Anti-HER2 antibodies and methods of use thereof

Abstract

In one embodiment, an antibody is provided that binds to subdomain II of human HER2. In another embodiment, an antibody is provided that comprises a light chain polypeptide paired with both a heavy chain polypeptide for binding to subdomain II of human HER2 and a heavy chain polypeptide for binding to subdomain IV of human HER2. In a further embodiment, an antibody is provided that binds to both subdomain II and subdomain IV of human HER2, comprising a common light chain polypeptide. Methods of treating cancer or treating brain metastasis of cancer using these antibodies are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 237,104, filed August 25, 2021, the disclosure of which is incorporated by reference in its entirety herein for all purposes. [Background technology]

[0002] background The treatment of brain metastases of cancers such as breast cancer currently represents a formidable clinical challenge. In breast cancer patients, the incidence of brain metastases is as high as 50%. Clinical data has shown that HER2-positive breast cancers are prone to metastasize to the brain. In particular, anti-HER2 therapies have proven useful in controlling extracranial tumors, but not intracranial lesions. The failure of these therapies to control metastatic lesions such as brain metastases of HER2-positive breast cancer is mainly due to the inability of therapeutic agents to cross the blood-brain barrier (BBB) ​​and access the brain parenchyma. Summary of the Invention

[0003] overview In one aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 89; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 90; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 91 An isolated antibody comprising one or more (e.g., one, two, or all three) complementarity determining regions (CDRs) selected from the group consisting of: below: X1 of SEQ ID NO:89 is not T; X2 of SEQ ID NO:89 is not F; X3 of SEQ ID NO:89 is not T; X1 in SEQ ID NO:90 is not N; X2 of SEQ ID NO:90 is not N; X3 of SEQ ID NO:90 is not S; X4 of SEQ ID NO:90 is not G; X5 of SEQ ID NO:90 is not G; X6 in SEQ ID NO:90 is not Q; X1 in SEQ ID NO:91 is not L; X2 of SEQ ID NO:91 is not G; X3 of SEQ ID NO:91 is not P; and X4 in SEQ ID NO:91 is not S The present invention provides an isolated antibody, wherein the antibody is at least one of:

[0004] In some embodiments, the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 89 and X1 is N, K, M, or H. In some embodiments, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 90 and X5 is Q. In some embodiments, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 90 and X6 is R, H, or T. In some embodiments, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 91 and X4 is W, F, D, L, or Y. In some embodiments, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 91 and X4 is L.

[0005] In some embodiments, the antibody comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 89; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 90, wherein X5 is Q; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 91, and X4 is L.

[0006] In some embodiments, the antibody comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 49 to 52, or having up to two amino acid substitutions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 49 to 52; (b) a heavy chain CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 6 and 53 to 55, or having up to two amino acid substitutions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 6 and 53 to 55; and (c) a heavy chain CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 8 and 56 to 59, or having up to two amino acid substitutions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 8 and 56 to 59.

[0007] In some embodiments, the antibody comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:4, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6; and (c) a heavy chain CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.

[0008] In some embodiments, the antibody comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.

[0009] In some embodiments, the antibody comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:6; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:7.

[0010] In some embodiments, the antibody comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:5; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:8.

[0011] In some embodiments, the antibody comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:6; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:8.

[0012] In some embodiments, the antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-3. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 1-3.

[0013] In a related aspect, the disclosure provides an isolated antibody heavy chain comprising one or more (e.g., one, two, or all three) of the above CDRs. In some embodiments, the antibody heavy chain comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-3. In some embodiments, the antibody heavy chain comprises a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 1-3.

[0014] In another aspect, the disclosure provides an isolated antibody comprising: (a) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13 or 14.

[0015] In some embodiments, the antibody further comprises one or more (e.g., one or both) CDRs selected from the group consisting of: (b) a light chain CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:11, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:11; and (c) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO:12.

[0016] In some embodiments, the antibody further comprises one or more (e.g., one or both) CDRs selected from the group consisting of: (b) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11; and (c) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12.

[0017] In some embodiments, the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 14.

[0018] In some embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 9-10. In some embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 9-10.

[0019] In a related aspect, the disclosure provides an isolated antibody light chain comprising one or more (e.g., one, two, or all three) of the above CDRs. In some embodiments, the antibody light chain comprises a light chain variable region comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs:9-10. In some embodiments, the antibody light chain comprises a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOs:9-10.

