Anti-HER2 polypeptides and methods of use thereof

Abstract

This provides a novel therapeutic agent that can target HER2 in the brain parenchyma, transcending the blood-brain barrier (BBB). [Solution] In some embodiments, anti-HER2 constructs (e.g., Fc polypeptide dimer-antibody variable region fusion proteins) retain effector function upon binding to HER2 but do not substantially reduce reticulocytes in vivo. The disclosure also relates to methods for transcytosis of anti-HER2 antibody variable regions across the blood-brain barrier to treat HER2-positive cancers and their metastatic lesions.

Description

[Background technology]

[0001] background The treatment of brain metastases from cancers such as breast cancer currently presents a significant clinical challenge. Brain metastases occur in approximately 50% of breast cancer patients. Clinical data indicate that HER2-positive breast cancer tends to metastasize to the brain. In particular, anti-HER2 therapy has proven effective 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 drugs to cross the blood-brain barrier (BBB) ​​and access the brain parenchyma. Therefore, there is a need for new therapies that can cross the BBB and target HER2 within the brain parenchyma. [Overview of the project]

[0002] overview In some embodiments, provided herein are Fc polypeptide dimer-antibody variable region fusion proteins comprising (a) an antibody variable region or an antigen-binding fragment thereof capable of binding to human epidermal growth factor receptor 2 (HER2), and (b) a modified Fc polypeptide dimer comprising a first Fc polypeptide comprising amino acid modifications (may include more than one) 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 may 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 fused to the CH1 domain (from N-terminus to C-terminus), then to the hinge region, then to the first Fc polypeptide, and / or the second heavy chain variable region can fused to the CH1 domain (from N-terminus to C-terminus), then to the hinge region, then to the second Fc polypeptide.

[0004] In some embodiments, the antibody variable region is: (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 CDR2 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 system includes one or more complementarity-determining regions (CDRs) selected from the group consisting of (d) a heavy chain CDR3 having up to two amino acid substitutions in a column, (e) a light chain CDR1 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 72 or having up to two amino acid substitutions in the amino acid sequence of SEQ ID NO: 72, (e) a light chain CDR2 having up to two amino acid substitutions in the amino acid sequence of SEQ ID NO: 73, and (f) a light chain CDR3 having up to two amino acid substitutions in the amino acid sequence of SEQ ID NO: 74.

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

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

[0007] In some embodiments, the antibody variable region includes one or more CDRs (e.g., all six) 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.

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

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

[0010] In some embodiments, the antibody variable region is: (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 CDR2 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 system includes one or more complementarity-determining regions (CDRs) selected from the group consisting of (d) a heavy chain CDR3 having up to two amino acid substitutions to the sequence, (e) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 253 or 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 to the amino acid sequence of SEQ ID NO: 254, and (f) a light chain CDR3 having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 255.

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

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

[0013] In some embodiments, the TfR binding site is located within a modified CH3 domain. In some embodiments, the modified CH3 domain is derived from a human IgG1, IgG2, IgG3, or IgG4 CH3 domain. In some embodiments, the modified CH3 domain includes substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 in a 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 has 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 The compound contains Asn, Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr at position 390, 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 includes an amino acid modification that reduces FcγR binding when the Fc polypeptide dimer binds to TfR (e.g., it has limited binding reduction or no binding reduction when it does not bind 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 include an amino acid modification that reduces FcγR binding (e.g., both polypeptides include Ala at positions 234 and 235).

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

[0016] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide includes an amino acid modification that increases the serum half-life. In some embodiments, the amino acid modification that increases the 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.

[0017] In some embodiments, according to EU numbering, the first Fc polypeptide further comprises the knob mutation T366W, and the second Fc polypeptide comprises the whole mutations T366S, L368A, and Y407V. 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 either SEQ ID NO: 67 or 68.

[0018] In other embodiments, provided herein is an Fc polypeptide dimer-antibody variable region fusion protein comprising: (a) an antibody variable region or antigen-binding fragment thereof capable of binding to human HER2; (b) a first Fc polypeptide comprising a modification and knob mutation T366W that creates a TfR binding site according to EU numbering; and (c) a second Fc polypeptide comprising the hole mutations T366S, L368A, and Y407V according to EU numbering, and not comprising a TfR binding site.

[0019] In some embodiments, the antibody variable region is: (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 CDR2 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 system includes one or more complementarity-determining regions (CDRs) selected from the group consisting of (d) a heavy chain CDR3 having up to two amino acid substitutions in a column, (e) a light chain CDR1 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 72 or having up to two amino acid substitutions in the amino acid sequence of SEQ ID NO: 72, (e) a light chain CDR2 having up to two amino acid substitutions in the amino acid sequence of SEQ ID NO: 73, and (f) a light chain CDR3 having up to two amino acid substitutions in the amino acid sequence of SEQ ID NO: 74.

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

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

[0022] In some embodiments, the antibody variable region includes one or more CDRs (e.g., all six) 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.

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

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

[0025] In some embodiments, the antibody variable region is: (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 CDR2 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 system includes one or more complementarity-determining regions (CDRs) selected from the group consisting of (d) a heavy chain CDR3 having up to two amino acid substitutions to the sequence, (e) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 253 or 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 to the amino acid sequence of SEQ ID NO: 254, and (f) a light chain CDR3 having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 255.

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

[0027] In some embodiments, the antibody variable region includes two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 256, and two light chain variable regions containing 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 containing one of the amino acid sequences 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 containing one of the amino acid sequences 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 containing 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 containing the amino acid sequence of SEQ ID NO: 57.

[0029] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 290. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

[0031] In other embodiments, provided herein is an Fc polypeptide dimer-antibody variable region fusion protein comprising: (a) an antibody variable region or antigen-binding fragment thereof capable of binding to human HER2; (b) a first Fc polypeptide comprising an amino acid modification N434S with or without modifications, knob mutations T366W and M428L, according to EU numbering, that create a TfR binding site; and (c) a second Fc polypeptide comprising whole mutations T366S, L368A and Y407V, according to EU numbering, and without a TfR binding site.

[0032] In some embodiments, the antibody variable region is: (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 CDR2 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 system includes one or more complementarity-determining regions (CDRs) selected from the group consisting of (d) a heavy chain CDR3 having up to two amino acid substitutions in a column, (e) a light chain CDR1 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 72 or having up to two amino acid substitutions in the amino acid sequence of SEQ ID NO: 72, (e) a light chain CDR2 having up to two amino acid substitutions in the amino acid sequence of SEQ ID NO: 73, and (f) a light chain CDR3 having up to two amino acid substitutions in the amino acid sequence of SEQ ID NO: 74.

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

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

[0035] In some embodiments, the antibody variable region includes one or more CDRs (e.g., all six) 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.

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

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

[0038] In some embodiments, the antibody variable region is: (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 CDR2 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 system includes one or more complementarity-determining regions (CDRs) selected from the group consisting of (d) a heavy chain CDR3 having up to two amino acid substitutions to the sequence, (e) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 253 or 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 to the amino acid sequence of SEQ ID NO: 254, and (f) a light chain CDR3 having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 255.

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

[0040] In some embodiments, the antibody variable region includes two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 256, and two light chain variable regions containing 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 containing one of the amino acid sequences 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 containing one of the amino acid sequences 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 containing 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 containing the amino acid sequence of SEQ ID NO: 57.

[0042] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing one of the amino acid sequences 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 containing 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 containing the amino acid sequence of SEQ ID NO: 293.

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

[0045] In some embodiments, the antibody variable region is: (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 CDR2 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 system includes one or more complementarity-determining regions (CDRs) selected from the group consisting of (d) a heavy chain CDR3 having up to two amino acid substitutions in a column, (e) a light chain CDR1 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 72 or having up to two amino acid substitutions in the amino acid sequence of SEQ ID NO: 72, (e) a light chain CDR2 having up to two amino acid substitutions in the amino acid sequence of SEQ ID NO: 73, and (f) a light chain CDR3 having up to two amino acid substitutions in the amino acid sequence of SEQ ID NO: 74.

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

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

[0048] In some embodiments, the antibody variable region includes one or more CDRs (e.g., all six) 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.

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

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

[0051] In some embodiments, the antibody variable region is: (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 CDR2 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 system includes one or more complementarity-determining regions (CDRs) selected from the group consisting of (d) a heavy chain CDR3 having up to two amino acid substitutions to the sequence, (e) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 253 or 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 to the amino acid sequence of SEQ ID NO: 254, and (f) a light chain CDR3 having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 255.

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

[0053] In some embodiments, the antibody variable region includes two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 256, and two light chain variable regions containing 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 containing one of the amino acid sequences 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 containing one of the amino acid sequences 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 containing 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 containing the amino acid sequence of SEQ ID NO: 57.

[0055] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 291. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

[0057] In other embodiments, provided herein is an Fc polypeptide dimer-antibody variable region fusion protein comprising: (a) an antibody variable region or antigen-binding fragment thereof capable of binding to human HER2; (b) a first Fc polypeptide comprising an amino acid modification N434S with or without modifications, knob mutations T366W and M428L, according to EU numbering, for creating a TfR binding site; and (c) a second Fc polypeptide comprising an amino acid modification N434S with or without hole mutations T366S, L368A and Y407V and M428L, according to EU numbering, and without a TfR binding site.

[0058] In some embodiments, the antibody variable region is: (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 CDR2 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 system includes one or more complementarity-determining regions (CDRs) selected from the group consisting of (d) a heavy chain CDR3 having up to two amino acid substitutions in a column, (e) a light chain CDR1 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 72 or having up to two amino acid substitutions in the amino acid sequence of SEQ ID NO: 72, (e) a light chain CDR2 having up to two amino acid substitutions in the amino acid sequence of SEQ ID NO: 73, and (f) a light chain CDR3 having up to two amino acid substitutions in the amino acid sequence of SEQ ID NO: 74.

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

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

[0061] In some embodiments, the antibody variable region includes one or more CDRs (e.g., all six) 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.

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

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

[0064] In some embodiments, the antibody variable region is: (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 CDR2 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 system includes one or more complementarity-determining regions (CDRs) selected from the group consisting of (d) a heavy chain CDR3 having up to two amino acid substitutions to the sequence, (e) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 253 or 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 to the amino acid sequence of SEQ ID NO: 254, and (f) a light chain CDR3 having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 255.

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

[0066] In some embodiments, the antibody variable region includes two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 256, and two light chain variable regions containing 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 containing one of the amino acid sequences 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 containing one of the amino acid sequences 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 containing 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 containing the amino acid sequence of SEQ ID NO: 57.

[0068] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing one of the amino acid sequences 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 containing 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 containing 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 reticulocyte reduction after administration of the Fc polypeptide dimer-antibody variable region fusion protein is less than the amount of reticulocyte reduction after administration of the control. In some embodiments, the control is the corresponding TfR-binding polypeptide dimer-antibody variable region fusion protein that has full effector function and / or does not contain mutations that reduce FcγR binding.

[0071] In other embodiments, 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 modifications for creating a TfR binding site. In some embodiments, the modified Fc polypeptide comprises one or more amino acid modifications that reduce FcγR binding when bound to TfR.

[0072] In some embodiments, the antibody heavy chain variable region includes one or more CDRs selected from the group consisting of (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) 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, and (c) 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. In some embodiments, the antibody heavy chain variable region includes one or more CDRs selected from the group consisting of (a) heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 69, (b) heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 70, and (c) heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 71. In some embodiments, the antibody heavy chain variable region includes the amino acid sequence of SEQ ID NO: 59.

[0073] In some embodiments, the antibody variable region includes one or more CDRs selected from the group consisting of (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) 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, and (c) 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. In some embodiments, the antibody variable region includes one or more CDRs selected from the group consisting of (a) heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 75, (b) heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 76, and (c) heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody heavy chain variable region contains the amino acid sequence of SEQ ID NO: 61.

[0074] In some embodiments, the antibody variable region includes one or more CDRs selected from the group consisting of (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) 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, and (c) 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. In some embodiments, the antibody variable region includes one or more CDRs selected from the group consisting of (a) heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 250, (b) heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 251, and (c) heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 252. In some embodiments, the antibody heavy chain variable region contains the amino acid sequence of SEQ ID NO: 256.

[0075] In some embodiments, the TfR binding site is located within a modified CH3 domain. In some embodiments, the modified CH3 domain is derived from a human IgG1, IgG2, IgG3, or IgG4 CH3 domain. In some embodiments, the modified CH3 domain includes substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 in a 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 has 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 The compound contains Asn, Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr at position 390, 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 when bound to TfR includes Ala at positions 234 and 235, according to EU numbering. In some embodiments, the modified Fc polypeptide further includes an amino acid modification that increases serum half-life. In some embodiments, the amino acid modification that increases serum half-life includes (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 includes the knob mutation T366W, according to EU numbering. In some embodiments, the modified Fc polypeptide includes the amino acid sequence of SEQ ID NO: 63.

[0077] In other embodiments, 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 a modification and knob mutation T366W that creates a TfR binding site according to EU numbering.

[0078] In some embodiments, the antibody heavy chain variable region includes one or more CDRs selected from the group consisting of (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) 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, and (c) 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. In some embodiments, the antibody heavy chain variable region includes one or more CDRs selected from the group consisting of (a) heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 69, (b) heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 70, and (c) heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 71. In some embodiments, the antibody heavy chain variable region includes the amino acid sequence of SEQ ID NO: 59.

[0079] In some embodiments, the antibody variable region includes one or more CDRs selected from the group consisting of (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) 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, and (c) 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. In some embodiments, the antibody variable region includes one or more CDRs selected from the group consisting of (a) heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 75, (b) heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 76, and (c) heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody heavy chain variable region contains the amino acid sequence of SEQ ID NO: 61.

[0080] In some embodiments, the antibody variable region includes one or more CDRs selected from the group consisting of (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) 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, and (c) 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. In some embodiments, the antibody variable region includes one or more CDRs selected from the group consisting of (a) heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 250, (b) heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 251, and (c) heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 252. In some embodiments, the antibody heavy chain variable region contains the amino acid sequence of SEQ ID NO: 256.

