbering scheme or SEQ ID NO: 99 can be easily determined.

[0287] Those skilled in the art will understand that CH3 domains of other immunoglobulin isotypes, such as IgM, IgA, IgE, IgD, etc., can be similarly modified by specifying amino acids in those domains that correspond to the amino acid positions described herein. Modifications can also be made to corresponding domains from immunoglobulins from other species, such as non-human primates, monkeys, mice, rats, rabbits, dogs, pigs, chickens, etc.

[0288] 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: 102), which corresponds to position 11 of the human TfR apical domain sequence set forth in SEQ ID NO: 103. SEQ ID NO: 103 corresponds to amino acids 198-378 of the human TfR-1 single protein sequence P02786 (SEQ ID NO: 102). 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: 102). 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%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%). In some embodiments, binding of transferrin to TfR is inhibited by less than about 20% (e.g., less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%). Exemplary CH3 domain polypeptides that exhibit this 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.

[0289] CH3 TfR binding set: 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 In some embodiments, the modified CH3 domain polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions at the set of amino acid positions including 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421, according to the EU numbering scheme (set CH3C). Exemplary substitutions that may be introduced at these positions are shown in Table 5. Additional substitutions are shown in Table 6. In some embodiments, the amino acid at position 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.

[0290] In certain embodiments, the modified CH3 domain polypeptide comprises: 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; an aliphatic amino acid, Gly, Ser, Thr, or Asn at position 389; Gly at position 390; His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr; an acidic amino acid, 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 an acidic amino acid at position 416; and / or an aromatic amino acid, His, or Lys, at position 421. The present invention also includes positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 selected from the group consisting of:

[0291] 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: 177-180. 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: 177-180. In some embodiments, such a modified CH3 domain polypeptide comprises amino acids 150-160 and / or 183-191 of any one of SEQ ID NOs: 177-180. In some embodiments, such a modified CH3 domain polypeptide comprises amino acids 150-160 and / or 183-196 of any one of SEQ ID NOs: 177-180.

[0292] 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: 99, provided that the percent identity excludes the set of positions 154, 156, 157, 158, 159, 160, 183, 186, and 191 (positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 according to the EU numbering scheme) of SEQ ID NO: 99. 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: 177-180.

[0293] 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 any one of SEQ ID NOs: 177-180, but does not include a sequence at positions 150, 154, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, provided that at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 positions corresponding to 60, 161, 162, 183, 184, 185, 186, 191, 194, and 196 (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.

[0294] 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: 177-180, and also has one of the following: 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, or an acidic amino acid at position 387; 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, 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; Ser, Trp, or Gly at position 426 at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 positions of

[0295] In additional 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: 177-180. 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: 177-180, or amino acids 153-194, or amino acids 153-199 of any one of SEQ ID NOs: 177-180.

[0296] In some embodiments, the polypeptide comprises any one of SEQ ID NOs: 177-180. In further embodiments, the polypeptide may 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: 177-180.

[0297] FcRn binding site The polypeptides described herein that can be transported across the BBB can additionally contain an FcRn-binding site. In some embodiments, the FcRn-binding site is within an altered Fc polypeptide or fragment thereof.

[0298] In some embodiments, the FcRn-binding site comprises a native FcRn-binding site. In some embodiments, the FcRn-binding site does not comprise amino acid changes 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 FcRn binding.

[0299] In some embodiments, the FcRn-binding site has one or more mutated, e.g., substituted, amino acid residues, and the mutation(s) increase or do not substantially reduce serum half-life (i.e., reduce serum half-life by 25% or less when assayed under identical conditions compared to an equivalent protein having wild-type residues at the mutated positions). In some embodiments, the FcRn-binding site has one or more substituted amino acid residues at positions 21-26, 198, and 203-206, the positions being determined with reference to SEQ ID NO:99.