[0020] In yet another aspect, the disclosure provides an isolated antibody comprising an antigen-binding site comprising: (a) a heavy chain CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 49 to 52, or having up to two amino acid substitutions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 49 to 52; (b) a heavy chain CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 6 and 53 to 55, or having up to two amino acid substitutions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 6 and 53 to 55; (c) a heavy chain CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 8 and 56 to 59, or having up to two amino acid substitutions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 8 and 56 to 59; (d) a light chain CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:11, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:11; (e) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 12; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13 or 14.

[0021] In some embodiments, the antigen binding site comprises: (a) a heavy chain CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:4, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6; (c) a heavy chain CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8; (d) a light chain CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:11, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:11; (e) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 12; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13 or 14.

[0022] In some embodiments, the antigen-binding site comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 to 3, and a light chain variable region comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 9 to 10. In some embodiments, the antigen-binding site comprises a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 3, and a light chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 9 to 10.

[0023] In some embodiments, the antibody further comprises a second antigen-binding site comprising one or more CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 16; (b) a heavy chain CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 17, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 17; and (c) a heavy chain CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:18, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:18.

[0024] In some embodiments, the second antigen-binding site comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 15. In some embodiments, the second antigen-binding site comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 15.

[0025] In some embodiments, the second antigen binding site further comprises one or more CDRs selected from the group consisting of: (a) a light chain CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:11, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:11; (b) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 12; and (c) a light chain CDR3 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 13 or 14.

[0026] In some embodiments, the second antigen-binding site comprises a light chain variable region comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of SEQ ID NOs: 9 to 10. In some embodiments, the second antigen-binding site comprises a light chain variable region comprising the sequence of any one of SEQ ID NOs: 9 to 10.

[0027] In some embodiments, the first and second antigen-binding sites comprise the same light chain CDR1, CDR2, and CDR3 sequences. In some embodiments, the antibody comprises heavy and light chain CDRs selected from the combinations listed in Table 1.

[0028] In a related aspect, the disclosure provides an isolated antibody comprising a heavy chain and a light chain selected from the combinations listed in Table 2.

[0029] In a further aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: (a) a first antigen-binding site against human epidermal growth factor receptor 2 (HER2) subdomain IV; (b) a second antigen-binding site against human HER2 subdomain II; and (c) a modified Fc polypeptide dimer comprising a first Fc polypeptide containing a modification that creates a TfR binding site. 1. An isolated antibody comprising: the light chain polypeptide sequence of the first antigen-binding site is identical to the light chain polypeptide sequence of the second antigen-binding site; Isolated antibodies are provided.

[0030] In another aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: (a) a first antigen-binding site against human HER2 subdomain II; (b) a second antigen-binding site against human HER2 subdomain IV; and (c) a modified Fc polypeptide dimer comprising a first Fc polypeptide containing a modification that creates a TfR binding site. 1. An isolated antibody comprising: the light chain polypeptide sequence of the first antigen-binding site is identical to the light chain polypeptide sequence of the second antigen-binding site; Isolated antibodies are provided.

[0031] In some embodiments, the first Fc polypeptide comprises a modified CH3 domain that comprises a TfR binding site, hi some embodiments, the modified CH3 domain is derived from a CH3 domain of human IgG1, IgG2, IgG3, or IgG4.

[0032] In some embodiments, the modified CH3 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions at a set of amino acid positions including 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 according to EU numbering. In some embodiments, the modified CH3 domain comprises Glu, Leu, Ser, Val, Trp, Tyr, or Gln at position 380, Leu, Tyr, Phe, Trp, Met, Pro, or Val at position 384, Leu, Thr, His, Pro, Asn, Val, or Phe at position 386, Val, Pro, Ile, or an acidic amino acid at position 387, Trp at position 388, an aliphatic amino acid, Gly, Ser, Thr, or an aliphatic amino acid at position 389 according to EU numbering. at position 390; 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.

[0033] In some embodiments, the first Fc polypeptide containing a modification that creates a TfR binding site binds to the apical domain of TfR.

[0034] In some embodiments, the first Fc polypeptide and the second Fc polypeptide each comprise a modification that promotes heterodimerization. In some embodiments, the first Fc polypeptide comprises a T366W substitution and the second Fc polypeptide comprises a T366S, L368A, and Y407V substitution according to EU numbering. In other embodiments, the first Fc polypeptide comprises a T366S, L368A, and Y407V substitution and the second Fc polypeptide comprises a T366W substitution according to EU numbering.