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

[0082] In other embodiments, 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, with or without an amino acid modification N434S that creates a TfR binding site according to EU numbering, a knob mutation T366W, and M428L.

[0083] In some embodiments, the antibody heavy chain variable region includes one or more CDRs selected from the group consisting of (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) 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, and (c) 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. In some embodiments, the antibody heavy chain variable region includes one or more CDRs selected from the group consisting of (a) heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 69, (b) heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 70, and (c) heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 71. In some embodiments, the antibody heavy chain variable region includes the amino acid sequence of SEQ ID NO: 59.

[0084] In some embodiments, the antibody variable region includes one or more CDRs selected from the group consisting of (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) 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, and (c) 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. In some embodiments, the antibody variable region includes one or more CDRs selected from the group consisting of (a) heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 75, (b) heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 76, and (c) heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody heavy chain variable region contains the amino acid sequence of SEQ ID NO: 61.

[0085] In some embodiments, the antibody variable region includes one or more CDRs selected from the group consisting of (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) 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, and (c) 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. In some embodiments, the antibody variable region includes one or more CDRs selected from the group consisting of (a) heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 250, (b) heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 251, and (c) heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 252. In some embodiments, the antibody heavy chain variable region contains the amino acid sequence of SEQ ID NO: 256.

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

[0087] In other embodiments, provided herein are pharmaceutical compositions comprising an Fc polypeptide dimer-antibody variable region fusion protein as described herein and a pharmaceutically acceptable carrier.

[0088] In other embodiments, provided herein are methods for transcytosis across the endothelium of an antibody variable region or its antigen-binding fragment 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 as described herein. In some embodiments, the endothelium is the blood-brain barrier (BBB).

[0089] In other embodiments, 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 the Fc polypeptide dimer-antibody variable region fusion protein described herein. In some embodiments, the cancer is HER2-positive cancer. 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. The HER2-positive cancer is metastatic cancer.

[0090] In other embodiments, provided herein are methods for treating brain metastases of HER2-positive cancer in a subject, the methods comprising administering to the subject a therapeutically effective amount of a composition comprising the 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, different combinations of Fc polypeptide dimer-antibody variable region fusion proteins (e.g., combinations of Fc polypeptide dimer-antibody variable region fusion proteins that bind to HER2 subdomains IV and II) 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 containing the amino acid sequence of SEQ ID NO: 59 and two light chain variable regions containing the amino acid sequence of SEQ ID NO: 60 (i.e., 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 containing the amino acid sequence of SEQ ID NO: 61 and two light chain variable regions containing the amino acid sequence of SEQ ID NO: 62 (i.e., anti-HER2 subdomain II Fc polypeptide dimer-antibody variable region fusion protein). In some embodiments, the anti-HER2 subdomain IV Fc polypeptide dimer-antibody variable region fusion protein may be administered alone or in combination with the anti-HER2 subdomain II Fc polypeptide dimer-antibody variable region fusion protein. In some embodiments, the anti-HER2 subdomain II Fc polypeptide dimer-antibody variable region fusion protein may be administered alone or in combination with the anti-HER2 subdomain IV Fc polypeptide dimer-antibody variable region fusion protein. In certain embodiments, the anti-HER2 subdomain IV Fc polypeptide dimer-antibody variable region fusion protein may be administered alone. In certain embodiments, the anti-HER2 subdomain II Fc polypeptide dimer-antibody variable region fusion protein may be administered alone.

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

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

[0094] In some embodiments, compositions comprising an Fc polypeptide dimer-antibody variable region fusion protein antagonistize HER2 activity. In 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 in a subject or for treating brain metastases of cancer, the method comprising administering to a subject a therapeutically effective amount of an anti-HER2 construct bound to (a) subdomain I or II of human HER2 and (b) the transferrin receptor (TfR), the anti-HER2 construct being therapeutically effective on its own for treating cancer.

[0096] In some embodiments of this model, the anti-HER2 construct includes an antibody variable region that binds to subdomain I or II of human HER2. The anti-HER2 construct may include a modified Fc polypeptide dimer comprising a first Fc polypeptide that includes modifications to create 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 model, the anti-HER2 construct includes 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 monotherapy. In some embodiments, the anti-HER2 construct is administered in combination with chemotherapy or radiotherapy.

[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 in a subject or for treating brain metastases of cancer, wherein the method comprises 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. This includes administering the drug to the target population. The first and / or second anti-HER2 construct also binds to TfR.

[0101] In some embodiments of this model, the first anti-HER2 construct, rather than the second anti-HER2 construct, binds to TfR. The first anti-HER2 construct can specifically bind to both TfR and HER2 on the same cell.

[0102] In some embodiments, the second anti-HER2 construct, rather than the first anti-HER2 construct, binds to TfR. The second anti-HER2 construct can specifically bind to both TfR and HER2 on the same cell.

[0103] In some embodiments of this model, the first and / or second 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 with modifications to create 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, each of which comprises heavy chain CDR1(CDR-H1), CDR H2, and CDR H3, and each of which comprises light chain CDR1(CDR-L1), CDR L2, and CDR L3. (1) CDR-H1 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 75, or contains a sequence with up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 75. (2) CDR-H2 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 76, or contains a sequence having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 76. (3) CDR-H3 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 77, or contains a sequence having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 77. (4) CDR-L1 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 78, or includes a sequence having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 78. (5) CDR-L2 contains a sequence having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 79. (6) CDR-L3 contains 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 the Fc polypeptide dimer-antibody variable region fusion protein that binds to subdomain II of HER2, each of the two heavy chain variable regions contains a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 61, and each of the two light chain variable regions contains a sequence having at least 90% sequence identity with 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 bound to subdomain I of HER2, each of the two heavy chain variable regions comprising heavy chain CDR1(CDR-H1), CDR H2, and CDR H3, and each of the two light chain variable regions comprising light chain CDR1(CDR-L1), CDR L2, and CDR L3. (1) CDR-H1 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 250, or contains a sequence having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 250. (2) CDR-H2 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 251, or includes a sequence having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 251. (3) CDR-H3 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 252, or includes a sequence having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 252. (4) CDR-L1 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 253, or includes a sequence having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 253. (5) CDR-L2 contains a sequence having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 254. (6) CDR-L3 contains 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 the Fc polypeptide dimer-antibody variable region fusion protein that binds to subdomain I of HER2, each of the two heavy chain variable regions contains 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 contains 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 includes heavy chain CDR1 (CDR-H1), CDR H2, and CDR H3, and each of the two light chain variable regions includes light chain CDR1 (CDR-L1), CDR L2, and CDR L3. (1) CDR-H1 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 69, or contains a sequence with up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 69. (2) CDR-H2 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 70, or contains a sequence having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 70. (3) CDR-H3 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 71, or contains a sequence having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 71. (4) CDR-L1 has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 72, or includes a sequence having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 72. (5) CDR-L2 contains a sequence having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 73. (6) CDR-L3 contains 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 contains 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 contains a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 60.

[0110] In some embodiments of this model, the TfR binding site in the Fc polypeptide of the Fc polypeptide dimer-antibody variable region fusion protein includes a modified CH3 domain. The modified CH3 domain may be derived from the human IgG1, IgG2, IgG3, or IgG4 CH3 domain. The modified CH3 domain may include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 in a 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 has 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 The compound contains Asn, Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr at position 390, 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 present 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 when bound 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 present disclosure, according to EU numbering, the first Fc polypeptide further comprises the knob mutation T366W, and the second Fc polypeptide in the Fc polypeptide dimer comprises the whole mutations T366S, L368A, and Y407V. 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 either SEQ ID NO: 67 or 68.

[0114] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein that binds to the subdomain II of human HER2 is (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, and 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 is (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) First heavy chain having the sequence of SEQ ID NO: 259, 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 the subdomain IV of human HER2 is (a) A first heavy chain having the sequence of sequence number 10, a second heavy chain having the sequence of sequence number 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) First heavy chain having the sequence of SEQ ID NO: 2, second heavy chain having the sequence of SEQ ID NO: 27 Includes.

[0117] In other embodiments of this model, the first and / or second anti-HER2 constructs include antibody-variable regions that bind to TfR. For example, the first anti-HER2 construct may 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 may 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 may 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 present 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 embodiment, the Disclosure features a method for reducing the level of TfR expression on the surface of cells by contacting cells with an anti-HER2 construct that binds to (a) subdomain I, II, or IV of human HER2 and (b) the transferrin receptor (TfR), wherein the anti-HER2 construct is effective alone in reducing the level of TfR expression on the surface of cells, and the anti-HER2 construct binds to both TfR and HER2 on the same cell. In this embodiment, the anti-HER2 construct may be any of the constructs described herein, such as an Fc polypeptide dimer-antibody variable region fusion protein (containing human HER2 comprising a first Fc polypeptide with modifications to create a TfR binding site and an antibody variable region capable of binding to the modified Fc polypeptide dimer) or an anti-HER2 bispecific construct (containing an antibody variable region capable of binding to human HER2 and an antibody variable region that binds to TfR).

[0120] In yet another embodiment, the Disclosure features a method for conjugating a construct to 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 to (a) subdomain I, II, or IV of human HER2 and (b) the transferrin receptor (TfR), wherein the anti-HER2 construct reduces the level of TfR expression on the cell surface of the cell when the cell is in contact with the anti-HER2 construct. In this embodiment, the construct that binds to TfR and human HER2 may be any of the constructs described herein, such as an Fc polypeptide dimer-antibody variable region fusion protein (containing human HER2 with a first Fc polypeptide that includes a modification to create a TfR binding site and an antibody variable region that can bind to the modified Fc polypeptide dimer) or an anti-HER2 bispecific construct (containing an antibody variable region that can bind to human HER2 and an antibody variable region that binds to TfR).

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

[0122] In some embodiments, the anti-HER2 construct includes an antibody variable region that binds to human HER2 subdomains I, II, or IV. In certain embodiments, the anti-HER2 construct may include an antibody variable region that binds to human HER2 subdomains I, II, or IV and a modified Fc polypeptide dimer that includes a first Fc polypeptide modified to create a TfR binding site. For example, the anti-HER2 construct may be an Fc polypeptide dimer-antibody variable region fusion protein. In yet another embodiment, the anti-HER2 construct may be a bispecific construct that includes an antibody variable region that binds to human HER2 and an antibody variable region that binds to TfR.

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

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

Brief Description of the Drawings

[0125] [Figure 1A] Shows Biacore (trademark) data. Binding affinities 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 are shown. [Figure 1B] Shows Biacore (trademark) data. Binding affinities to human apical hTfR using HER2_DIV-35.23.1.1cisLALA and HER2_DII-35.23.1.1cisLALA are shown. [Figure 2] Growth inhibition assay of BT474 cells using the WST1 reagent, showing inhibition of cancer cell growth on day 6 by anti-HER2_DIV, HER2_DIV-35.23.1.1cisLALA, the combination of anti-HER2_DIV and anti-HER2_DII, and the combination of HER2_DIV-35.23.1.1cisLALA and HER2_DII-35.23.1.1cisLALA. [Figure 3] 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] Growth inhibition assay of BT474 cells using the WST1 reagent, showing the response of cancer cell growth on day 6 by anti-HER2_DIV, HER2_DIV-35.23.1.1cisLALA, the combination of anti-HER2_DIV and anti-HER2_DII, and the combination of HER2_DIV-35.23.1.1cisLALA and HER2_DII-35.23.1.1cisLALA in the presence of neuregulin-1. [Figure 5]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, anti-HER2_DIV and anti-HER2_DII combinations, and HER2_DIV-35.23.1.1cisLALA and HER2_DII-35.23.1.1cisLALA combinations in the presence of Neureglin-1 are shown. [Figure 6] This shows ADCC activity in SK-BR-3 cells using HER2_DIV-35.23.1.1cisLALA. [Figure 7A] This study demonstrates the in vivo antitumor efficacy of ATV:HER2-DIV and ATV:HER2-DII in a BT474 xenograft tumor model in SCID mice. Compared to anti-HER2-DIV + anti-HER2-DII, animals treated with ATV:HER2-DIV + ATV:HER2-DII showed significantly higher inhibition of tumor growth. [Figure 7B] This study demonstrates the in vivo antitumor efficacy of ATV:HER2-DIV and ATV:HER2-DII in a BT474 xenograft tumor model in SCID mice. It also shows dose-response relationships using 3, 10, and 20 mg / kg of each test product, indicating that ATV:HER2-DIV + ATV:HER2-DII is more potent than anti-HER2-DIV + anti-HER2-DII. [Figure 7C] This study demonstrates 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 the mechanism by which HER2 targeting can suppress the PI3K / Akt signaling pathway activated in HER2+ tumors. "ATV" refers to TfR-binding Fc polypeptide. [Figure 8A] This shows plasma exposure, brain uptake, and brain-to-plasma ratio in TfRmu / hu KI mice treated with ATV:HER2-DIV and ATV:HER2-DII. [Figure 8B] This shows plasma exposure, brain uptake, and brain-to-plasma ratio in TfRmu / hu KI mice treated with ATV:HER2-DIV and ATV:HER2-DII. [Figure 8C] This shows plasma exposure, brain uptake, and brain-to-plasma ratio in TfRmu / hu KI mice treated with ATV:HER2-DIV and ATV:HER2-DII. [Figure 9A] We demonstrate growth inhibition assays of BT474 and OE19 cells using ATV:HER2-DIV, showing that ATV:HER2-DIV increased the antiproliferative effect compared to anti-HER2-DIV in anti-HER2-DIV resistant HER2+ cancer cell lines. [Figure 9B] We demonstrate growth inhibition assays of BT474 and OE19 cells using ATV:HER2-DIV, showing that ATV:HER2-DIV increased the antiproliferative effect compared to anti-HER2-DIV in anti-HER2-DIV resistant HER2+ cancer cell lines. [Figure 10A] This study demonstrates growth inhibition assays of BT474 and OE19 cells using ATV:HER2-DII, showing that ATV:HER2-DII exhibits superior growth inhibition compared to anti-HER2-DII, while ATV:ctrl and anti-TfR, with or without the anti-HER2-DII group, have the least effect in HER2+ cancer cell lines. [Figure 10B] This study demonstrates growth inhibition assays of BT474 and OE19 cells using ATV:HER2-DII, showing that ATV:HER2-DII exhibits superior growth inhibition compared to anti-HER2-DII, while ATV:ctrl and anti-TfR, with or without the anti-HER2-DII group, have the least effect in HER2+ cancer cell lines. [Figure 11] This study demonstrates a growth inhibition assay of OE19 cells using ATV:HER2-DI, showing that ATV:HER2-DI exhibits superior growth inhibition compared to anti-HER2-DI in HER2+ cancer cell lines. [Figure 12]We present a growth inhibition assay using ATV:HER2-DIV + ATV:HER2-DII in BT474 cells lacking NRG1, demonstrating that the ATV:HER2-DIV + ATV:HER2-DII combination is more potent in inhibiting growth than anti-HER2-DIV + anti-HER2-DII combinations with or without NRG1. [Figure 13A] This 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 the downregulation of cell surface TfR expression under internalization conditions (30 minutes at 37°C). [Figure 13B] This 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 the downregulation of cell surface TfR expression under internalization conditions (30 minutes at 37°C). [Figure 13C] This 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 the downregulation of cell surface TfR expression under internalization conditions (30 minutes at 37°C). [Modes for carrying out the invention]