[0300] In some embodiments, the FcRn-binding site comprises one or more mutations relative to a native human IgG sequence that extend the serum half-life of the altered 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, as determined with reference to SEQ ID NO: 99 (positions corresponding 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 position 21, 22, 24, 25, 26, 77, 78, 79, 81, 82, 84, 155, 156, 157, 159, 198, 203, 204, or 206, as determined with reference to SEQ ID NO: 99 (positions corresponding 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, as determined with reference to SEQ ID NO: 99 (positions corresponding to positions 252, 254, and 256 using EU numbering). In some embodiments, the mutations are M22Y, S24T, and T26E, numbered with reference to SEQ ID NO: 99. In some embodiments, the modified Fc polypeptide described herein further comprises the mutations M22Y, S24T, and T26E. In some embodiments, the mutations are introduced at one or two of positions 198 and 204 (corresponding to positions 428 and 434 using EU numbering), as determined with reference to SEQ ID NO: 99. In some embodiments, the mutations are M198L and N204S, numbered with reference to SEQ ID NO: 99. In some embodiments, the modified Fc polypeptide described herein further comprises 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 (corresponding to T77, E150, and N204, numbered with reference to SEQ ID NO: 99), according to EU numbering.In some embodiments, the mutations are T307Q and N434A (SEQ ID NO: 99, T77Q and N204A). In some embodiments, the modified Fc polypeptide comprises mutations T307A, E380A, and N434A (SEQ ID NO: 99, T77A, E150A, and N204A). In some embodiments, the modified Fc polypeptide comprises substitutions at positions T250 and M428 (corresponding to T20 and M198, numbered with reference to SEQ ID NO: 99). In some embodiments, the Fc polypeptide comprises mutations T250Q and / or M428L (SEQ ID NO: 99, T20Q and M198L). In some embodiments, the modified Fc polypeptide comprises substitutions at positions M428 and N434 (corresponding to M198 and N204, numbered with reference to SEQ ID NO: 99). In some embodiments, the modified Fc polypeptide comprises the substitutions M428L and N434S (corresponding to M198L and N204S, numbered with reference to SEQ ID NO: 99). In some embodiments, the modified Fc polypeptide comprises the substitutions N434S or N434A (corresponding to N204S or N204A, numbered with reference to SEQ ID NO: 99).

[0301] V. Mutations that reduce effector function or FCΓR binding The Fc polypeptides provided herein (e.g., modified to bind TfR and initiate transport across the BBB) can also include additional mutations to reduce effector function. As described herein, by introducing both a TfR binding site and mutations that reduce TfR-mediated FcγR binding to the same Fc polypeptide of an Fc polypeptide dimer, it was possible to reduce effector function upon TfR binding, resulting in TfR binding without a substantial reduction in reticulocytes, yet still maintain and exhibit a level of effector function (e.g., ADCC or CDC) when the Fc polypeptide dimer is fused to a therapeutic Fab and binds to the Fab's target antigen.

[0302] In some embodiments, an Fc polypeptide comprising an altered 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 effector function, including, but not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and cytotoxic T cells.

[0303] Examples of effector functions include, but are not limited to, C1q binding and CDC, Fc receptor binding, ADCC, antibody-dependent cell-mediated phagocytosis (ADCP), downregulation 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 upon binding to the appropriate Fc receptors present on immune system cells, and native human IgG1, IgG2, IgG3, and IgG4 can induce ADCP function upon binding to the appropriate Fc receptors present on immune cells.

[0304] In some embodiments, the Fc polypeptides having a TfR-binding site described herein may contain additional modifications that reduce effector function, i.e., reduce effector function upon TfR binding. Having reduced effector function upon TfR binding of an Fc polypeptide dimer is desirable because reticulocytes also have TfR on the cell surface, leading to reduced reticulocyte counts. As described in detail herein, Fc polypeptide dimers 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, exhibit TfR binding without a substantial reduction in reticulocyte counts, yet maintain a level of effector function (e.g., ADCC) when the Fc polypeptide dimer is fused to a therapeutic Fab and binds to the Fab's target antigen. Having effector function when the Fc polypeptide dimer is fused to a therapeutic Fab that binds to the Fab's target antigen is desirable, for example, in cancer therapy (e.g., brain cancer therapy).

[0305] Exemplary Fc polypeptide mutations that modulate effector function include, but are not limited to, substitutions in the CH2 domain, e.g., at positions corresponding to positions 4 and 5 of SEQ ID NO: 99 (positions 234 and 235 according to the EU numbering scheme). In some embodiments, the substitutions in the modified CH2 domain include Ala at positions 4 and 5 of SEQ ID NO: 99. In some embodiments, the substitutions in the modified CH2 domain include Ala at positions 4 and 5 of SEQ ID NO: 99 and Gly at position 99 of SEQ ID NO: 99.