[0035] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise a modification that reduces TfR-mediated effector function. In some embodiments, the modification that reduces effector function is a substitution of L234A and L235A according to EU numbering. In certain embodiments, the first Fc polypeptide specifically binds to TfR and comprises a substitution of L234A and L235A. In certain embodiments, the first Fc polypeptide further comprises a substitution of P329G or P329S according to EU numbering. In certain embodiments, the second Fc polypeptide comprises Leu at positions 234 and 235 and Proline at position 329 according to EU numbering. In other embodiments, the second Fc polypeptide specifically binds to TfR and comprises a substitution of L234A and L235A. In certain embodiments, the second Fc polypeptide further comprises a substitution of P329G or P329S according to EU numbering. In a specific embodiment, the first Fc polypeptide comprises Leu at positions 234 and 235 and a proline at position 329 according to EU numbering.

[0036] In some embodiments, the hinge region, or a portion thereof, is linked to the N-terminus of the first Fc polypeptide and / or the second Fc polypeptide.

[0037] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence selected from the group consisting of SEQ ID NOs: 71-86 and 98-100. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence selected from the group consisting of SEQ ID NOs: 71-73, 85, and 99-100. In other embodiments, the first Fc polypeptide or the second Fc polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence selected from the group consisting of SEQ ID NOs: 74-84, 86, and 98.

[0038] In some embodiments of this antibody, the first antigen-binding site comprises the amino acid sequence of SEQ ID NO: 15; the second antigen-binding site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 60-70; the first Fc polypeptide containing a modification that creates a TfR-binding site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-84, 86, and 98; and the light chain polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In some embodiments, the antibody further comprises a second Fc polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-73, 85, and 99-100.

[0039] In other embodiments of this antibody, the first antigen-binding site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 60-70; the second antigen-binding site comprises the amino acid sequence of SEQ ID NO: 15; the first Fc polypeptide containing a modification that creates a TfR-binding site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-84, 86, and 98; and the light chain polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In some embodiments, the antibody further comprises a second Fc polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-73, 85, and 99-100.

[0040] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or an I332E substitution according to EU numbering. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or an I332E substitution are capable of enhancing HER2-mediated effector function.

[0041] In some embodiments of the antibody, (a) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (b) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (c) the first Fc polypeptide comprises an S239D and I332E substitution, and the second Fc polypeptide comprises an S239D substitution, according to EU numbering; (d) the second Fc polypeptide comprises a S239D substitution according to EU numbering; (e) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (f) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (g) the first Fc polypeptide comprises an S239D and an I332E substitution, and the second Fc polypeptide comprises an I332E substitution, according to EU numbering; (h) the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (i) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; (j) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; (k) the first Fc polypeptide comprises an S239D and I332E substitution, and the second Fc polypeptide comprises an S239D and I332E substitution, according to EU numbering; (l) the second Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E; (m) the first Fc polypeptide comprises a S239D substitution, according to EU numbering; (n) the first Fc polypeptide comprises a I332E substitution, according to EU numbering; or (o) The first Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E.

[0042] In certain embodiments of this antibody, (a) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (b) the first Fc polypeptide comprises an S239D and I332E substitution, and the second Fc polypeptide comprises an S239D substitution, according to EU numbering; (c) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (d) the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (e) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; or (f) the first Fc polypeptide comprises an I332E substitution, according to EU numbering.

[0043] In certain embodiments of this antibody, (a) a first Fc polypeptide comprises a I332E substitution and a serine at position 239, and a second Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, according to EU numbering; (b) the first Fc polypeptide comprises an S239D and an I332E substitution, and the second Fc polypeptide comprises an S239D substitution and an isoleucine at position 332, according to EU numbering; (c) the first Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, and the second Fc polypeptide comprises a I332E substitution and a serine at position 239, according to EU numbering; (d) the first Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, and the second Fc polypeptide comprises a I332E substitution and a serine at position 239, according to EU numbering; (e) the first Fc polypeptide comprises an S239D substitution and an isoleucine at position 332, and the second Fc polypeptide comprises an S239D and an I332E substitution, according to EU numbering; or (f) the first Fc polypeptide comprises a I332E substitution and a serine at position 239, and the second Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, according to EU numbering.