[0126] Detailed explanation I. Introduction We have developed anti-HER2 constructs that can cross the blood-brain barrier (BBB). Generally, these anti-HER2 constructs include an antibody variable region (e.g., subdomain I, II, or IV of human HER2) that can bind to human HER2. In some embodiments, the anti-HER2 construct includes an antibody variable region that can bind to HER2 fused to a modified Fc polypeptide that has been engineered to include a non-native TfR binding site, and is also referred to herein as an "Fc polypeptide dimer-antibody variable region fusion protein." For example, an Fc polypeptide dimer-antibody variable region fusion protein may include an anti-HER2 Fab fused to a modified Fc polypeptide dimer that includes a TfR binding site.

[0127] In other embodiments, the anti-HER2 construct includes 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] Accordingly, this disclosure relates in part to Fc polypeptide dimer-antibody variable region fusion proteins and fragments thereof that bind to HER2 subdomain IV, subdomain II, or subdomain I, bind to TfR, and have reduced effector function (e.g., ADCC or CDC) upon binding to TfR, but still retain and exhibit levels of effector function (e.g., ADCC or CDC) when the Fc polypeptide dimer-antibody variable region fusion protein binds to HER2. This disclosure also relates in part to methods for delivering anti-HER2 therapeutic constructs across the BBB to treat HER2-positive cancer, as well as metastases of HER2-positive cancer. Surprisingly, as described herein, the use of combinations of Fc polypeptide dimer-antibody variable region fusion proteins targeting HER2 subdomains IV and II has been found to be more effective in inhibiting breast cancer cell growth than the use of combinations of anti-HER2 subdomain IV and anti-HER2 subdomain II antibodies. Furthermore, in some embodiments, the use of a combination of Fc polypeptide dimer-antibody variable region fusion proteins targeting HER2 subdomains IV and I is more effective in inhibiting breast cancer cell growth than the use of a combination of anti-HER2 subdomain IV and anti-HER2 subdomain I antibodies. Furthermore, in some embodiments, the use of a combination of Fc polypeptide dimer-antibody variable region fusion proteins targeting HER2 subdomains II and I is more effective in inhibiting breast cancer cell growth than the use of a combination of anti-HER2 subdomain II and anti-HER2 subdomain I antibodies.

[0129] Furthermore, using Fc polypeptide dimer-antibody variable region fusion proteins targeting HER2 subdomain IV, subdomain II, or subdomain I individually is more effective in inhibiting breast cancer cell growth than using anti-HER2 subdomain IV antibodies, anti-HER2 subdomain II antibodies, or anti-HER2 subdomain I antibodies, respectively.

[0130] In other embodiments, the disclosure also relates in part to the use of an anti-HER2 construct comprising an antibody variable region capable of binding to HER2 (e.g., subdomains I, II, or IV of human HER2) and an antibody variable region capable of binding to TfR in order to inhibit breast cancer cell growth. Using such an anti-HER2 construct having bispecificity to HER2 (e.g., subdomains 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, unless the content clearly indicates otherwise, the singular forms "a," "an," and "the" refer to multiple objects. Therefore, for example, a reference to "polypeptide" may include two or more such molecules.

[0132] As used herein, the terms “about” and “approximately” mean, when used to modify a quantity specified by a number or range, that a reasonable deviation from the number, as well as from a value known to those skilled in the art, such as ±20%, ±10%, or ±5%, falls within the intended meaning of the enumerated 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, which is a member of the human epidermal growth factor receptor (HER / EGFR / ERBB) family. Amplification or overexpression of HER2 plays a significant role in the development and progression of certain aggressive types of cancer, including breast cancer. Non-exclusive examples of human HER2 nucleotide sequences are defined in GenBank reference numbers NP_001005862, NP_001289936, NP_001289937, NP_001289938, and NP_004448. Non-restrictive examples of human HER2 peptide sequences are defined in GenBank reference numbers NP_001005862, NP_001276865, NP_001276866, NP_001276867, and NP_004439.

[0134] The extracellular domain of HER2, containing approximately 600 amino acids, includes 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 cells, the cells are referred to as "HER2-positive" or "HER2+". The level of HER2 amplification or overexpression in HER2-positive cells 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 a greater degree of expression.

[0136] HER2 testing methods include immunohistochemistry (IHC), fluorescence insight hybridization (FISH), ELISA, and RNA quantification (e.g., of HER2 expression), such as RT-PCR and microarray analysis. HER2 testing may 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) subdomains I, II, or IV of human HER2 and (b) the transferrin receptor (TfR). An anti-HER2 construct may include 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 with modifications to create 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. Anti-HER2 constructs described herein can bind to subdomains 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 human HER2 subdomains I, II, or IV, 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 containing at least a CH2 domain and / or a CH3 domain, and may include at least a portion of a hinge region. Generally, Fc polypeptides do not contain a variable region.

[0140] A "modified Fc polypeptide" refers to an Fc polypeptide that has at least one mutation, e.g., substitution, deletion, or insertion, compared to a wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retains the overall Ig fold or structure of the 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 an interaction between two CH3 antibody constant domains. In some embodiments, two Fc polypeptides may also dimerize via one or more disulfide bonds formed between the hinge domains of two dimerized Fc domain monomers. An Fc polypeptide dimer may be a wild-type Fc polypeptide dimer or a modified Fc polypeptide dimer. A wild-type Fc polypeptide dimer is formed by the dimerization of two wild-type Fc polypeptides. An Fc polypeptide dimer may 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, a modified Fc polypeptide dimer comprises two modified Fc polypeptides. A modified Fc polypeptide dimer may be a homodimer (i.e., comprising two identical modified Fc polypeptides) or a heterodimer (i.e., comprising two different Fc polypeptides, with at least one of the two Fc polypeptides being 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 defined in Sequence ID No. 102. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee, accession number XP_003310238.1; rhesus macaque, NP_001244232.1; dog, NP_001003111.1; cattle, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP_990587.1). The term “transferrin receptor” also includes exemplary reference sequences, such as allele variants of the human sequence encoded by the gene at the transferrin receptor protein 1 locus. The full-length TfR protein includes a short N-terminal intracellular domain, a transmembrane domain, 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 defined as sequence number 103.

[0144] As used herein, the terms “Fcγ receptor” or “FcγR” refer to a single type of Fc receptor, classified based on the type of antibody they recognize. FcγRs include several members such as FcγRI(CD64), FcγRIIA(CD32), FcγRIIB(CD32), FcγRIIIA(CD16a), and FcγRIIIB(CD16b), which differ in their antibody affinity due to their different molecular structures. FcγRs are important for binding to the Fc portion of IgG class antibodies and inducing phagocytosis of opsonized microorganisms. FcγRs are found on the cell surface of immune system cells. FcγRs are involved in inducing effector functions of the immune system 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 “reducing FcγR binding” refers to a modified Fc polypeptide or modified Fc polypeptide dimer containing a mutation in the CH3 domain of the modified Fc polypeptide, the mutation reducing the affinity of the modified Fc polypeptide to 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 that does not contain the mutation reducing FcγR binding (e.g., a wild-type Fc polypeptide dimer). FcγR binding can be measured, for example, using a surface plasmon resonance (SPR) method (e.g., the Biacore® system). Alternatively, FcγR binding can be measured using functional assays, such as ADCC assays (e.g., in vivo or in vitro assays of cell death) as described herein. The 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 a limited reduction (e.g., a reduction of less than 25%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1%) or no reduction when not bound to TfR.

[0146] Where further described herein, a modified Fc polypeptide dimer may comprise a first Fc polypeptide having both a TfR binding site and a mutation that reduces FcγR binding upon TfR binding, 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 may have an overall reduced affinity for FcγR. On the other hand, when not bound to TfR, there may be a limited reduction in FcγR binding (e.g., as described above) or no reduction at all.

[0147] The term "FcRn" refers to the fetal Fc receptor. Binding of Fc polypeptides to FcRn reduces the clearance of Fc polypeptides and increases their 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 a region of an Fc polypeptide that binds to FcRn. In human IgG, when numbered using the EU numbering scheme, FcRn binding sites include 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, “natural FcRn binding site” refers to a region of an Fc polypeptide that binds to FcRn and has the same amino acid sequence as the region of a naturally occurring Fc polypeptide that binds to FcRn.

[0150] As used herein, the terms “substantially no reduction in reticulocytes” or “substantially no reduction in reticulocytes in vivo” mean that the reduction in reticulocytes (e.g., reduction of bone marrow reticulocytes or circulating reticulocytes) caused by the effector-function-positive TfR-binding Fc polypeptide dimer or the Fc polypeptide dimer-antibody variable region fusion protein described herein, which includes the effector-function-positive TfR-binding Fc polypeptide dimer, is compared to a control, e.g., a control having full effector function and / or reducing FcγR binding. This means that the reduction in reticulocytes (e.g., bone marrow reticulocytes or circulating reticulocytes) caused by an antibody containing a corresponding TfR-binding Fc dimer or Fc polypeptide dimer-antibody variable region fusion protein that does not contain mutations that reduce FcγR binding, or an antibody that has full effector function and / or does not contain mutations that reduce FcγR binding, is less than (e.g., less than 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1%).

[0151] The terms "substantially no reduction in reticulocytes" or "substantially no reduction in reticulocytes in vivo" mean that the amount or percentage of remaining reticulocytes (e.g., remaining reticulocytes in bone marrow or circulating reticulocytes) after administration of an effector-function-positive TfR-binding Fc polypeptide dimer or an Fc polypeptide dimer-antibody variable region fusion protein described herein, including an effector-function-positive TfR-binding Fc polypeptide dimer, is greater than that of a control (e.g., one with full effector function and / or FcγR binding). This can also mean exceeding the amount or percentage of remaining reticulocytes (e.g., remaining reticulocytes in the bone marrow or circulation) after administration of a corresponding TfR-binding Fc dimer or Fc polypeptide dimer-antibody variable region fusion protein that does not contain mutations that reduce FcγR binding, or an antibody that has full effector function and / or contains a corresponding TfR-binding Fc dimer that does not contain mutations that reduce FcγR binding (e.g., exceeding at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the total).

[0152] The amount or percentage of reticulocytes (e.g., bone marrow or circulating reticulocytes), or the amount or percentage of remaining reticulocytes (e.g., bone marrow or remaining circulating reticulocytes), is determined by human TfR knock-in (TfR) which is engineered to replace mouse TfR with human apical domain / mouse chimeric TfR protein. ms / hu KI) Mouse (for example, human TfR apical domain knock-in mouse ("hTfR アピカル Measurements can be made in "knock-in mice" or in non-human primates such as cynomolgus monkeys. Measurements can be made by intravenous administration of a modified Fc dimer or control, for example, 25-50 mg / kg (e.g., TfR). ms / hu In KI mice, circulating reticulocytes can be measured at 24 hours post-administration by cytochemical reactions using the Advia 120 hematology system, as described herein. Bone marrow reticulocytes can be measured using Ter119, as described herein. + hCD71 hi , and FSClow FACS sorting can be used to measure the grouping of a population.

[0153] As used herein, the terms “CH3 domain” and “CH2 domain” refer to immunoglobulin constant region domain polypeptides. In the context of IgG antibodies, the CH3 domain polypeptide refers to the amino acid segment from approximately position 341 to approximately position 447, numbered according to the EU numbering scheme, and the CH2 domain polypeptide refers to the amino acid segment from approximately position 231 to approximately 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, where the CH2 domain is numbered from 1 to 110 and the CH3 domain is numbered from 1 to 107, according to the IMGT Scientific chart numbering (IMGT website). The CH2 and CH3 domains are part of the Fc region of immunoglobulins. In the context of IgG antibodies, the Fc region refers to the amino acid segment from approximately position 231 to approximately position 447, numbered according to the EU numbering scheme. As used herein, the term “Fc region” may also include at least a portion of the hinge region of an antibody. An exemplary hinge region sequence is defined in SEQ ID NO: 104.

[0154] The term "variable region" refers to a domain in the antibody heavy or light chain that originates from germline variable (V) genes, diversity (D) genes, or binding (J) genes (and does not originate from stationary (Cμ and Cδ) gene segments) and gives the antibody its specificity for binding to an antigen. Typically, an antibody variable region contains four conserved "framework" regions interspersed with three hypervariable "complementarity-determining regions."