[0306] Additional Fc polypeptide mutations that modulate effector function include, but are not limited to, one or more substitutions at positions 238, 265, 269, 270, 297, 327, and 329 (EU numbering scheme, corresponding to positions 8, 35, 39, 40, 67, 97, and 99, numbered with reference to SEQ ID NO: 99). Exemplary substitutions include (as numbered in the EU numbering scheme): position 329 may have a mutation in which proline is substituted with glycine or arginine or an amino acid residue of sufficient size to disrupt the Fc / Fcγ receptor interface formed between proline 329 of Fc and tryptophan residues Trp 87 and Trp 110 of FcγRIII. Additional exemplary substitutions include S228P, E233P, L235E, N297A, N297D, and P331S. Multiple substitutions may also be present, such as L234A and L235A in the human IgG1 Fc region; L234A, L235A, and P329G in the human IgG1 Fc region; S228P and L235E in the human IgG4 Fc region; L234A and G237A in the human IgG1 Fc region; L234A, L235A, and G237A in the human IgG1 Fc region; V234A and G237A in the human IgG2 Fc region; L235A, G237A, and E318A in the human IgG4 Fc region; and S228P and L236E in the human IgG4 Fc region. In some embodiments, the Fc polypeptide may have one or more amino acid substitutions that modulate ADCC, for example, substitutions at positions 298, 333, and / or 334 in the Fc region according to the EU numbering scheme.

[0307] 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.

[0308] In certain embodiments, an Fc polypeptide having a TfR-binding site may be modified to reduce effector function, i.e., reduce FcγR binding. In some embodiments, an Fc polypeptide having a TfR-binding site may comprise mutations L234A and L235A (EU numbering scheme, corresponding to positions 4 and 5 numbered with reference to SEQ ID NO: 99). In other embodiments, an Fc polypeptide having a TfR-binding site may comprise mutations L234A, L235A, and P329G (EU numbering scheme, corresponding to positions 4, 5, and 99 numbered with reference to SEQ ID NO: 99).

[0309] VI. Measuring Effector Function or FcγR Binding Methods for analyzing binding affinity, binding kinetics, and cross-reactivity between an Fc polypeptide or Fc polypeptide dimer and an FcγR are known in the art. These methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore™ (GE Healthcare, Piscataway, NJ)), equilibrium exclusion binding assays (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), BioLayer interferometry (e.g., Octet® (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to determine binding affinity and / or cross-reactivity. Methods for performing ELISA assays are known in the art. In some embodiments, surface plasmon resonance (SPR) is used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, equilibrium exclusion binding assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, BioLayer interferometry assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity.

[0310] 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, such as peripheral blood mononuclear cells (e.g., natural killer (NK) cells, T cells, and B cells). FcγR-bearing effector cells recognize and bind to the Fc region of antibodies bound to target cells. Thus, antibodies confer specificity to target cell killing. CDC is initiated when C1q, the initiating component of the classical complement pathway, binds to the Fc region of target-bound antibodies. ADCC and CDC activity can be determined in standard in vivo or in vitro assays of cell killing. Methods for determining ADCC and CDC activity are available in the art. In some embodiments, the method comprises: 51 This may involve labeling target cells with a radioactive substance such as Cr or a fluorescent dye such as calcein AM. The labeled cells can be incubated with the antibody and effector cells, and killing of the target cells by ADCC or CDC can be detected by the emission of radioactivity or fluorescence.

[0311] Other assays for measuring ADCC and CDC activity include, for example, lactate dehydrogenase (LDH) release assays. When the cell membrane is somehow compromised or damaged, 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 medium can then be quantified by measuring the amount of released LDH using a colorimetric or fluorometric LDH cytotoxicity assay.

[0312] VII. Additional mutations in the FC region containing the modified CH3 domain polypeptide The Fc polypeptides provided herein (e.g., modified to bind to TfR and initiate transport across the BBB) may also include additional mutations that, for example, increase serum stability or serum half-life, modulate effector function, affect glycosylation, reduce immunogenicity in humans, and / or provide knob and hole heterodimerization of the Fc polypeptide.

[0313] 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).

[0314] The modified Fc polypeptides described herein may also have other mutations introduced outside of the specified set of amino acids, for example, to affect glycosylation, increase serum half-life, or, for the CH3 domain, to provide knob and hole heterodimerization of polypeptides comprising the modified CH3 domain. Generally, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding depression ("hole") at the interface of a second polypeptide, such that the protrusion can be positioned in the depression to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary depression of identical or similar size to the protrusion is created in 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 at positions in the polypeptide that do not have an adverse effect on binding of the modified CH3 domain to TfR.