[0044] In some embodiments, the antibody comprises two heavy chains and two light chains. In certain embodiments, the antibody comprises a heavy chain and a light chain selected from the combinations listed in Table 2. In certain embodiments, the first heavy chain comprises a V H and an Fc sequence, and the second heavy chain comprises a V selected from the combination of Table 4. H and an Fc sequence. In certain embodiments, the first heavy chain comprises a V H and an Fc sequence, and the second heavy chain comprises a V H and an Fc sequence.

[0045] In another aspect, the disclosure provides a pharmaceutical composition comprising any of the antibodies described herein and a pharma- ceutically acceptable carrier.

[0046] In another aspect, the disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding an antibody described herein.

[0047] In another aspect, the present disclosure provides a vector comprising a polynucleotide of the preceding aspect.

[0048] In another aspect, the disclosure provides a host cell comprising the polynucleotide or vector.

[0049] In another aspect, the disclosure provides a method for treating cancer or treating brain metastasis of cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or pharmaceutical composition thereof described herein.

[0050] In some embodiments, the antibody is administered in combination with chemotherapy or radiation therapy. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is a breast cancer. In some embodiments, the cancer is a HER2 positive cancer. [Brief description of the drawings]

[0051] [Figure 1] FIG. 1 is a schematic diagram showing an exemplary bispecific antibody having a first antigen-binding site against human HER2 subdomain IV ("anti-HER2_D4") and a second antigen-binding site against human HER2 subdomain II ("anti-HER2_D2"), where the first and second antigen-binding sites comprise identical light chain polypeptides and an Fc polypeptide dimer comprising a TfR binding site and a first Fc polypeptide having a knob mutation and a second Fc polypeptide having a hole mutation. [Diagram 2] The results of the growth inhibition assay on ZR-75-30 cells are shown, as well as the IC50 and maximum % growth inhibition values ​​of different antibodies in Table 12. [Diagram 3] A and B show in vivo antitumor activity tumor in a single dose study with ATV:CLC bispecific antibodies in two human cell line derived xenograft models. A: BT-474; B: OE19. [Figure 4] A and B show in vivo antitumor activity tumor in a single dose study with ATV:CLC bispecific antibodies in two human cell line derived xenograft models. A: BT-474; B: OE19. [Diagram 5] A and B show in vivo antitumor activity in a multi-dose study with an ATV:CLC bispecific antibody in two human cell line-derived xenograft models. A: BT-474; B: OE19. [Figure 6] 1 shows brain uptake of ATV:CLC bispecific antibody. [Figure 7] A and B show IHC brain distribution of CLC bispecific antibody. [Figure 8] 1 shows plasma PK in a single dose study with an ATV:CLC bispecific antibody in cynomolgus monkeys. [Figure 9] 1 shows ADCC of the ATV:CLC bispecific antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Detailed Description I. Introduction Described herein are anti-HER2 bispecific antibodies that utilize a common light chain approach, i.e., two antigen-binding domains that are paired with the same light chain but still retain separate specificities. The use of a common light chain prevents mispairing of light chains, thereby facilitating the manufacture of these bispecific antibodies. In some embodiments, the bispecific antibody comprises a first antigen-binding site for human HER2 subdomain IV and a second antigen-binding site for human HER2 subdomain II, and the light chain polypeptide sequence of the first antigen-binding site is identical to the light chain polypeptide sequence of the second antigen-binding site. In another embodiment, the bispecific antibody comprises a first antigen-binding site for human HER2 subdomain II and a second antigen-binding site for human HER2 subdomain IV, and the light chain polypeptide sequence of the first antigen-binding site is identical to the light chain polypeptide sequence of the second antigen-binding site.

[0053] Furthermore, previous therapies have failed to control brain metastasis of HER2-positive breast cancer, mainly due to the inability of the therapeutics to cross the blood-brain barrier (BBB) ​​and access the brain parenchyma. Thus, there is a need for new therapeutics that can cross the BBB and target HER2 in the brain parenchyma. We have previously described the use of transferrin receptor (TfR) binding as a method to enable BBB delivery across brain endothelium, as TfR expression is highly expressed on brain endothelial cells and can enable BBB delivery by receptor-mediated transcytosis. Interestingly, TfR is highly expressed in various cancers, including HER2-positive breast cancer. The mechanism by which cancer cells acquire increased TfR expression may be related to tumor cell proliferation and increased metabolic demands, such as iron uptake. Indeed, a publicly available microarray dataset has demonstrated a correlation between TfR expression and breast cancer prognosis (Miller et al., Cancer Res. 71:6728, 2011). There are also several reports on the use of TfR as a pharmacological target for various types of cancer.