[0155] The terms “wild-type,” “natural,” and “naturally occurring” with respect to CH3 or CH2 domains are used herein to refer to domains having naturally occurring sequences.

[0156] As used herein, the term “variant” with respect to a mutant polypeptide or mutant polynucleotide is to be used interchangeably with “variant.” A variant with respect to a given wild-type CH3 or CH2 domain reference sequence may include naturally occurring allelic variants. A “non-naturally occurring” CH3 or CH2 domain refers to a variant or mutant domain that is not present in cells in nature and is produced by genetic modification, for example, using genetic manipulation or mutagenesis techniques of a natural 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 may include substitutions, insertions, and deletions.

[0157] The term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids.

[0158] Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analogs" refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimes" refer to chemical compounds that have a different structure from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[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 herein by either their commonly known three-letter symbols or 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 polymers of amino acid residues in a single chain. These terms apply to amino acid polymers, as well as naturally occurring amino acid polymers and unnaturally occurring amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids. Amino acid polymers may consist entirely of L-amino acids, entirely of D-amino acids, or mixtures of L and D amino acids.

[0162] As used herein, the term “protein” refers to either a single-chain polypeptide, a dimer (i.e., two), or a polymer (i.e., three or more). The single-chain polypeptides of a protein may be linked by covalent bonds, such as disulfide bonds, or by 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 in another amino acid that can be classified as having similar characteristics. Examples of classifications of conserved amino acid groups defined in this way include the "charged / polar group" containing 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" containing Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and the "aliphatic group" containing 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, a 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, a group of aromatic or cyclic amino acids can be subdivided into subgroups including a "nitrogen ring subgroup" containing Pro, His, and Trp; and a "phenyl subgroup" containing Phe and Tyr. In yet another example, an aliphatic group can be subdivided into subgroups, for example, an "aliphatic nonpolar subgroup" containing Val, Leu, Gly, and Ala; and an "aliphatic micropolar subgroup" containing Met, Ser, Thr, and Cys. Examples of the classification of conservative mutations include, but are not limited to, amino acid substitutions of amino acids within the above subgroups, such as Lys for Arg or vice versa, where a positive charge may be maintained; Glu for Asp or vice versa, where a negative charge may be maintained; Ser for Thr or vice versa, where a free -OH group may be maintained; and Gln for Asn or vice versa, where a free -NH2 group may be maintained.In some embodiments, hydrophobic amino acids are substituted for naturally occurring hydrophobic amino acids in the active site, for example, in order to preserve hydrophobicity.

[0164] In the context of two or more polypeptide sequences, the term “identical” or “identity” percentage refers to two or more sequences or subsequences that, when compared and aligned for maximum match across a comparison window or specified region, are identical, 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, when 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 compared to the candidate sequences. Alignment can be performed using various methods available to those skilled in the art, such as visual alignment or using publicly available software that employs 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 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 context of a given amino acid residue in a polypeptide sequence refer to the position of the residue of the designated reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. 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 aligned to a reference sequence need not be of the same length as the reference sequence.

[0167] As used herein, the terms "specifically binds to" or "selectively binds 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 for a longer duration than it binds to structurally distinct targets. 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 greater affinity for a particular target, e.g., TfR or FcγR, compared to an irrelevant 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., e.g., TfR or FcγR) refer to, for example, an equilibrium dissociation constant K -4 of 10 -5 M or less (e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M) for the target, e.g., e.g., TfR or FcγR. DThis can be expressed by molecules having the following characteristics. In some embodiments, the modified CH3 domain polypeptide specifically binds to an epitope on TfR that is conserved between species (e.g., structurally conserved between species), such as one conserved between non-human primates and the human species (e.g., structurally conserved between non-human primates and the human species). In some embodiments, the polypeptide may bind exclusively to human TfRs.

[0168] As used herein, the term “binding affinity” refers to the strength of a non-covalent interaction between two molecules, for example, a single binding site on a polypeptide and the target to which it binds, for example, TfR. Therefore, unless otherwise indicated or evident from the context, this term may refer to a 1:1 interaction between a polypeptide and its target. Binding affinity is defined by the equilibrium dissociation constant (K). D It can be quantified by measuring the coupling rate constant (k a ,time -1 M -1 The dissociation rate constant (k) divided by ) d ,time -1 ) refers to K D This can be determined by measuring the dynamics of complex formation and dissociation, for example, by 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” can reflect not only formal binding affinity, such as a 1:1 interaction between a polypeptide and its target, but also K, which can reflect strong binding. D This includes the apparent affinity that is calculated.

[0169] The terms “antigen-binding region” and “antigen-binding fragment” are used interchangeably herein and refer to one or more fragments of an antibody variable region that possess 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 VL, VH, CL, and CH1 domains), F(ab')2 fragments (bivalent fragments containing two Fab fragments linked by disulfide crosslinks at a hinge region), single-chain Fv (scFv), disulfide-linked Fv (dsFv), complementarity-determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), nanobodies, diabodies, and other forms described in Spiess et al., Mol.Immun.67(2015)95-106, which are incorporated herein by reference.

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

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

[0172] The amino acid sequences of the CDR and framework region 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, the CDR is determined according to the Contact definition. See MacCallum et al., J.Mol.Biol., 262:732-745 (1996). In some embodiments, the CDR is determined by a combination of the Kabat, Chothia, and Contact CDR definitions.

[0173] Where used interchangeably in this specification, 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] Terms such as “treatment” and “to treat” are used herein to mean, in general, obtaining a desired pharmacological and / or physiological effect. “To treat” or “treatment” may refer to any indication of successful treatment or improvement of cancer (e.g., HER2-positive and / or metastatic cancer), including any objective or subjective parameters 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 improvement of the patient’s physical or mental health. Treatment or improvement of symptoms may be based on objective or subjective parameters. The effect of treatment may be compared to an untreated individual or a pool of individuals, or to the same patient at different points in time before or during treatment.

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

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

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

[0178] III. FC polypeptide dimer-antibody variable region fusion protein In some embodiments, the disclosure provides an Fc polypeptide dimer-antibody variable region fusion protein capable of binding to human epidermal growth factor 2 (HER2) and modified to bind to the transferrin receptor (TfR), thereby 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 protein provided herein retains effector function upon binding to HER2 but has reduced effector function upon binding to TfR. In this configuration, the Fc polypeptide dimer-antibody variable region fusion protein can transport the anti-HER2 antibody variable region (e.g., forming part of the Fab domain) across the BBB without substantial reduction in reticulocytes (including TfR on the cell surface), and 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-positive, cis-configured Fc polypeptide dimer-antibody variable region fusion proteins, where 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 when bound to TfR. In these embodiments, the other Fc polypeptide in the Fc polypeptide dimer does not include either a TfR binding site or a modification that substantially reduces FcγR binding. The trans configuration of the 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 includes a modification that, for example, reduces effector function when bound to TfR. Modified Fc polypeptide dimers having a cis configuration rather than a trans configuration can reduce reticulocyte reduction in 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 reticulocyte reduction after administration of the Fc polypeptide dimer-antibody variable region fusion protein is less than the amount of reticulocyte reduction after administration of the control. In some embodiments, the control is the corresponding TfR-binding polypeptide dimer-antibody variable region fusion protein that has full effector function and / or does not contain mutations that reduce FcγR binding. In some cases, the control is an Fc polypeptide dimer-antibody variable region fusion protein in which the first Fc polypeptide contains an amino acid sequence defined as SEQ ID NOs: 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., the first Fc polypeptide specifically binds to TfR containing a TfR binding site but does not contain LALA substitutions or other modifications that reduce FcγR binding), and the second Fc polypeptide does not contain any modifications that reduce the TfR binding site or FcγR binding.

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

[0182] In some embodiments, the first and / or second Fc polypeptide includes an amino acid modification that increases the serum half-life. In some embodiments, the first Fc polypeptide includes an amino acid modification that increases the serum half-life. In some embodiments, the second Fc polypeptide includes an amino acid modification that increases the serum half-life. In some embodiments, the first and second Fc polypeptides include an amino acid modification that increases the serum half-life. In some embodiments, the amino acid modification that increases the 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.

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

[0184] Anti-HER2 variable region anti-HER2_DIV In some embodiments, the antibody variable region includes 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 includes 2, 3, 4, 5, or all 6 of (a) to (f). In some embodiments, the antibody variable region includes heavy chain CDR1 of (a), heavy chain CDR2 of (b), and heavy chain CDR3 of (c). In some embodiments, the antibody variable region includes light chain CDR1 of (d), light chain CDR2 of (e), and light chain CDR3 of (f). In some embodiments, a CDR having up to two amino acid substitutions has one amino acid substitution relative to the reference sequence. In some embodiments, a CDR having up to two amino acid substitutions has two amino acid substitutions relative to the reference sequence. In some embodiments, the up to two amino acid substitutions are conservative substitutions.

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

[0187] In some embodiments, the antibody variable region includes 2, 3, 4, 5, or all 6 of (a) to (f). In some embodiments, the antibody variable region includes heavy chain CDR1 of (a), heavy chain CDR2 of (b), and heavy chain CDR3 of (c). In some embodiments, the antibody variable region includes light chain CDR1 of (d), light chain CDR2 of (e), and light chain CDR3 of (f).

[0188] In some embodiments, the antibody variable region includes (a)(i) at least 75% sequence identity to SEQ ID NO: 59 (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), and (ii) a heavy CDR-H1, CDR-H2, and CDR-H3 which are identical to SEQ ID NOs: 69, 70, and 71, respectively. A light chain variable region and / or (b)(i) at least 75% sequence identity to sequence number 60 (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) and (ii) a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 which are identical to sequence numbers 72, 73, and 74, respectively.

[0189] In some embodiments, the antibody variable region includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 59 (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). In some embodiments, the antibody variable region includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 59.

[0190] In some embodiments, the antibody variable region includes a light chain variable region containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 60 (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). In some embodiments, the antibody variable region includes a light chain variable region containing the amino acid sequence of SEQ ID NO: 60.

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

[0192] anti-HER2_DII In some embodiments, the antibody variable region includes 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 includes 2, 3, 4, 5, or all 6 of (a) to (f). In some embodiments, the antibody variable region includes heavy chain CDR1 of (a), heavy chain CDR2 of (b), and heavy chain CDR3 of (c). In some embodiments, the antibody variable region includes light chain CDR1 of (d), light chain CDR2 of (e), and light chain CDR3 of (f). In some embodiments, a CDR having up to two amino acid substitutions has one amino acid substitution relative to the reference sequence. In some embodiments, a CDR having up to two amino acid substitutions has two amino acid substitutions relative to the reference sequence. In some embodiments, the up to two amino acid substitutions are conservative substitutions.

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

[0195] In some embodiments, the antibody variable region includes 2, 3, 4, 5, or all 6 of (a) to (f). In some embodiments, the antibody variable region includes heavy chain CDR1 of (a), heavy chain CDR2 of (b), and heavy chain CDR3 of (c). In some embodiments, the antibody variable region includes light chain CDR1 of (d), light chain CDR2 of (e), and light chain CDR3 of (f).

[0196] In some embodiments, the antibody variable region includes (a)(i) at least 75% sequence identity to SEQ ID NO: 61 (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), and (ii) a heavy CDR-H1, CDR-H2, and CDR-H3 which are identical to SEQ ID NOs: 75, 76, and 77, respectively. A light chain variable region and / or (b)(i) at least 75% sequence identity to sequence number 62 (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) and (ii) a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 which are identical to sequence numbers 78, 79, and 80, respectively.

[0197] In some embodiments, the antibody variable region includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 61 (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).

[0198] In some embodiments, the antibody variable region includes a light chain variable region containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 62 (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).

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

[0200] Anti-HER2_DI In some embodiments, the antibody variable region is: (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 CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 252, or having the same amino acid substitutions to the amino acid sequence of SEQ ID NO: 252 (d) a heavy chain CDR3 having up to two amino acid substitutions to the amino acid sequence of (sequence number 253), a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of (sequence number 253) or up to two amino acid substitutions to the amino acid sequence of (sequence number 253), a light chain CDR2 having up to two amino acid substitutions to the amino acid sequence of (sequence number 254), and a light chain CDR3 having up to two amino acid substitutions to the amino acid sequence of (sequence number 255).

[0201] In some embodiments, the antibody variable region includes 2, 3, 4, 5, or all 6 of (a) to (f). In some embodiments, the antibody variable region includes heavy chain CDR1 of (a), heavy chain CDR2 of (b), and heavy chain CDR3 of (c). In some embodiments, the antibody variable region includes light chain CDR1 of (d), light chain CDR2 of (e), and light chain CDR3 of (f). In some embodiments, a CDR having up to two amino acid substitutions has one amino acid substitution relative to the reference sequence. In some embodiments, a CDR having up to two amino acid substitutions has two amino acid substitutions relative to the reference sequence. In some embodiments, the up to two amino acid substitutions are conservative substitutions.

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

[0203] In some embodiments, the antibody variable region includes 2, 3, 4, 5, or all 6 of (a) to (f). In some embodiments, the antibody variable region includes heavy chain CDR1 of (a), heavy chain CDR2 of (b), and heavy chain CDR3 of (c). In some embodiments, the antibody variable region includes light chain CDR1 of (d), light chain CDR2 of (e), and light chain CDR3 of (f).

[0204] In some embodiments, the antibody variable region includes (a)(i) at least 75% sequence identity to SEQ ID NO: 256 (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), and (ii) a heavy CDR-H1, CDR-H2, and CDR-H3 which are identical to SEQ ID NOs: 250, 251, and 252, respectively. A light chain variable region and / or (b)(i) at least 75% sequence identity to sequence number 257 (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) and (ii) a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 which are identical to sequence numbers 253, 254, and 255, respectively.

[0205] In some embodiments, the antibody variable region includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 256 (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). In some embodiments, the antibody variable region includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 256.