[0315] In one exemplary embodiment of the knobs and holes approach to dimerization, the position corresponding to position 136 of SEQ ID NO:99 of the first Fc polypeptide subunit to be dimerized has a tryptophan instead of the native threonine, and the second Fc polypeptide subunit of the dimer has a valine instead of the native tyrosine at the position corresponding to position 177 of SEQ ID NO:99. The second subunit of the Fc polypeptide may further comprise a substitution in which the native threonine at the position corresponding to position 136 of SEQ ID NO:99 is substituted with serine, and the native leucine at the position corresponding to position 138 of SEQ ID NO:99 is substituted with alanine.

[0316] The modified Fc polypeptides described herein can also be engineered to include other modifications for heterodimerization, such as electrostatic manipulation of contact residues within the naturally charged CH3-CH3 interface, or hydrophobic patch modifications.

[0317] In some embodiments, modifications to enhance serum half-life may be introduced. 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:99, a Thr at position corresponding to 24 of SEQ ID NO:99, and a Glu at position corresponding to position 26 of SEQ ID NO:99. Alternatively, the modified Fc polypeptides described herein may comprise M198L and N204S substitutions numbered with reference to SEQ ID NO:99. Alternatively, the modified Fc polypeptides described herein may comprise N204S or N204A substitutions numbered with reference to SEQ ID NO:99.

[0318] Exemplary Fc Polypeptides Containing Additional Mutations The modified Fc polypeptides described herein (e.g., any one of clones CH3C.35.23.3, CH3C.35.23.4, CH3C.35.23, and CH3C.35.23.1.1) may include additional mutations, including knob mutations (e.g., T136W numbered with reference to SEQ ID NO:99), hole mutations (e.g., T136S, L138A, and Y177V numbered with reference to SEQ ID NO:99), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) numbered with reference to SEQ ID NO:99), and / or mutations that increase serum stability or serum half-life (e.g., (i) M22Y, S24T, and T26E numbered with reference to SEQ ID NO:99, or (ii) N204S with or without M198L numbered with reference to SEQ ID NO:99).

[0319] In some embodiments, a modified Fc polypeptide described herein (e.g., any one of clones CH3C.35.23.3, CH3C.35.23.4, CH3C.35.23, and CH3C.35.23.1.1) may have a knob mutation (e.g., T136W, numbered with reference to SEQ ID NO: 99) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 177-180. In some embodiments, a modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 177-180 may be modified to have a knob mutation.

[0320] In some embodiments, a modified Fc polypeptide described herein (e.g., any one of clones CH3C.35.23.3, CH3C.35.23.4, CH3C.35.23, and CH3C.35.23.1.1) can have a knob mutation (e.g., T136W numbered with reference to SEQ ID NO:99), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) numbered with reference to SEQ ID NO:99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:177-180. In some embodiments, a modified Fc polypeptide having the sequence of any one of SEQ ID NOs:177-180 can be modified to have a knob mutation and a mutation that modulates effector function.

[0321] In some embodiments, a modified Fc polypeptide described herein (e.g., any one of clones CH3C.35.23.3, CH3C.35.23.4, CH3C.35.23, and CH3C.35.23.1.1) may have a knob mutation (e.g., T136W numbered with reference to SEQ ID NO: 99), a mutation that increases serum stability or serum half-life (e.g., (i) M22Y, S24T, and T26E numbered with reference to SEQ ID NO: 99, or (ii) N204S, with or without M198L numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 177-180. In some embodiments, a modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 177-180 may be modified to have a knob mutation and a mutation that increases serum stability or serum half-life.

[0322] In some embodiments, the modified Fc polypeptides described herein (e.g., any one of clones CH3C.35.23.3, CH3C.35.23.4, CH3C.35.23, and CH3C.35.23.1.1) may have a knob mutation (e.g., T136W numbered with reference to SEQ ID NO: 99), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) numbered with reference to SEQ ID NO: 99), a mutation that increases serum stability or serum half-life (e.g., (i) M22Y, S24T, and T26E numbered with reference to SEQ ID NO: 99, or (ii) N204S with or without M198L numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 177-180. In some embodiments, a modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 177-180 may be modified to have knob mutations, mutations that modulate effector function, and mutations that increase serum stability or serum half-life.