[0054] In some embodiments, the anti-HER2 bispecific antibody comprises one or more modified Fc polypeptides (i.e., TfR-binding Fc polypeptides) that specifically bind to a BBB receptor, e.g., TfR. In some embodiments, the anti-HER2 bispecific antibody is capable of transport across the BBB. In some embodiments, the anti-HER2 bispecific antibodies that bind to both HER2 and TfR described herein may provide additional anti-tumor effects when binding to HER2-positive tumor cells that also express high levels of TfR, compared to other therapeutic agents that only bind to HER2. Specifically, these antibodies may bind to both TfR and HER2 simultaneously, thereby increasing their potency and / or efficacy.

[0055] II. Definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to an "antibody" optionally includes a combination of two or more such molecules, and the like.

[0056] As used herein, the terms "about" and "approximately," when used to modify a quantity stated in a numerical value or range, indicate that reasonable variations from that numerical value and values ​​known to one of ordinary skill in the art, such as ±20%, ±10%, or ±5%, are within the intended meaning of the stated value.

[0057] The terms "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 a tyrosine receptor kinase protein encoded by the ERBB2 gene in humans, a member of the human epidermal growth factor receptor (HER / EGFR / ERBB) family. Amplification or overexpression of HER2 plays a prominent role in the development and progression of certain aggressive types of cancer, including breast cancer. Non-limiting examples of human HER2 nucleotide sequences are set forth in GenBank Reference Nos. NP_001005862, NP_001289936, NP_001289937, NP_001289938, and NP_004448. Non-limiting examples of human HER2 peptide sequences are set forth in GenBank Reference Nos. NP_001005862, NP_001276865, NP_001276866, NP_001276867, and NP_004439.

[0058] The extracellular domain of HER2, which contains approximately 600 amino acids, contains four subdomains (subdomains I, II, III, and IV). Subdomains I and III form the ligand-binding site. Cysteine-rich subdomains II and IV are involved in receptor homodimerization and heterodimerization. Anti-HER2 antibodies may bind to specific subdomains (e.g., subdomain II and / or subdomain IV).

[0059] As used herein, the terms "anti-HER2_D2" or "anti-HER2_D4" refer to antibodies that bind to subdomains II or IV, respectively, of human HER2.

[0060] As used herein, the term "antibody" refers to a protein having an immunoglobulin fold and specifically binds to an antigen through its variable region. The term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single chain antibodies, multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, and human antibodies. As used herein, the term "antibody" also includes antibody fragments that retain antigen-binding specificity, including, but not limited to, Fab, F(ab')2, Fv, scFv, and bivalent scFv. Antibodies may contain light chains, which are classified as either kappa or lambda. Antibodies may contain heavy chains, which are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0061] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The "variable light chain" (VLC) is L ) and "variable heavy chain" (V H ) refer to these light and heavy chains respectively.

[0062] The term "variable region" or "variable domain" refers to the domain in an antibody heavy or light chain that is derived from germline variable (V), diversity (D), or joining (J) genes (and not from constant (Cμ and Cδ) gene segments) and confers to the antibody its antigen-binding specificity. Typically, antibody variable regions contain four conserved "framework" regions interspersed with three hypervariable "complementarity determining regions".

[0063] The term "complementarity determining region" or "CDR" refers to the three hypervariable regions in each chain that interrupt the four framework regions established by the light and heavy chain variable regions. The CDRs are primarily responsible for antibody binding to an epitope of an antigen. The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also usually identified by the chain in which a particular CDR is located. Thus, the V H CDR3 or CDR-H3 is located in the variable region of the antibody heavy chain in which it is found, whereas V L CDR1 or CDR-L1 is the CDR1 from the variable region of the antibody light chain in which it is found.

[0064] The "framework regions" or "FRs" of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, functions to position and align the CDRs in three-dimensional space. Framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBASE2" germline variable gene sequence database for human and mouse sequences.