[0206] In some embodiments, the antibody variable region includes a light chain variable region containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 257 (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). In some embodiments, the antibody variable region includes a light chain variable region containing the amino acid sequence of SEQ ID NO: 257.

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

[0208] Exemplary Fc polypeptide dimer-antibody variable region fusion protein The Fc polypeptide dimer-antibody variable region fusion proteins described herein may contain any combination of the above anti-HER2 variable regions. In some embodiments, the first Fc polypeptide comprises a TfR binding site containing a modified CH3 domain. Non-limiting examples of modified CH3 domains that may be used in compositions and methods are described in the section titled “TfR-binding Fc polypeptides” herein. In some embodiments, according to EU numbering, the first Fc polypeptide further comprises the knob mutation T366W, and the second Fc polypeptide comprises the whole mutations T366S, L368A, and Y407V. 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 one of the amino acid sequences of SEQ ID NO: 67 and 68.

[0209] In some embodiments, according to EU numbering, the first Fc polypeptide further comprises the whole mutations T366S, L368A, and Y407V, and the second Fc polypeptide comprises the knob mutation T366W. 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 either SEQ ID NO: 65 or 66.

[0210] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises (a) an antibody variable region or its antigen-binding fragment capable of binding to HER2 (e.g., human HER2); (b) a first Fc polypeptide comprising, according to EU numbering, modifications that create a TfR binding site, amino acid modifications L234A and L235A, and knob mutation T366W; and (c) a second Fc polypeptide comprising, according to EU numbering, hole mutations T366S, L368A, and Y407V, and not comprising 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 290. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

[0214] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises (a) an antibody variable region or antigen-binding fragment thereof that can bind to HER2 (e.g., human HER2); (b) a first Fc polypeptide comprising, according to EU numbering, a modification, amino acid modifications L234A and L235A, knob mutation T366W, and amino acid modification N434S with or without M428L to create a TfR binding site; and (c) a second Fc polypeptide comprising, according to EU numbering, hole mutations T366S, L368A, and Y407V, and without a TfR binding site.

[0215] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 290. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

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

[0219] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 291. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

[0222] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises (a) an antibody variable region or its antigen-binding fragment capable of binding to HER2 (e.g., human HER2); (b) a first Fc polypeptide comprising, according to EU numbering, a modification, amino acid modifications L234A and L235A, knob mutation T366W, and amino acid modification N434S with or without M428L, which create a TfR binding site; and (c) a second Fc polypeptide comprising, according to EU numbering, a hole mutation T366S, L368A, and Y407V, and amino acid modification N434S with or without M428L, but without a TfR binding site.

[0223] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 291. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

[0226] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises (a) an antibody variable region or its antigen-binding fragment capable of binding to HER2 (e.g., human HER2); (b) a first Fc polypeptide comprising, according to EU numbering, modifications for creating a TfR binding site, amino acid modifications L234A and L235A, and whole mutations T366S, L368A, and Y407V; and (c) a second Fc polypeptide comprising, according to EU numbering, a knob mutation T366W and not comprising a TfR binding site.

[0227] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 294. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

[0230] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises (a) an antibody variable region or antigen-binding fragment thereof that can bind to HER2 (e.g., human HER2); (b) a first Fc polypeptide comprising, according to EU numbering, a modification, amino acid modifications L234A and L235A, whole mutations T366S, L368A, and Y407V, and amino acid modification N434S with or without M428L to create a TfR binding site; and (c) a second Fc polypeptide comprising, according to EU numbering, a knob mutation T366W and without a TfR binding site.

[0231] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 294. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

[0234] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises (a) an antibody variable region or its antigen-binding fragment capable of binding to HER2 (e.g., human HER2); (b) a first Fc polypeptide comprising, according to EU numbering, modifications that create a TfR binding site, amino acid modifications L234A and L235A, and whole mutations T366S, L368A, and Y407V; and (c) a second Fc polypeptide comprising, according to EU numbering, the knob mutation T366W, and amino acid modification N434S with or without M428L, and without a TfR binding site.

[0235] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 295. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

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

[0239] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 295. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

[0242] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises (a) an antibody variable region or its antigen-binding fragment capable of binding to HER2 (e.g., human HER2), (b) a first Fc polypeptide comprising modifications and knob mutations T366W 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 not comprising a TfR binding site.

[0243] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 290. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

[0246] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises (a) an antibody variable region or its antigen-binding fragment capable of binding to HER2 (e.g., human HER2), (b) a first Fc polypeptide comprising an amino acid modification N434S with or without modifications, knob mutations T366W and M428L, according to EU numbering, to create a TfR binding site, and (c) a second Fc polypeptide comprising whole mutations T366S, L368A and Y407V, according to EU numbering, and without a TfR binding site.

[0247] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 or its antigen-binding fragment capable of binding to HER2 (e.g., human HER2), (b) a first Fc polypeptide comprising a modification and knob mutation T366W that creates a TfR binding site according to EU numbering, and (c) a second Fc polypeptide comprising amino acid modification N434S with or without the whole mutations T366S, L368A, and Y407V, and M428L, according to EU numbering, and without a TfR binding site.

[0251] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 291. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

[0254] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises (a) an antibody variable region or its antigen-binding fragment capable of binding to HER2 (e.g., human HER2), (b) a first Fc polypeptide comprising an amino acid modification N434S with or without modifications, knob mutations T366W and M428L, according to EU numbering, to create a TfR binding site, and (c) a second Fc polypeptide comprising an amino acid modification N434S with or without hole mutations T366S, L368A and Y407V, and M428L, according to EU numbering, and without a TfR binding site.

[0255] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 or its antigen-binding fragment capable of binding to HER2 (e.g., human HER2), (b) a first Fc polypeptide comprising modifications to create a TfR binding site according to EU numbering, and hole mutations T366S, L368A, and Y407V, and (c) a second Fc polypeptide comprising a knob mutation T366W according to EU numbering, and not comprising a TfR binding site.

[0259] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 294. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

[0262] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises (a) an antibody variable region or its antigen-binding fragment capable of binding to HER2 (e.g., human HER2); (b) a first Fc polypeptide comprising, according to EU numbering, a modification, a whole mutation T366S, L368A, and Y407V, and an amino acid modification N434S with or without M428L to create a TfR binding site; and (c) a second Fc polypeptide comprising, according to EU numbering, a knob mutation T366W and without a TfR binding site.

[0263] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 53. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 58.

[0265] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 294. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

[0266] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises (a) an antibody variable region or its antigen-binding fragment capable of binding to HER2 (e.g., human HER2), (b) a first Fc polypeptide comprising a modification that creates a TfR binding site according to EU numbering, and whole mutations T366S, L368A, and Y407V, and (c) a second Fc polypeptide comprising an amino acid modification N434S with or without a knob mutation T366W, and M428L, according to EU numbering, and without a TfR binding site.

[0267] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 295. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

[0270] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises (a) an antibody variable region or its antigen-binding fragment capable of binding to HER2 (e.g., human HER2), (b) a first Fc polypeptide comprising an amino acid modification N434S with or without modifications, whole mutations T366S, L368A, and Y407V, and M428L, according to EU numbering, that create a TfR binding site, and (c) a second Fc polypeptide comprising an amino acid modification N434S with or without knob mutations T366W and M428L, according to EU numbering, and without a TfR binding site.

[0271] In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing 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 containing 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 containing one of the amino acid sequences 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 containing the amino acid sequence of SEQ ID NOs. 295. In some embodiments, the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains containing the amino acid sequence of SEQ ID NOs. 293.

[0274] antibody heavy chain In other embodiments, the foregoing provides an antibody heavy chain. 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 including modifications to create a TfR binding site. The antibody heavy chain may include any of the anti-HER2 variable heavy chain CDRs and / or heavy chain variable region sequences in the section titled “Anti-HER2 Variable Region” above. The modified Fc polypeptide may include any of the TfR binding sites (e.g., modified CH3 domains) described herein and / or any of the modifications described herein that increase serum half-life or decrease FcγR binding (e.g., when bound to TfR).

[0275] In some embodiments, the modified Fc polypeptide further comprises the 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 the whole 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 fragment thereof, and (b) a modified Fc polypeptide comprising modifications, amino acid modifications L234A and L235A, and knob mutation T366W, according to EU numbering, for creating a TfR binding site. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences of SEQ ID NOs: 2, 10, 18, and 82. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences of SEQ ID NOs: 30, 38, 46, and 90. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences 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 fragment thereof, and (b) a modified Fc polypeptide, which, according to EU numbering, includes modifications to create a TfR binding site, amino acid modifications L234A and L235A, knob mutation T366W, and amino acid modification N434S with or without M428L. In some embodiments, the antibody heavy chain comprises one amino acid sequence from SEQ ID NOs: 4, 12, 20, and 84. In some embodiments, the antibody heavy chain comprises one amino acid sequence from SEQ ID NOs: 32, 40, 48, and 92. In some embodiments, the antibody heavy chain comprises one amino acid sequence from 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 fragment thereof, and (b) a modified Fc polypeptide, including modifications to create a TfR binding site according to EU numbering, amino acid modifications L234A and L235A, and whole mutations T366S, L368A, and Y407V. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences of SEQ ID NOs. 6, 14, 22, and 86. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences of SEQ ID NOs. 34, 42, 50, and 94. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences 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 fragment thereof, and (b) a modified Fc polypeptide, including modifications to create a TfR binding site according to EU numbering, amino acid modifications L234A and L235A, hole mutations T366S, L368A, and Y407V, and amino acid modification N434S with or without M428L. In some embodiments, the antibody heavy chain comprises one amino acid sequence from SEQ ID NOs: 8, 16, 24, and 88. In some embodiments, the antibody heavy chain comprises one amino acid sequence from SEQ ID NOs: 36, 44, 52, and 96. In some embodiments, the antibody heavy chain comprises one amino acid sequence from 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 fragment thereof, and (b) a modified Fc polypeptide containing a modification and knob mutation T366W that creates a TfR binding site according to EU numbering. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences of SEQ ID NOs: 1, 9, 17, and 81. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences of SEQ ID NOs: 29, 37, 45, and 89. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences 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, according to EU numbering, a modification, a knob mutation T366W, and an amino acid modification N434S with or without M428L to create a TfR binding site. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences of SEQ ID NOs: 3, 11, 19, and 83. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences of SEQ ID NOs: 31, 39, 47, and 91. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences 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 fragment thereof, and (b) a modified Fc polypeptide containing modifications to create a TfR binding site according to EU numbering, as well as the hole mutations T366S, L368A, and Y407V. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences of SEQ ID NOs. 5, 13, 21, and 85. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences of SEQ ID NOs. 33, 41, 49, and 93. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences 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 fragment thereof, and (b) a modified Fc polypeptide comprising, according to EU numbering, a modification, a hole mutation T366S, L368A, and Y407V, and an amino acid modification N434S with or without M428L to create a TfR binding site. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences of SEQ ID NOs. 7, 15, 23, and 87. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences of SEQ ID NOs. 35, 43, 51, and 95. In some embodiments, the antibody heavy chain comprises one of the amino acid sequences of SEQ ID NOs. 264, 272, 280, and 288.

[0284] IV. TFR-binding FC polypeptide 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 includes a modified human Ig CH3 domain, such as an IgG CH3 domain. The CH3 domain may be from any IgG subtype, i.e., IgG1, IgG2, IgG3, or IgG4. In the context of IgG antibodies, the CH3 domain refers to a segment of amino acids numbered according to the EU numbering scheme, from approximately position 341 to approximately position 447. For the purpose of identifying the corresponding set of amino acid positions for TfR binding, the positions in the CH3 domain are determined by 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 by reference to the EU numbering scheme or SEQ ID NO: 99, i.e., an amino acid is considered a substitution for an amino acid at its corresponding position in the EU numbering scheme or SEQ ID NO: 99.

[0286] As described above, the set of residues in the CH3 domain that can be modified is numbered herein with reference to the EU numbering scheme or Sequence ID No. 99. Any CH3 domain, e.g., IgG1, IgG2, IgG3, or IgG4 CH3 domain, may have modifications, e.g., amino acid substitutions, in one or more sets of residues corresponding to residues at recognized positions in the EU numbering scheme or Sequence ID No. 99. The respective positions in the IgG1, IgG2, IgG3, and IgG4 sequences corresponding to any given position in the EU numbering scheme or Sequence ID No. 99 can be easily determined.

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

[0288] In one embodiment, a modified CH3 domain polypeptide that specifically binds to TfR binds to the apical domain of TfR via an epitope containing 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 defined 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, the modified CH3 domain polypeptide binds to the apical domain of TfR via an epitope containing 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 can bind to TfR without blocking or otherwise inhibiting the binding of transferrin to the receptor. In some embodiments, the binding of transferrin to TfR is substantially uninhibited. In some embodiments, the 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, the 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 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 1%). Exemplary CH3 domain polypeptides exhibiting this binding specificity include polypeptides having 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 bond sets: 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 In some embodiments, the modified CH3 domain polypeptide includes substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 in a 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 a particular embodiment, the modified CH3 domain polypeptide is as follows: 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 acidic amino acids at position 387; Trp at position 388; aliphatic amino acids, 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 at position 413, Ala, Ser, Leu, Thr, Pro, Ile, or His, 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 at position 421, His, or Lys Includes positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, selected from the range.

[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 any one of the amino acids 111-217 of SEQ ID NOs. 177-180. In some embodiments, such a modified CH3 domain polypeptide contains amino acids 154-160 and / or 183-191 of any one of the amino acids 177-180. In some embodiments, such a modified CH3 domain polypeptide contains amino acids 150-160 and / or 183-191 of any one of the amino acids 177-180. In some embodiments, the modified CH3 domain polypeptide contains amino acids 150-160 and / or 183-196 of any one of the amino acids 177-180.