[0323] In some embodiments, a modified Fc polypeptide described herein (e.g., any one of clones CH3C.35.23.3, CH3C.35.23.4, CH3C.35.23, and CH3C.35.23.1.1) may have hole mutations (e.g., T136S, L138A, and Y177V, numbered with reference to SEQ ID NO: 99) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 177-180. In some embodiments, a modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 177-180 may be modified to have hole mutations.

[0324] In some embodiments, a modified Fc polypeptide described herein (e.g., any one of clones CH3C.35.23.3, CH3C.35.23.4, CH3C.35.23, and CH3C.35.23.1.1) can have hole mutations (e.g., T136S, L138A, and Y177V numbered with reference to SEQ ID NO:99), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) numbered with reference to SEQ ID NO:99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:177-180. In some embodiments, a modified Fc polypeptide having the sequence of any one of SEQ ID NOs:177-180 can be modified to have hole mutations and mutations that modulate effector function.

[0325] In some embodiments, a modified Fc polypeptide described herein (e.g., any one of clones CH3C.35.23.3, CH3C.35.23.4, CH3C.35.23, and CH3C.35.23.1.1) may have a whole mutation (e.g., T136S, L138A, and Y177V numbered with reference to SEQ ID NO: 99), a mutation that increases serum stability or serum half-life (e.g., (i) M22Y, S24T, and T26E numbered with reference to SEQ ID NO: 99, or (ii) N204S with or without M198L numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 177-180. In some embodiments, a modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 177-180 may be modified to have a whole mutation and a mutation that increases serum stability or serum half-life.

[0326] In some embodiments, the modified Fc polypeptides described herein (e.g., any one of clones CH3C.35.23.3, CH3C.35.23.4, CH3C.35.23, and CH3C.35.23.1.1) comprise a hole mutation (e.g., T136S, L138A, and Y177V, numbered with reference to SEQ ID NO: 99), a mutation that modulates effector function (e.g., L4A, L5A, and / or L6A, numbered with reference to SEQ ID NO: 99), or a mutation that modulates effector function (e.g., L4A, L5A, and / or L6A, numbered with reference to SEQ ID NO: 99). P99G (e.g., L4A and L5A), mutations that increase serum stability or serum half-life (e.g., (i) M22Y, S24T, and T26E numbered with reference to SEQ ID NO: 99, or (ii) N204S with or without M198L numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 177-180. In some embodiments, a modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 177-180 may be modified to have a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life.

[0327] Clone CH3C.35.23.3 In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T136W, numbered with reference to SEQ ID NO: 99) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 105. In some embodiments, clone CH3C.35.23.3 with the knob mutation has the sequence of SEQ ID NO: 105.

[0328] In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T136W, numbered with reference to SEQ ID NO: 99), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A), numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 106 or 107. In some embodiments, clone CH3C.35.23.3 having a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO: 106 or 107.

[0329] In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T136W, numbered with reference to SEQ ID NO: 99), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E, numbered with reference to SEQ ID NO: 99), at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 108. 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: 108.

[0330] In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T136W, numbered with reference to SEQ ID NO: 99), a mutation that increases serum stability or serum half-life (e.g., N204S, with or without M198L, numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 109. 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: 109.

[0331] In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T136W numbered with reference to SEQ ID NO:99), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) numbered with reference to SEQ ID NO:99), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E numbered with reference to SEQ ID NO:99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:110 or 111. 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:110 or 111.

[0332] In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T136W numbered with reference to SEQ ID NO: 99), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) numbered with reference to SEQ ID NO: 99), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 112 or 113. 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: 112 or 113.

[0333] In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T136S, L138A, and Y177V, numbered with reference to SEQ ID NO: 99) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 114. In some embodiments, clone CH3C.35.23.3 has whole mutations and the sequence of SEQ ID NO: 114.

[0334] In some embodiments, clone CH3C.35.23.3 can have hole mutations (e.g., T136S, L138A, and Y177V numbered with reference to SEQ ID NO:99), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) numbered with reference to SEQ ID NO:99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:115 or 116. In some embodiments, clone CH3C.35.23.3 with hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:115 or 116.

[0335] In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T136S, L138A, and Y177V, numbered with reference to SEQ ID NO:99), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E, numbered with reference to SEQ ID NO:99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 117. 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:117.

[0336] In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T136S, L138A, and Y177V, numbered with reference to SEQ ID NO: 99), mutations that increase serum stability or serum half-life (e.g., N204S, with or without M198L, numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 118. 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: 118.