[0065] The amino acid sequences of the CDRs and framework regions can be determined using various definitions known in the art, such as Kabat, Chothia, the International ImMunoGeneTics database (IMGT), AbM, and observed antigen contact ("Contact"). In some embodiments, the CDRs are determined according to the Contact definition. See MacCallum et al., J. Mol. Biol., 262:732-745, 1996. In some embodiments, the CDRs are determined by a combination of the Kabat, Chothia, and / or Contact CDR definitions.

[0066] The term "epitope" refers to the area or region of a molecule, e.g., an antigen, to which the CDR of an antibody specifically binds, and may include a few amino acids, or a portion of a few amino acids, e.g., 5 or 6, or more, e.g., 20 or more amino acids, or a portion of those amino acids. In some cases, the epitope includes non-protein components, e.g., derived from carbohydrates, nucleic acids, or lipids. In some cases, the epitope is a three-dimensional portion. Thus, for example, if the target is a protein, the epitope may consist of contiguous amino acids (e.g., a linear epitope) or may consist of amino acids from different parts of the protein that are brought into close proximity by protein folding (e.g., a discontinuous or conformational epitope).

[0067] As used herein, the phrase "recognizes an epitope," when used in reference to an antibody, means that the CDRs of the antibody interact with or specifically bind to the antigen at the epitope or a portion of the antigen that contains the epitope.

[0068] A "humanized antibody" is a chimeric immunoglobulin derived from a non-human source (e.g., mouse) that contains minimal sequence derived from the non-human immunoglobulin outside the CDRs. In general, a humanized antibody contains at least one (e.g., two) variable domains, the CDR regions of which correspond substantially to those of a non-human immunoglobulin and the framework regions of which correspond substantially to those of a human immunoglobulin sequence. In some cases, certain framework region residues of the human immunoglobulin can be replaced with corresponding residues from the non-human species, e.g., to improve specificity, affinity, and / or serum half-life. A humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), usually of a human immunoglobulin sequence. Methods for humanizing antibodies are known in the art.

[0069] A "human antibody" or "fully human antibody" is an antibody that has human heavy and light chain sequences, typically derived from human germline genes. In some embodiments, the antibody is produced by human cells, by non-human animals utilizing the human antibody repertoire (e.g., transgenic mice genetically engineered to express human antibody sequences), or by phage display platforms.

[0070] The term "specifically binds" refers to a molecule (e.g., an antibody as described herein) that binds to an epitope or target in a sample with higher affinity, higher avidity, and / or longer duration than it binds to another epitope or non-target compound (e.g., a structurally distinct antigen). In some embodiments, a molecule that specifically binds to an epitope or target is one that binds to an epitope or target with at least 5-fold higher affinity, e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, or 10,000-fold or more affinity, than it binds to another epitope or non-target compound. As used herein, the terms "specific binding," "specifically binds," or "is specific" for a particular epitope or target refers to, for example, the equilibrium dissociation constant K for the epitope or target to which it binds. D But, for example, 10 -4 M or less, e.g., 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M. One of skill in the art will recognize that a molecule that specifically binds to a target from one species may also specifically bind to an ortholog of that target.

[0071] The term "binding affinity" is used herein to refer to the strength of a non-covalent interaction between two molecules, for example, an antibody as described herein, and an antigen. Thus, for example, unless otherwise indicated or clear from the context, the term may refer to a 1:1 interaction between an antibody and an antigen. Binding affinity is measured by the equilibrium dissociation constant (K D ), which can be quantified by measuring the binding rate constant (k a ,time -1 M -1 ) divided by the dissociation rate constant (k d ,time -1 ) refers to D can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) techniques (e.g., Biacore™ systems); equilibrium exclusion techniques such as KinExA®, and BioLayer interferometry (e.g., using the ForteBio® Octet platform). As used herein, "binding affinity" refers not only to formal binding affinity, which reflects a 1:1 interaction between an antibody and an antigen, but also to K, which can reflect strong binding. D The apparent affinity is also included, where the affinity is calculated as

[0072] As used herein, "transferrin receptor" or "TfR" refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is set forth in SEQ ID NO: 150. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee, accession number XP_003310238.1; rhesus monkey, NP_001244232.1; dog, NP_001003111.1; cow, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP_990587.1). The term "transferrin receptor" also encompasses allelic variants of exemplary reference sequences encoded by genes at the transferrin receptor protein 1 chromosomal locus, e.g., human sequences. The full-length transferrin receptor protein includes a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain.