[0292] In some embodiments, the modified CH3 domain polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to amino acids 111–217 of SEQ ID NO: 99, provided that the identity percentage does not include the set of positions 154, 156, 157, 158, 159, 160, 183, 186, and 191 of SEQ ID NO: 99 (positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 according to the EU numbering scheme). In some embodiments, the modified CH3 domain polypeptide includes amino acids 154–160 and / or amino acids 183–191 as defined 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 with respect to positions 150, 154, 156, 157, 158, 159, 1 The conditions are 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 replaced.

[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: 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 acidic amino acids at position 387; aromatic amino acids at position 388, e.g., Trp; 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; 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 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 Including at least positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0295] In additional embodiments, the TfR-binding polypeptide comprises amino acids 157-194, 153-194, or 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 any one of amino acids 157-194, 153-194, or 153-199 of any one of SEQ ID NOs. 177-180.

[0296] In some embodiments, the polypeptide comprises one of sequence numbers 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 one of sequence numbers 177-180.

[0297] FcRn binding site Polypeptides described herein that can be transported across the BBB may additionally include an FcRn binding site. In some embodiments, the FcRn binding site is located within a modified Fc polypeptide or a fragment thereof.

[0298] In some embodiments, the FcRn binding site includes a native FcRn binding site. In some embodiments, the FcRn binding site does not involve 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, for example, a human IgG binding site. In some embodiments, the FcRn binding site includes modifications that alter the 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) either increase or substantially reduce the serum half-life (i.e., reduce the serum half-life by 25% or less when assayed under identical conditions compared to a protein equivalent with 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 of which are determined by reference to SEQ ID NO: 99.

[0300] In some embodiments, the FcRn binding site includes one or more mutations in the native human IgG sequence that extend the serum half-life of the modified polypeptide. In some embodiments, the mutation, e.g., substitution, is introduced at one or more of the following positions, determined by reference to SEQ ID NO: 99: 14-27, 49-54, 77-87, 153-160, and 198-205 (the positions correspond to positions 244-257, 279-284, 307-317, 383-390, and 428-435 using EU numbering). In some embodiments, one or more mutations are introduced at positions 21, 22, 24, 25, 26, 77, 78, 79, 81, 82, 84, 155, 156, 157, 159, 198, 203, 204, or 206, determined by reference to Sequence ID No. 99 (the positions correspond to positions 251, 252, 254, 255, 256, 307, 308, 309, 311, 312, 314, 385, 386, 387, 389, 428, 433, 434, or 436 using EU numbering). In some embodiments, the mutations are introduced at one, two, or three of positions 22, 24, and 25 (corresponding to positions 252, 254, and 256 using EU numbering), determined by reference to Sequence ID No. 99. 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 mutations M22Y, S24T, and T26E. In some embodiments, the mutation is introduced at one or two of positions 198 and 204 (corresponding to positions 428 and 434 using EU numbering), 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 mutation N204S having or not having M198L. In some embodiments, the modified Fc polypeptide includes substitution 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 includes mutations T307A, E380A, and N434A (SEQ ID NO: 99, T77A, E150A, and N204A). In some embodiments, the modified Fc polypeptide includes substitutions at positions T250 and M428 (numbered with reference to SEQ ID NO: 99, corresponding to T20 and M198). In some embodiments, the Fc polypeptide includes mutations T250Q and / or M428L (SEQ ID NO: 99, T20Q, and M198L). In some embodiments, the modified Fc polypeptide includes substitutions at positions M428 and N434 (numbered with reference to SEQ ID NO: 99, corresponding to M198 and N204). In some embodiments, the modified Fc polypeptide includes substitutions M428L and N434S (corresponding to M198L and N204S, numbered with reference to SEQ ID NO: 99). In some embodiments, the modified Fc polypeptide includes N434S or N434A substitutions (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 to TfR and initiate transport across the BBB) may also include additional mutations to reduce effector function. As described herein, by introducing both TfR binding sites and mutations that reduce TfR-mediated FcγR binding of the Fc polypeptide dimer to the same Fc polypeptide, it was possible to reduce effector function upon TfR binding and bring about TfR binding without substantial reduction in reticulocytes, while still maintaining and exhibiting levels of effector function (e.g., ADCC or CDC) when the Fc polypeptide dimer fuses to therapeutic Fab and binds to the target antigen of Fab.

[0302] In some embodiments, Fc polypeptides containing a modified CH3 domain possess effector function, i.e., the ability to induce a specific biological function upon binding to an Fc receptor expressed on effector cells that mediate effector function. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granule 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 appropriate Fc receptors present on immune system cells, while native human IgG1, IgG2, IgG3, and IgG4 can induce ADCP function upon binding to appropriate Fc receptors present on immune cells.

[0304] In some embodiments, the Fc polypeptides having TfR binding sites described herein may include additional modifications that reduce effector function, i.e., reduce effector function upon TfR binding. Having reduced effector function of the Fc polypeptide dimer upon TfR binding is desirable because it leads to reduced reticulocyte reduction, as reticulocytes also have TfR on their cell surface. As described in detail herein, Fc polypeptide dimers in a cis configuration, i.e., Fc polypeptide dimers having both a TfR binding site and mutations that reduce effector function on the same Fc polypeptide as the Fc polypeptide dimer, exhibit TfR binding without substantial reticulocyte reduction, yet still maintain a level of effector function (e.g., ADCC) when the Fc polypeptide dimer fuses to therapeutic Fab and binds to the Fab's target antigen. Having effector function when the Fc polypeptide dimer fuses to 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 at positions corresponding to, for example, positions 4 and 5 of SEQ ID NO: 99 (positions 234 and 235 according to the EU numbering scheme). In some embodiments, substitutions in the modified CH2 domain include Ala at positions 4 and 5 of SEQ ID NO: 99. In some embodiments, 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 (corresponding to positions 8, 35, 39, 40, 67, 97, and 99, numbered with reference to SEQ ID NO: 99, in the EU numbering scheme). Exemplary substitutions (as numbered in the EU numbering scheme) include, a mutation in which position 329 is replaced by a glycine or arginine or amino acid residue of sufficient size to disrupt the Fc / Fcγ receptor interface formed between proline 329 of Fc and the tryptophan residues Trp 87 and Trp 110 of FcγRIII. Additional exemplary substitutions include S228P, E233P, L235E, N297A, N297D, and P331S. Multiple substitutions, 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 may also be present. In some embodiments, the Fc polypeptide may have one or more amino acid substitutions modulating 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 linkage and / or CDC.

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

[0309] VI. Measure effector function or FcγR binding. Methods for analyzing the binding affinity, binding kinetics, and cross-reactivity between Fc polypeptides or Fc polypeptide dimers and 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)), binding equilibrium exclusion (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, binding equilibrium exclusion is used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, BioLayer interference assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity.

[0310] ADCC is a type of immune response in which an antibody binds to an antigen on the surface of a pathogenic or tumorigenic target cell and identifies the antibody for destruction by effector cells, such as peripheral blood mononuclear cells (e.g., natural killer (NK) cells, T cells, and B cells). Effector cells with FcγR recognize and bind to the Fc region of the antibody bound to the target cell. Thus, the antibody confers specificity to the death of the target cell. CDC is initiated when C1q, the initiating component of the classical complement pathway, binds to the Fc region of the target-binding antibody. ADCC and CDC activity can be determined in standard in vivo or in vitro assays of cell death. Methods for determining ADCC and CDC activity are available in the art. In some embodiments, the method is: 51 This may involve labeling target cells with radioactive materials such as Cr, or fluorescent dyes such as calcein AM. Labeled cells can be incubated with antibodies and effector cells, and the death of 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 damaged or injured in any way, LDH, a soluble yet 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 amounts of viable and dead cells in the medium can then be quantified by measuring the amount of released LDH using a colorimetric or fluorescence-quantified 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 human immunogenicity, and / or provide knob and whole 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 the corresponding wild-type Fc polypeptide (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide).

[0314] The modified Fc polypeptides described herein may also have other mutations introduced outside of a particular set of amino acids to, for example, affect glycosylation, increase serum half-life, or, with respect to the CH3 domain, provide knob and hole heterodimerization of polypeptides containing the modified CH3 domain. Generally, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding recess ("hole") at the interface of a second polypeptide, such that the protrusion can be positioned in a recess to promote heterodimerization and prevent homodimerization. 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 recess of the same or similar size to the protrusion is created at the interface of the second polypeptide by replacing a larger amino acid side chain with a smaller one (e.g., alanine or threonine). Such additional mutations are located in a polypeptide at a position that does not adversely affect the binding of the modified CH3 domain to TfR.

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

[0316] The modified Fc polypeptides described herein may also be manipulated 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 the serum half-life may be introduced. For example, in some embodiments, the modified Fc polypeptide described herein includes a CH2 domain containing Tyr at position 22 of SEQ ID NO: 99, Thr at position 24 of SEQ ID NO: 99, and Glu at position 26 of SEQ ID NO: 99. Alternatively, the modified Fc polypeptide described herein may include M198L and N204S substitutions numbered with reference to SEQ ID NO: 99. Alternatively, the modified Fc polypeptide described herein may include N204S or N204A substitutions numbered with reference to SEQ ID NO: 99.

[0318] Exemplary Fc polypeptides including 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., N204S, having or not having (i) M22Y, S24T, and T26E, numbered with reference to SEQ ID NO: 99, or (ii) M198L, numbered with reference to SEQ ID NO: 99).

[0319] In some embodiments, the 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 at least 85% identity, at least 90% identity, or at least 95% identity to a knob mutation (e.g., T136W, numbered with reference to SEQ ID NO: 99) and any one of the sequences of SEQ ID NOs. 177-180. In some embodiments, a modified Fc polypeptide having any one of the sequences 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) may have at least 85% identity, at least 90% identity, or at least 95% identity to any one of sequences from sequence numbers 177-180, including a knob mutation (e.g., T136W, numbered with reference to sequence number 99), a mutation that modulates effector function (e.g., L4A, L5A, and / or P99G (e.g., L4A and L5A), numbered with reference to sequence number 99), and 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 having or not having M198L, numbered with reference to SEQ ID NO: 99), and at least 85% identity, at least 90% identity, or at least 95% identity to any one of the sequences of SEQ ID NOs. 177-180. In some embodiments, a modified Fc polypeptide having any one of the sequences 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 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 at least 85% identity, at least 90% identity, or at least 95% identity to any one of the sequences of sequence numbers 177-180, including knob mutations (e.g., T136W, numbered with reference to SEQ ID NO: 99), mutations that modulate effector function (e.g., L4A, L5A, and / or P99G, numbered with reference to SEQ ID NO: 99 (e.g., L4A and L5A)), mutations that increase serum stability or serum half-life (e.g., N204S having or not having (i) M22Y, S24T, and T26E, numbered with reference to SEQ ID NO: 99, or (ii) M198L, numbered with reference to SEQ ID NO: 99), and any one of the sequences of sequence numbers 177-180. In some embodiments, a modified Fc polypeptide having any one of the sequences of sequence numbers 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, 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 hole mutation (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 any one of the sequences of SEQ ID NOs. 177-180. In some embodiments, a modified Fc polypeptide having any one of the sequences of SEQ ID NOs. 177-180 may be modified to have a hole mutation.

[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) may have a hole mutation (e.g., T136S, L138A, and Y177V, numbered with reference to SEQ ID NO: 99), a mutation that modifies 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 any one of the sequences of SEQ ID NOs. 177-180. In some embodiments, a modified Fc polypeptide having any one of the sequences of SEQ ID NOs. 177-180 may be modified to have a hole mutation and a mutation that modulates effector function.

[0325] 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 at least 85% identity, at least 90% identity, or at least 95% identity to any one of sequences from sequence numbers 177 to 180, including 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 having or not having (i) M22Y, S24T, and T26E, numbered with reference to SEQ ID NO: 99, or (ii) M198L, numbered with reference to SEQ ID NO: 99), and sequence numbers 177 to 180. In some embodiments, a modified Fc polypeptide having any one of the sequences 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) are 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, numbered with reference to SEQ ID NO: 99, and / or The modified Fc polypeptide may have at least 85% identity, at least 90% identity, or at least 95% identity to any one of the sequences of sequence numbers 177-180. In some embodiments, the modified Fc polypeptide having any one of the sequences of sequence numbers 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 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 SEQ ID NO: 105. In some embodiments, clone CH3C.35.23.3 having the knob mutation has the sequence of SEQ ID NO: 105.

[0328] In some embodiments, clone CH3C.35.23.3 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), 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 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., M22Y, S24T, and T26E, numbered with reference to SEQ ID NO: 99), and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 108. In some embodiments, clone CH3C.35.23.3 having 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 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., N204S, having or not having M198L, numbered with reference to SEQ ID NO: 99), and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 109. In some embodiments, clone CH3C.35.23.3 having 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 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)), 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 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)), a mutation that increases serum stability or serum half-life (e.g., N204S having or not having 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 may have a hole mutation (e.g., T136S, L138A, and Y177V, numbered with reference to SEQ ID NO: 99) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 114. In some embodiments, clone CH3C.35.23.3 has a hole mutation and the sequence of SEQ ID NO: 114.

[0334] In some embodiments, clone CH3C.35.23.3 may have hole mutations (e.g., T136S, L138A, and Y177V, numbered with reference to SEQ ID NO: 99), effector-modulating mutations (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 having hole mutations and effector-modulating mutations has the sequence of SEQ ID NO: 115 or 116.

[0335] In some embodiments, clone CH3C.35.23.3 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., M22Y, S24T, and T26E, numbered with reference to SEQ ID NO: 99), and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 117. In some embodiments, clone CH3C.35.23.3 having a whole mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO: 117.

[0336] In some embodiments, clone CH3C.35.23.3 may have a hole 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., N204S, having or not having M198L, numbered with reference to SEQ ID NO: 99), and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 118. In some embodiments, clone CH3C.35.23.3 having a hole mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO: 118.