[0337] In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T136S, L138A, and Y177V numbered with reference to SEQ ID NO:99), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) numbered with reference to SEQ ID NO:99), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E numbered with reference to SEQ ID NO:99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:119 or 120. In some embodiments, clone CH3C.35.23.3 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:119 or 120.

[0338] In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T136S, L138A, and Y177V numbered with reference to SEQ ID NO:99), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) numbered with reference to SEQ ID NO:99), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L numbered with reference to SEQ ID NO:99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:121 or 122. In some embodiments, clone CH3C.35.23.3 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:121 or 122.

[0339] Clone CH3C.35.23.4 In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T136W, numbered with reference to SEQ ID NO: 99) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 123. In some embodiments, clone CH3C.35.23.4 with the knob mutation has the sequence of SEQ ID NO: 123.

[0340] In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T136W, numbered with reference to SEQ ID NO: 99), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A), numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 124 or 125. In some embodiments, clone CH3C.35.23.4 having a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO: 124 or 125.

[0341] In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T136W, numbered with reference to SEQ ID NO: 99), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E, numbered with reference to SEQ ID NO: 99), at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 126. 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: 126.

[0342] In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T136W, numbered with reference to SEQ ID NO: 99), a mutation that increases serum stability or serum half-life (e.g., N204S, with or without M198L, numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 127. 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: 127.

[0343] In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T136W numbered with reference to SEQ ID NO: 99), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) numbered with reference to SEQ ID NO: 99), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 128 or 129. 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: 128 or 129.

[0344] In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T136W numbered with reference to SEQ ID NO: 99), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) numbered with reference to SEQ ID NO: 99), a mutation that increases serum stability or serum half-life (e.g., N204S with or without M198L numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 130 or 131. 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: 130 or 131.

[0345] In some embodiments, clone CH3C.35.23.4 can have hole mutations (e.g., T136S, L138A, and Y177V, numbered with reference to SEQ ID NO: 99) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 132. In some embodiments, clone CH3C.35.23.4 with hole mutations has the sequence of SEQ ID NO: 132.

[0346] In some embodiments, clone CH3C.35.23.4 can have hole mutations (e.g., T136S, L138A, and Y177V, numbered with reference to SEQ ID NO:99), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A), numbered with reference to SEQ ID NO:99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:133 or 134. In some embodiments, clone CH3C.35.23.4 having hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:133 or 134.

[0347] In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T136S, L138A, and Y177V, numbered with reference to SEQ ID NO: 99), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E, numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 135. 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: 135.

[0348] In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T136S, L138A, and Y177V, numbered with reference to SEQ ID NO: 99), mutations that increase serum stability or serum half-life (e.g., N204S, with or without M198L, numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 136. 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: 136.

[0349] In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T136S, L138A, and Y177V numbered with reference to SEQ ID NO:99), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) numbered with reference to SEQ ID NO:99), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E numbered with reference to SEQ ID NO:99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:137 or 138. In some embodiments, clone CH3C.35.23.4 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO:137 or 138.

[0350] In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T136S, L138A, and Y177V numbered with reference to SEQ ID NO:99), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A) numbered with reference to SEQ ID NO:99), mutations that increase serum stability or serum half-life (e.g., N204S with or without M198L numbered with reference to SEQ ID NO:99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 139 or 140. In some embodiments, clone CH3C.35.23.4 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability or serum half-life has the sequence of SEQ ID NO: 139 or 140.

[0351] Clone CH3C.35.23 In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T136W, numbered with reference to SEQ ID NO: 99) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 141. In some embodiments, clone CH3C.35.23 with the knob mutation has the sequence of SEQ ID NO: 141.

[0352] In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T136W, numbered with reference to SEQ ID NO: 99), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A), numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 142 or 143. In some embodiments, clone CH3C.35.23 having a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO: 142 or 143.

[0353] In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T136W, numbered with reference to SEQ ID NO: 99), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E, numbered...