[0073] As used herein, the term "Fc polypeptide" refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide characterized by an Ig fold as a structural domain. An Fc polypeptide comprises a constant region sequence comprising at least a CH2 domain and / or a CH3 domain, and may include at least a portion of the hinge region, but does not include the variable region.

[0074] A "modified Fc polypeptide" refers to an Fc polypeptide that has at least one mutation, e.g., a substitution, deletion, or insertion, relative to a wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retains the overall Ig fold or structure of a native Fc polypeptide.

[0075] As used herein, "FcRn" refers to the fetal Fc receptor. Binding of an Fc polypeptide to FcRn reduces the clearance and extends the serum half-life of the Fc polypeptide. The human FcRn protein is a heterodimer consisting of a protein approximately 50 kDa in size similar to major histocompatibility (MHC) class I proteins and β2-microglobulin, approximately 15 kDa in size.

[0076] As used herein, "FcRn binding site" refers to a region of an Fc polypeptide that binds to FcRn. In human IgG, the FcRn binding site, as numbered by the EU index, includes 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:95.

[0077] As used herein, a "native FcRn-binding site" refers to a region of an Fc polypeptide that binds to FcRn and has the same amino acid sequence as a region of a naturally occurring Fc polypeptide that binds to FcRn.

[0078] As used herein, the terms "CH3 domain" and "CH2 domain" refer to immunoglobulin constant region domain polypeptides. For purposes of this application, a CH3 domain polypeptide refers to the segment of amino acids from about position 341 to about position 447 as numbered according to the EU numbering scheme, and a CH2 domain polypeptide refers to the segment of amino acids from about position 231 to about position 340 as numbered according to the EU numbering scheme, not including the sequence of the hinge region. Polypeptides of CH2 and CH3 domains may also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, where the numbering of the CH2 domain is 1-110 and the numbering of the CH3 domain is 1-107 according to the numbering of the IMGT Scientific chart (IMGT website). The CH2 and CH3 domains are part of the Fc region of an immunoglobulin. Fc region, as used herein, refers to the segment of amino acids from about 231 to about 447, numbered according to the EU numbering scheme, but may also include at least a portion of the hinge region of an antibody. An exemplary hinge region sequence is the human IgG1 hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO:96).

[0079] The terms "wild-type," "native," and "naturally occurring" as used with respect to a CH3 or CH2 domain refer to a domain having a sequence that occurs in nature.

[0080] As used herein, the term "mutant" when used in reference to a mutant polypeptide or mutant polynucleotide is used interchangeably with "variant". Variants with respect to a given wild-type CH3 or CH2 domain reference sequence can include naturally occurring allelic variants. A "non-natural" CH3 or CH2 domain refers to a polynucleotide or polypeptide variant or mutant domain of a natural CH3 or CH2 domain that does not occur in a natural cell and is created by genetic modification, for example, using genetic engineering or mutagenesis techniques. A "variant" includes any domain that contains at least one amino acid mutation with respect to the wild type. Mutations can include substitutions, insertions, and deletions.

[0081] The term "isolated" as used with respect to a nucleic acid or protein indicates that the nucleic acid or protein is essentially free of other cellular components with which it is naturally associated. It is preferably in a homogeneous state. Purity and homogeneity are usually measured using analytical chemistry techniques, such as electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high performance liquid chromatography). In some embodiments, an isolated nucleic acid or protein is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure.

[0082] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof. "Amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid."Amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0083] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. An amino acid polymer can contain entirely L-amino acids, entirely D-amino acids, or a mixture of L- and D-amino acids.

[0084] As used herein, the term "protein" refers to either a single polypeptide chain or a dimer (i.e., two) or multimer (i.e., three or more). The single polypeptide chains of a protein can be linked by covalent bonds, e.g., disulfide bonds, or by non-covalent interactions.

[0085] The terms "polynucleotide" and "nucleic acid" refer interchangeably to a chain of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. Examples of polynucleotides contemplated herein include single- and double-stranded DNA, single- and double-stranded RNA, and hybrid molecules having a mixture of single- and double-stranded DNA and RNA.