[0337] In some embodiments, clone CH3C.35.23.3 may have 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 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: 119 or 120. In some embodiments, clone CH3C.35.23.3 having a hole 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: 119 or 120.

[0338] In some embodiments, clone CH3C.35.23.3 may have 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 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 having or not having 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 a hole 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: 121 or 122.

[0339] Clone CH3C.35.23.4 In some embodiments, clone CH3C.35.23.4 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 SEQ ID NO: 123. In some embodiments, clone CH3C.35.23.4 having the knob mutation has the sequence of SEQ ID NO: 123.

[0340] In some embodiments, clone CH3C.35.23.4 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), 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 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., M22Y, S24T, and T26E, numbered with reference to SEQ ID NO: 99), and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 126. In some embodiments, clone CH3C.35.23.4 having 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 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., N204S, having or not having M198L, numbered with reference to SEQ ID NO: 99), and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 127. In some embodiments, clone CH3C.35.23.4 having 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 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)), 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 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)), a mutation that increases serum stability or serum half-life (e.g., N204S having or not having 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 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 SEQ ID NO: 132. In some embodiments, clone CH3C.35.23.4 having hole mutations has the sequence of SEQ ID NO: 132.

[0346] In some embodiments, clone CH3C.35.23.4 may have hole mutations (e.g., T136S, L138A, and Y177V, numbered with reference to SEQ ID NO: 99), effector-modulating mutations (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 effector-modulating mutations has the sequence of SEQ ID NO: 133 or 134.

[0347] In some embodiments, clone CH3C.35.23.4 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., M22Y, S24T, and T26E, numbered with reference to SEQ ID NO: 99), and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 135. In some embodiments, clone CH3C.35.23.4 having a whole mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO: 135.

[0348] In some embodiments, clone CH3C.35.23.4 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., N204S, having or not having M198L, numbered with reference to SEQ ID NO: 99), and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 136. In some embodiments, clone CH3C.35.23.4 having a whole mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO: 136.

[0349] In some embodiments, clone CH3C.35.23.4 may have 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 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: 137 or 138. In some embodiments, clone CH3C.35.23.4 having a hole 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: 137 or 138.

[0350] In some embodiments, clone CH3C.35.23.4 may have 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 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 having or not having 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 a hole 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: 139 or 140.

[0351] Clone CH3C.35.23 In some embodiments, clone CH3C.35.23 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 SEQ ID NO: 141. In some embodiments, clone CH3C.35.23 having the knob mutation has the sequence of SEQ ID NO: 141.

[0352] In some embodiments, clone CH3C.35.23 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), and at least 85%, at least 90%, 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 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., M22Y, S24T, and T26E, numbered with reference to SEQ ID NO: 99), and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 144. In some embodiments, clone CH3C.35.23 having a knob mutation and a mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO: 144.

[0354] In some embodiments, clone CH3C.35.23 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., N204S, having or not having M198L, numbered with reference to SEQ ID NO: 99), and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 145. In some embodiments, clone CH3C.35.23 having both the knob mutation and the mutation that increases serum stability or serum half-life has the sequence of SEQ ID NO: 145.

[0355] In some embodiments, clone CH3C.35.23 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)), 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...

Claims

1. (a) an antibody variable region capable of binding to human epidermal growth factor receptor 2 (HER2), or an antigen-binding fragment thereof, (b) A modified Fc polypeptide dimer comprising a first Fc polypeptide that includes a modification for creating a TfR binding site An Fc polypeptide dimer-antibody variable region fusion protein containing this protein.

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

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

4. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 3, wherein the antibody variable region binds to subdomain IV of human HER2.

5. The antibody variable region is (a) Heavy chain CDR1 having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 69, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 69, (b) Heavy chain CDR2 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 70, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 70, (c) Heavy chain CDR3 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 71, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO:

71. (d) Light chain CDR1 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 72, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 72 (e) Light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 73, (f) Light chain CDR3 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 74 The Fc polypeptide dimer-antibody variable region fusion protein according to claim 4, comprising one or more complementarity-determining regions (CDRs) selected from the group consisting of the following.

6. The antibody variable region is (a) Heavy chain CDR1 containing the amino acid sequence described in SEQ ID NO: 69, (b) Heavy chain CDR2 containing the amino acid sequence described in SEQ ID NO: 70, (c) Heavy chain CDR3 containing the amino acid sequence described in SEQ ID NO: 71, (d) Light chain CDR1 containing the amino acid sequence described in Sequence ID No. 72, (e) Light chain CDR2 containing the amino acid sequence described in Sequence ID No. 73, and (f) Light chain CDR3 containing the amino acid sequence described in Sequence ID No. 74 The Fc polypeptide dimer-antibody variable region fusion protein according to claim 5, comprising one or more CDRs selected from the group consisting of the above.

7. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 4 to 6, wherein the antibody variable region comprises two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 59 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

60.

8. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 3, wherein the antibody variable region binds to subdomain II of human HER2.

9. The antibody variable region is (a) Heavy chain CDR1 having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 75, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 75 (b) Heavy chain CDR2 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 76, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 76, (c) Heavy chain CDR3 having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 77, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 77 (d) Light chain CDR1 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 78, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO:

78. (e) Light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 79, (f) Light chain CDR3 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 80 The Fc polypeptide dimer-antibody variable region fusion protein according to claim 8, comprising one or more CDRs selected from the group consisting of the above.

10. The antibody variable region is (a) Heavy chain CDR1 containing the amino acid sequence described in SEQ ID NO: 75, (b) Heavy chain CDR2 containing the amino acid sequence described in SEQ ID NO: 76, (c) Heavy chain CDR3 containing the amino acid sequence described in SEQ ID NO: 77, (d) Light chain CDR1 containing the amino acid sequence described in Sequence ID No. 78, (e) Light chain CDR2 containing the amino acid sequence described in Sequence ID No. 79, and (f) Light chain CDR3 containing the amino acid sequence described in Sequence ID No. 80 The Fc polypeptide dimer-antibody variable region fusion protein according to claim 9, comprising one or more CDRs selected from the group consisting of the following.

11. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 8 to 10, wherein the antibody variable region comprises two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 61 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

62.

12. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 3, wherein the antibody variable region binds to subdomain I of human HER2.

13. The antibody variable region is (a) Heavy chain CDR1 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 250, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 250, (b) Heavy chain CDR2 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 251, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 251, (c) Heavy chain CDR3 having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 252, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 252, (d) Light chain CDR1 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 253, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO:

253. (e) Light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 254, (f) Light chain CDR3 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 255 The Fc polypeptide dimer-antibody variable region fusion protein according to claim 12, comprising one or more CDRs selected from the group consisting of the following.

14. The antibody variable region is (a) Heavy chain CDR1 containing the amino acid sequence described in SEQ ID NO: 250, (b) Heavy chain CDR2 containing the amino acid sequence described in SEQ ID NO: 251, (c) Heavy chain CDR3 containing the amino acid sequence described in SEQ ID NO: 252, (d) Light chain CDR1 containing the amino acid sequence described in Sequence ID No. 253, (e) Light chain CDR2 containing the amino acid sequence described in Sequence ID No. 254, and (f) Light chain CDR3 containing the amino acid sequence described in Sequence ID No. 255 The Fc polypeptide dimer-antibody variable region fusion protein according to claim 13, comprising one or more CDRs selected from the group consisting of the following.

15. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 12 to 14, wherein the antibody variable region comprises two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 256 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

257.

16. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 1 to 15, wherein the TfR binding site is located within the CH3 domain.

17. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 16, wherein the modified CH3 domain is derived from a human IgG1, IgG2, IgG3, or IgG4 CH3 domain.

18. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 16 or 17, wherein the modified CH3 domain includes substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 in a set of amino acid positions including 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 according to EU numbering.

19. The modified CH3 domain, according to EU numbering, has 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, His, Gln, Leu, L An Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 16 to 18, comprising ys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr at position 413, an acidic amino acid, Ala, Ser, Leu, Thr, Pro, Ile, or His at position 415, Glu, Ser, Asp, Gly, Thr, Pro, Gln, or Arg at position 416, Thr, Arg, Asn, or an acidic amino acid, and / or an aromatic amino acid, His, or Lys at position 421.

20. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 1 to 19, wherein the Fc polypeptide dimer-antibody variable region fusion protein binds to the apical domain of TfR.

21. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 1 to 20, comprising an amino acid modification that reduces FcγR binding when the first Fc polypeptide is bound to TfR.

22. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 21, wherein the amino acid modification includes A at positions 234 and 235 according to EU numbering.

23. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 1 to 22, wherein the first Fc polypeptide and / or second Fc polypeptide present in the Fc polypeptide dimer comprises an amino acid modification that increases the serum half-life.

24. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 23, wherein the amino acid modification that increases the serum half-life comprises, according to EU numbering, (i) Leu at position 428 and Ser at position 434, or (ii) Ser or Ala at position 434.

25. An Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 1 to 24, wherein, in accordance with EU numbering, the first Fc polypeptide further comprises the knob mutation T366W, and the second Fc polypeptide present in the Fc polypeptide dimer comprises the whole mutations T366S, L368A, and Y407V.

26. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 25, wherein the first Fc polypeptide comprises the amino acid sequence of SEQ ID NO:

63.

27. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 25 or 26, wherein the second Fc polypeptide comprises one of the amino acid sequences of SEQ ID NOs. 67 and 68.

28. (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 and knob mutation T366W that creates a TfR binding site in accordance with EU numbering, (c) A second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V in accordance with EU numbering, and not containing a TfR binding site. An Fc polypeptide dimer-antibody variable region fusion protein containing this protein.

29. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 28, wherein the antibody variable region comprises two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 59 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

60.

30. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 28, wherein the antibody variable region comprises two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 61 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

62.

31. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 28, wherein the antibody variable region comprises two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 256 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

257.

32. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 28 or 29, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 1, 9, 17, and 81.

33. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 28 or 29, wherein the first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering.

34. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 33, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 2, 10, 18, and 82.

35. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 28, 29, or 32-34, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain having the amino acid sequence of SEQ ID NO:

27.

36. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 28, 29, or 32-35, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains having the amino acid sequence of SEQ ID NO:

57.

37. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 28 or 30, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 29, 37, 45, and 89.

38. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 28 or 30, wherein the first Fc polypeptide further comprises amino acid modified L234A and L235A.

39. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 38, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 30, 38, 46, and 90.

40. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 28, 30, or 37-39, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain containing the amino acid sequence of SEQ ID NO:

55.

41. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 28, 30, or 37-40, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains having the amino acid sequence of SEQ ID NO:

58.

42. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 28 or 31, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 258, 266, 274, and 282.

43. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 28 or 31, wherein the first Fc polypeptide further comprises amino acid modified L234A and L235A.

44. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 43, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 259, 267, 275, and 283.

45. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 28, 31, and 42-44, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain having the amino acid sequence of SEQ ID NO:

291.

46. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 28, 31, and 42-45, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains having the amino acid sequence of SEQ ID NO:

293.

47. (a) an antibody variable region capable of binding to human HER2, or an antigen-binding fragment thereof, (b) A first Fc polypeptide comprising, in accordance with EU numbering, a modification to create a TfR binding site, a knob mutation T366W, and an amino acid modification N434S with or without M428L, (c) A second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V in accordance with EU numbering, and not containing a TfR binding site. An Fc polypeptide dimer-antibody variable region fusion protein containing this protein.

48. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 47, wherein the antibody variable region comprises two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 59 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

60.

49. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 47, wherein the antibody variable region comprises two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 61 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

62.

50. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 47, wherein the antibody variable region comprises two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 256 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

257.

51. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 47 or 48, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 3, 11, 19, and 83.

52. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 47 or 48, wherein the first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering.

53. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 52, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 4, 12, 20, and 84.

54. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 47, 48, and 51-53, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain having the amino acid sequence of SEQ ID NO:

27.

55. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 47, 48, and 51-54, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains having the amino acid sequence of SEQ ID NO:

57.

56. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 47 or 49, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 31, 39, 47, and 91.

57. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 47 or 49, wherein the first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering.

58. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 57, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 32, 40, 48, and 92.

59. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 47, 49, or 56-58, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain having the amino acid sequence of SEQ ID NO:

55.

60. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 47, 49, or 56-59, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains having the amino acid sequence of SEQ ID NO:

58.

61. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 47 or 50, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 260, 268, 276, and 284.

62. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 47 or 50, wherein the first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering.

63. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 62, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 261, 269, 277, and 285.

64. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 47, 50, or 61-63, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain having the amino acid sequence of SEQ ID NO:

290.

65. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 47, 50, or 61-64, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains having the amino acid sequence of SEQ ID NO:

293.

66. (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 and knob mutation T366W that creates a TfR binding site in accordance with EU numbering, (c) A second Fc polypeptide comprising, in accordance with EU numbering, the whole mutations T366S, L368A, and Y407V, and the amino acid modification N434S with or without M428L, and without a TfR binding site. An Fc polypeptide dimer-antibody variable region fusion protein containing this protein.

67. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 66, wherein the antibody variable region comprises two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 59 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

60.

68. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 66, wherein the antibody variable region comprises two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 61 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

62.

69. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 66, wherein the antibody variable region comprises two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 256 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

257.

70. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 66 or 67, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 1, 9, 17, and 81.

71. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 66 or 67, wherein the first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering.

72. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 71, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 2, 10, 18, and 82.

73. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 66, 67, or 70-72, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain having the amino acid sequence of SEQ ID NO:

28.

74. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 66, 67, or 70-73, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains having the amino acid sequence of SEQ ID NO:

57.

75. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 66 or 68, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences among SEQ ID NOs: 29, 37, 45, and 89.

76. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 66 or 68, wherein the first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering.

77. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 76, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 30, 38, 46, and 90.

78. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 66, 68, or 75-77, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain having the amino acid sequence of SEQ ID NO:

56.

79. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 66, 68, or 75-78, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains having the amino acid sequence of SEQ ID NO:

58.

80. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 66 or 69, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 258, 266, 274, and 282.

81. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 66 or 69, wherein the first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering.

82. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 80, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 259, 267, 275, and 283.

83. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 66, 69, or 80-82, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain having the amino acid sequence of SEQ ID NO:

291.

84. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 66, 69, or 80-83, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains having the amino acid sequence of SEQ ID NO:

293.

85. (a) an antibody variable region capable of binding to human HER2, or an antigen-binding fragment thereof, (b) A first Fc polypeptide comprising, in accordance with EU numbering, a modification to create a TfR binding site, a knob mutation T366W, and an amino acid modification N434S with or without M428L, (c) A second Fc polypeptide comprising, in accordance with EU numbering, the whole mutations T366S, L368A, and Y407V, and the amino acid modification N434S with or without M428L, and without a TfR binding site. An Fc polypeptide dimer-antibody variable region fusion protein containing this protein.

86. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 85, wherein the antibody variable region comprises two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 59 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

60.

87. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 85, wherein the antibody variable region comprises two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 61 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

62.

88. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 85, wherein the antibody variable region comprises two antibody heavy chain variable regions containing the amino acid sequence of SEQ ID NO: 256 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

257.

89. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 85 or 86, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 3, 11, 19, and 83.

90. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 85 or 86, wherein the first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering.

91. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 90, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 4, 12, 20, and 84.

92. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 85, 86, or 89-91, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain containing the amino acid sequence of SEQ ID NO:

28.

93. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 85, 86, or 89-92, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains having the amino acid sequence of SEQ ID NO:

57.

94. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 85 or 87, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 31, 39, 47, and 91.

95. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 85 or 87, wherein the first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering.

96. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 95, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences of SEQ ID NOs: 32, 40, 48, and 92.

97. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 85, 87, or 94-96, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain containing the amino acid sequence of SEQ ID NO:

56.

98. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 85, 87, or 94-97, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains having the amino acid sequence of SEQ ID NO:

58.

99. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 85 or 88, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences among SEQ ID NOs: 260, 268, 276, and 284.

100. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 85 or 88, wherein the first Fc polypeptide further comprises amino acid modifications L234A and L235A according to EU numbering.

101. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 100, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a first heavy chain having one of the amino acid sequences among SEQ ID NOs: 261, 269, 277, and 285.

102. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 85, 88, and 99-101, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises a second heavy chain having the amino acid sequence of SEQ ID NO:

291.

103. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 85, 88, and 99-101, wherein the Fc polypeptide dimer-antibody variable region fusion protein comprises two light chains having the amino acid sequence of SEQ ID NO:

293.

104. The Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 1 to 103, wherein the modified Fc polypeptide dimer does not substantially reduce reticulocytes.

105. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 104, wherein the amount of reticulocytes decreased after administration of the Fc polypeptide dimer-antibody variable region fusion protein is less than the amount of reticulocytes decreased after control administration.

106. The Fc polypeptide dimer-antibody variable region fusion protein according to claim 105, 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 mutations that reduce FcγR binding.

107. (a) The variable region of the heavy chain of an anti-human HER2 antibody, or a fragment thereof, (b) Modified Fc polypeptide including a modification that creates a TfR binding site and Antibody heavy chains, including those mentioned above.

108. The antibody heavy chain according to claim 107, wherein the modified Fc polypeptide comprises one or more amino acid modifications that reduce FcγR binding when bound to TfR.

109. The antibody heavy chain variable region is (a) Heavy chain CDR1 having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 69, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 69, (b) Heavy chain CDR2 having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 70, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 70, (c) Heavy chain CDR3 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 71, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO:

71. The antibody heavy chain according to claim 107 or 108, comprising one or more CDRs selected from the group consisting of the following.

110. The antibody heavy chain variable region is (a) Heavy chain CDR1 containing the amino acid sequence described in SEQ ID NO: 69, (b) Heavy chain CDR2 containing the amino acid sequence described in SEQ ID NO: 70, (c) Heavy chain CDR3 containing the amino acid sequence described in SEQ ID NO: 71 The antibody heavy chain according to claim 107 or 108, comprising one or more CDRs selected from the group consisting of the following.

111. The antibody heavy chain according to any one of claims 107 to 110, wherein the antibody heavy chain variable region includes the amino acid sequence of SEQ ID NO:

59.

112. The antibody variable region is (a) Heavy chain CDR1 having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 75, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 75 (b) Heavy chain CDR2 having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 76, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 76, (c) Heavy chain CDR3 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 77, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO:

77. The antibody heavy chain according to claim 107 or 108, comprising one or more CDRs selected from the group consisting of the following.

113. The antibody variable region is (a) Heavy chain CDR1 containing the amino acid sequence described in SEQ ID NO: 75, (b) Heavy chain CDR2 containing the amino acid sequence described in SEQ ID NO: 76, (c) Heavy chain CDR3 containing the amino acid sequence described in SEQ ID NO: 77 The antibody heavy chain according to claim 107 or 108, comprising one or more CDRs selected from the group consisting of the following.

114. The antibody heavy chain according to claim 107, 108, 112, or 113, wherein the antibody heavy chain variable region includes the amino acid sequence of SEQ ID NO:

61.

115. The antibody variable region is (a) Heavy chain CDR1 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 250, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 250, (b) Heavy chain CDR2 having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 251, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 251, (c) Heavy chain CDR3 having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 252, or having up to two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO:

252. The antibody heavy chain according to claim 107 or 108, comprising one or more CDRs selected from the group consisting of the following.

116. The antibody variable region is (a) Heavy chain CDR1 containing the amino acid sequence described in SEQ ID NO: 250, (b) Heavy chain CDR2 comprising the amino acid sequence described in SEQ ID NO: 251, (c) Heavy chain CDR3 containing the amino acid sequence described in SEQ ID NO: 252 The antibody heavy chain according to claim 107 or 108, comprising one or more CDRs selected from the group consisting of the following.

117. The antibody heavy chain according to claim 107, 108, 115, or 116, wherein the antibody heavy chain variable region includes the amino acid sequence of SEQ ID NO:

256.

118. The antibody heavy chain according to any one of claims 107 to 117, wherein the TfR binding site comprises a modified CH3 domain.

119. The antibody heavy chain according to claim 118, wherein the modified CH3 domain is derived from a human IgG1, IgG2, IgG3, or IgG4 CH3 domain.

120. The antibody heavy chain according to claim 118 or 119, wherein the modified CH3 domain includes substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 in a set of amino acid positions including 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 according to EU numbering.

121. The modified CH3 domain, according to EU numbering, has 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, His at position 390. The antibody heavy chain according to any one of claims 118 to 120, comprising, , Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr at position 413, an acidic amino acid, Ala, Ser, Leu, Thr, Pro, Ile, or His at position 415, Glu, Ser, Asp, Gly, Thr, Pro, Gln, or Arg at position 416, Thr, Arg, Asn, or an acidic amino acid, and / or an aromatic amino acid, His, or Lys at position 421.

122. The antibody heavy chain according to any one of claims 108 to 121, wherein the amino acid modification that reduces FcγR binding when bound to TfR comprises Ala at positions 234 and 235 according to EU numbering.

123. The antibody heavy chain according to any one of claims 107 to 122, wherein the modified Fc polypeptide further comprises an amino acid modification that increases the serum half-life.

124. The antibody heavy chain according to claim 123, wherein the amino acid modification that increases the serum half-life comprises, according to EU numbering, (i) Leu at position 428 and Ser at position 434, or (ii) Ser or Ala at position 434.

125. The antibody heavy chain according to any one of claims 107 to 124, wherein the modified Fc polypeptide further comprises the knob mutation T366W according to EU numbering.

126. The antibody heavy chain according to claim 125, wherein the modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO:

63.

127. (a) The variable region of the heavy chain of an anti-human HER2 antibody, or a fragment thereof, (b) Modified Fc polypeptides containing modifications and knob mutations T366W that create a TfR binding site in accordance with EU numbering. Antibody heavy chains, including those mentioned above.

128. The antibody heavy chain according to claim 127, wherein the antibody heavy chain variable region includes the amino acid sequence of SEQ ID NO:

59.

129. The antibody heavy chain according to claim 127, wherein the antibody heavy chain variable region includes the amino acid sequence of SEQ ID NO:

61.

130. The antibody heavy chain according to claim 127, wherein the antibody heavy chain variable region includes the amino acid sequence of SEQ ID NO:

256.

131. The antibody heavy chain according to claim 128, wherein the antibody heavy chain comprises one amino acid sequence from sequence numbers 1, 9, 17, and 81.

132. The antibody heavy chain according to claim 129, wherein the antibody heavy chain comprises one amino acid sequence from among SEQ ID NOs: 29, 37, 45, and 89.

133. The antibody heavy chain according to claim 130, wherein the antibody heavy chain comprises one amino acid sequence from among SEQ ID NOs. 258, 266, 274, and 282.

134. The antibody heavy chain according to any one of claims 127 to 133, further comprising amino acid modifications L234A and L235A according to EU numbering.

135. The antibody heavy chain according to claim 134, wherein the antibody heavy chain comprises one of the amino acid sequences of SEQ ID NOs: 2, 10, 18, and 82.

136. The antibody heavy chain according to claim 134, wherein the antibody heavy chain comprises one amino acid sequence from among SEQ ID NOs: 30, 38, 46, and 90.

137. The antibody heavy chain according to claim 134, wherein the antibody heavy chain comprises one amino acid sequence from among SEQ ID NOs: 259, 267, 275, and 283.

138. (a) The variable region of the heavy chain of an anti-human HER2 antibody, or a fragment thereof, (b) Modified Fc polypeptides comprising, in accordance with EU numbering, a modification to create a TfR binding site, a knob mutation T366W, and an amino acid modification N434S with or without M428L, and Antibody heavy chains, including those mentioned above.

139. The antibody heavy chain according to claim 138, wherein the antibody heavy chain variable region includes the amino acid sequence of SEQ ID NO:

59.

140. The antibody heavy chain according to claim 138, wherein the antibody heavy chain variable region includes the amino acid sequence of SEQ ID NO:

61.

141. The antibody heavy chain according to claim 138, wherein the antibody heavy chain variable region includes the amino acid sequence of SEQ ID NO:

256.

142. The antibody heavy chain according to claim 139, wherein the antibody heavy chain comprises one amino acid sequence from among SEQ ID NOs: 3, 11, 19, and 83.

143. The antibody heavy chain according to claim 140, wherein the antibody heavy chain comprises one amino acid sequence from among SEQ ID NOs: 31, 39, 47, and 91.

144. The antibody heavy chain according to claim 140, wherein the antibody heavy chain comprises one amino acid sequence from among SEQ ID NOs: 260, 268, 276, and 284.

145. The antibody heavy chain according to any one of claims 138 to 144, further comprising amino acid modifications L234A and L235A according to EU numbering.

146. The antibody heavy chain according to claim 145, wherein the antibody heavy chain comprises one of the amino acid sequences of SEQ ID NOs: 4, 12, 20, and 84.

147. The antibody heavy chain according to claim 145, wherein the antibody heavy chain comprises one amino acid sequence from among SEQ ID NOs: 32, 40, 48, and 92.

148. The antibody heavy chain according to claim 145, wherein the antibody heavy chain comprises one amino acid sequence from among SEQ ID NOs: 261, 269, 277, and 285.

149. A pharmaceutical composition comprising an Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 1 to 106, and a pharmaceutically acceptable carrier.

150. A method for transcytosis across the endothelium of an antibody variable region or an antigen-binding fragment thereof that can bind to human HER2, the method comprising contacting the endothelium with a composition comprising an Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 1 to 106.

151. The method according to claim 150, wherein the endothelium is BBB.

152. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising an Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 1 to 106.

153. The method according to claim 152, wherein the cancer is HER2-positive cancer.

154. The method according to claim 153, wherein the HER2-positive cancer is HER2-positive breast cancer.

155. The method according to claim 153, wherein the HER2-positive cancer is HER2-positive gastric adenocarcinoma and / or HER2-positive esophagogastric junction adenocarcinoma.

156. The method according to any one of claims 153 to 155, wherein the HER2-positive cancer is metastatic cancer.

157. A method for treating brain metastases of HER2-positive cancer in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising an Fc polypeptide dimer-antibody variable region fusion protein according to any one of claims 1 to 106.

158. The method according to claim 157, wherein the HER2-positive cancer is HER2-positive breast cancer.

159. The method according to claim 157, wherein the HER2-positive cancer is HER2-positive gastric adenocarcinoma and / or HER2-positive esophagogastric junction adenocarcinoma.

160. The method according to any one of claims 152 to 159, wherein the composition comprising the Fc polypeptide dimer-antibody variable region fusion protein antagonistizes HER2 activity.

161. The first Fc polypeptide dimer-antibody variable region fusion protein and the 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 containing the amino acid sequence of SEQ ID NO: 59 and two light chain variable regions containing 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 containing the amino acid sequence of SEQ ID NO: 61 and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

62. The method according to any one of claims 152 to 160.

162. The first Fc polypeptide dimer-antibody variable region fusion protein and the 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 containing the amino acid sequence of SEQ ID NO: 59 and two light chain variable regions containing 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 containing the amino acid sequence of SEQ ID NO: 256, and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

257. The method according to any one of claims 152 to 160.

163. The first Fc polypeptide dimer-antibody variable region fusion protein and the 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 containing the amino acid sequence of SEQ ID NO: 61 and two light chain variable regions containing 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 containing the amino acid sequence of SEQ ID NO: 256, and two light chain variable regions containing the amino acid sequence of SEQ ID NO:

257. The method according to any one of claims 152 to 160.

164. The method according to any one of claims 152 to 163, wherein the subject has not been previously treated with anti-HER2 therapy and / or chemotherapy for metastatic disease.

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