Claims

1. (a) an antibody variable region, or an antigen-binding fragment thereof, capable of binding to human epidermal growth factor receptor 2 (HER2); (b) a modified Fc polypeptide dimer comprising a first Fc polypeptide comprising a modified CH3 domain that binds to the transferrin receptor (TfR); and 1. An Fc polypeptide dimer-antibody variable region fusion protein comprising: the modified CH3 domain comprises: Glu, Leu, or Trp at position 380; Leu, Tyr, Phe, Trp, or Met at position 384; Leu, Thr, His, Pro, Asn, or Phe at position 386; Val, Pro, or Glu at position 387; Trp at position 388; Ala, Val, Gly, Ser, or Thr at position 389; Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, or Thr at position 390; Glu, Asp, Ala, Ser, Leu, Thr, or Pro at position 413; Glu or Ser at position 415; Thr, Asn, Glu, or Asp at position 416, and Phe, Tyr, or His at position 421 wherein said positions are according to EU numbering.

2. 2. The Fc polypeptide dimer-antibody variable region fusion protein of claim 1, wherein the antibody variable region forms part of a Fab domain.

3. The Fc polypeptide dimer-antibody variable region fusion protein of claim 1 or 2, wherein the antibody variable region binds to subdomain IV, II, or I of human HER2.

4. the antibody variable region binds to subdomain IV of human HER2; and (a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 69; (b) a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 70; (c) a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 71; (d) a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 72; (e) a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 73; and (f) The Fc polypeptide dimer-antibody variable region fusion protein of claim 3, comprising a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

74.

5. The Fc polypeptide dimer-antibody variable region fusion protein of claim 4, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO:59 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO:

60.

6. the antibody variable region binds to subdomain II of human HER2; and (a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 75; (b) a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 76; (c) a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77; (d) a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78; (e) a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79; and (f) a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

80. The Fc polypeptide dimer-antibody variable region fusion protein of claim 3, comprising:

7. The Fc polypeptide dimer-antibody variable region fusion protein of claim 6, wherein the antibody variable region comprises two antibody heavy chain variable regions comprising the amino acid sequence of SEQ ID NO: 61 and two antibody light chain variable regions comprising the amino acid sequence of SEQ ID NO:

62.

8. the modified CH3 domain is derived from a human IgG1, IgG2, IgG3, or IgG4 CH3 domain; and the Fc polypeptide dimer-antibody variable region fusion protein binds to the apical domain of the TfR; The Fc polypeptide dimer-antibody variable region fusion protein according to claim 1 or 2.

9. 9. The Fc polypeptide dimer-antibody variable region fusion protein of claim 8, wherein the modified CH3 domain comprises Glu or Trp at position 380, Tyr or Phe at position 384, Thr or Asn at position 386, Glu at position 387, Trp at position 388, Ser, Ala, Thr, or Val at position 389, Asn or Ser at position 390, Ser or Thr at position 413, Glu or Ser at position 415, Glu at position 416, and Phe or Tyr at position 421.

10. The Fc polypeptide dimer-antibody variable region fusion protein of claim 1 or 2, wherein the first Fc polypeptide comprises an amino acid modification that reduces FcγR binding when bound to TfR.

11. 11. The Fc polypeptide dimer-antibody variable region fusion protein of claim 10, wherein said amino acid modifications include Ala at positions 234 and 235 according to EU numbering.

12. 3. The Fc polypeptide dimer-antibody variable region fusion protein of claim 1 or 2, wherein the first Fc polypeptide further comprises knob mutation T366W and the second Fc polypeptide present in the Fc polypeptide dimer comprises hole mutations T366S, L368A, and Y407V, according to EU numbering.

13. 13. The Fc polypeptide dimer-antibody variable region fusion protein of claim 12, wherein the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO:

124.

14. The Fc polypeptide dimer-antibody variable region fusion protein of claim 13, wherein the second Fc polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 67 and 68.

15. The Fc polypeptide dimer-antibody variable region fusion protein of claim 12, wherein the second Fc polypeptide does not comprise a TfR binding site.

16. 16. The Fc polypeptide dimer-antibody variable region fusion protein of claim 15, comprising a first heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 1, 9, 17, and 81.

17. 16. The Fc polypeptide dimer-antibody variable region fusion protein of claim 15, wherein the first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering.

18. 18. The Fc polypeptide dimer-antibody variable region fusion protein of claim 17, comprising a first heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 2, 10, 18, and 82.

19. (a) the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO:27, and two light chains comprising the amino acid sequences of SEQ ID NO:57; (b) the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 29, 37, 45, and 89; (c) the first Fc polypeptide further comprises amino acid modifications L234A and L235A, and comprises an amino acid sequence selected from SEQ ID NOs: 30, 38, 46, and 90; or (d) the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 55, and two light chains comprising the amino acid sequence of SEQ ID NO:

58. The Fc polypeptide dimer-antibody variable region fusion protein of claim 15.

20. the modified Fc polypeptide dimer does not substantially reduce reticulocytes; or the amount of reticulocytes reduced after administration of the Fc polypeptide dimer-antibody variable region fusion protein is less than the amount of reticulocytes reduced after administration of a control; the control is a corresponding TfR-binding polypeptide dimer-antibody variable region fusion protein that has full effector function and / or does not contain a mutation that reduces FcγR binding; The Fc polypeptide dimer-antibody variable region fusion protein according to claim 1 or 2.

21. (a) an anti-human HER2 antibody heavy chain variable region, or an antigen-binding fragment thereof, which is used in combination with an anti-human HER2 antibody light chain variable region; (b) a modified Fc polypeptide comprising a modified CH3 domain that binds to the transferrin receptor (TfR); and An antibody heavy chain comprising: the modified CH3 domain comprises: Glu, Leu, or Trp at position 380; Leu, Tyr, Phe, Trp, or Met at position 384; Leu, Thr, His, Pro, Asn, or Phe at position 386; Val, Pro, or Glu at position 387; Trp at position 388; Ala, Val, Gly, Ser, or Thr at position 389; Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, or Thr at position 390; Glu, Asp, Ala, Ser, Leu, Thr, or Pro at position 413; Glu or Ser at position 415; Thr, Asn, Glu, or Asp at position 416, and Phe, Tyr, or His at position 421 wherein said positions are according to EU numbering.

22. 22. The antibody heavy chain of claim 21, wherein the modified Fc polypeptide comprises one or more amino acid modifications that reduce FcγR binding when bound to TfR.

23. the antibody heavy chain variable region (a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 69; (b) a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 70; and (c) a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

71. Including, the antibody heavy chain (d) a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 72; (e) a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 73; and (f) a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

74. and for use in combination with a light chain variable region comprising 23. An antibody heavy chain according to claim 21 or 22.

24. The antibody heavy chain of claim 21 or 22, wherein the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

59.

25. the antibody variable region (a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 75; (b) a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 76; and (c) a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

77. Including, the antibody heavy chain (d) a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78; (e) a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79; and (f) a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

80. and for use in combination with a light chain variable region comprising 23. An antibody heavy chain according to claim 21 or 22.

26. The antibody heavy chain of claim 21 or 22, wherein the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

61.

27. 23. The antibody heavy chain of claim 21 or 22, wherein the modified CH3 domain is derived from a human IgG1, IgG2, IgG3, or IgG4 CH3 domain.

28. 28. The antibody heavy chain of claim 27, wherein the modified CH3 domain comprises Glu or Trp at position 380, Tyr or Phe at position 384, Thr or Asn at position 386, Glu at position 387, Trp at position 388, Ser, Ala, Thr, or Val at position 389, Asn or Ser at position 390, Ser or Thr at position 413, Glu or Ser at position 415, Glu at position 416, and Phe or Tyr at position 421.

29. 23. The antibody heavy chain of claim 22, wherein the amino acid modifications that reduce FcγR binding when bound to TfR comprise Ala at position 234 and at position 235 according to EU numbering.

30. 23. The antibody heavy chain of claim 21 or 22, wherein the modified Fc polypeptide further comprises the knob mutation T366W according to EU numbering.

31. 31. The antibody heavy chain of claim 30, wherein the modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO:

124.

32. 31. The antibody heavy chain of claim 30, comprising an amino acid sequence selected from SEQ ID NOs: 1, 9, 17, 81, 29, 37, 45, and 89.

33. 33. The antibody heavy chain of claim 32, comprising an amino acid sequence selected from SEQ ID NOs: 2, 10, 18, 82, 30, 38, 46, and 90.

34. A pharmaceutical composition comprising the Fc polypeptide dimer-antibody variable region fusion protein of any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

35. 21. A pharmaceutical composition for use in a method for transcytosis across an endothelium of an antibody variable region capable of binding to human HER2, or an antigen-binding fragment thereof, comprising an Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 1 to 20, wherein the method comprises contacting the endothelium with the pharmaceutical composition.

36. 36. The pharmaceutical composition of claim 35, wherein the endothelium is the blood-brain barrier.

37. A pharmaceutical composition for treating cancer in a subject, comprising the Fc polypeptide dimer-antibody variable region fusion protein of any one of claims 1 to 20.

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