[0086] The term "conservative substitution" or "conservative variation" refers to an alteration that results in the replacement of one amino acid with another amino acid that can be classified as having similar characteristics. Examples of classifications of conservative amino acid groups defined in this way include the "charged / polar group" including Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H), the "aromatic group" including Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H), and the "aliphatic group" including Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine ​​or C). Within each group, subgroups may also be identified. For example, the group of charged or polar amino acids can be subdivided into subgroups including the "positively charged subgroup" consisting of Lys, Arg, and His, the "negatively charged subgroup" consisting of Glu and Asp, and the "polar subgroup" consisting of Asn and Gln. In another example, the aromatic or cyclic group can be subdivided into subgroups including the "nitrogen ring subgroup" consisting of Pro, His, and Trp, and the "phenyl subgroup" consisting of Phe and Tyr. In yet another example, the aliphatic group can be subdivided into subgroups, such as the "aliphatic non-polar subgroup" consisting of Val, Leu, Gly, and Ala, and the "aliphatic slightly polar subgroup" consisting of Met, Ser, Thr, and Cys. Examples of conservative mutation classes include amino acid substitutions of amino acids within the above subgroups, such as, but not limited to, Lys for Arg or vice versa, which can maintain a positive charge; Glu for Asp or vice versa, which can maintain a negative charge; Ser for Thr or vice versa, which can maintain a free -OH; and Gln for Asn or vice versa, which can maintain a free -NH2.In some embodiments, a hydrophobic amino acid is substituted for a naturally occurring hydrophobic amino acid, e.g., at the active site, to preserve the hydrophobicity.

[0087] The terms "identical" or percent "identity" in the context of two or more polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues, e.g., that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more identical over a particular region or over a designated region when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0088] In the sequence comparison of polypeptides, one amino acid sequence usually serves as a reference sequence and is compared with the candidate sequence. Alignment can be performed by various methods available to those skilled in the art, such as visual alignment, or by using publicly available software with known algorithms to achieve maximum alignment. Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters used for alignment to achieve maximum alignment can be determined by those skilled in the art. For the purpose of this application, for the sequence comparison of polypeptide sequences, the BLASTP algorithm standard protein BLAST is used to align two protein sequences with default parameters.

[0089] The phrases "corresponding to," "determined in accordance with," or "numbered in accordance with," when used in the context of identifying a given amino acid residue in a polypeptide sequence, refer to the position of the residue in a particular reference sequence when a given amino acid sequence is maximally aligned and compared to that reference sequence. Thus, for example, an amino acid residue in a modified Fc polypeptide "corresponds to" an amino acid in SEQ ID NO:95 if that residue matches the amino acid in SEQ ID NO:95 when optimally aligned against SEQ ID NO:95. A polypeptide that is aligned to a reference sequence need not be the same length as the reference sequence.

[0090] The terms "subject," "individual," and "patient," used interchangeably herein, 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.

[0091] The terms "treatment", "treat" and the like are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. "Treating" 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 parameter, such as alleviation, remission, improvement of patient survival, increase in survival time or rate, attenuation of symptoms or making the disease more tolerable to the patient, slowing down in the rate of degeneration or decline, or improving the patient's physical or mental health. The treatment or improvement of symptoms can be based on objective or subjective parameters. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient at different time points before or during treatment.

[0092] The term "pharmaceutical acceptable excipient" refers to non-active pharmaceutical ingredients, such as, but not limited to, buffers, carriers, or preservatives, that are biologically or pharmacologically compatible for use in humans or animals.

[0093] As used herein, a "therapeutic amount" or "therapeutically effective amount" of a molecule (e.g., an antibody described herein) is an amount of the molecule that treats, alleviates, relieves, or reduces the severity of a symptom of a disease in a subject.

[0094] The term "administering" refers to a method of delivering a molecule or composition to a desired biological site of action. These methods include, but are not limited to, topical, parenteral, intravenous, intradermal, intramuscular, intrathecal, colonic, rectal, or intraperitoneal delivery. In one embodiment, the antibodies described herein are administered intravenously.

[0095] III. Anti-HER2 antibody In one aspect, an antibody is provided that binds to both subdomain II and subdomain IV of human HER2, which comprises a common light chain polypeptide. In some embodiments, one or both of the Fc polypeptides of the antibody are modified Fc polypeptides (e.g., modified to promote TfR binding and / or improve heterodimerization of the Fc polypeptides). A schematic diagram of such a bispecific antibody is shown in Figure 1.

[0096] In some embodiments, the anti-HER2 antibody is (a) a first antigen-binding site against human HER2 subdomain IV; (b) a second antigen-binding site against human HER2 subdomain II; and (c) a modified Fc polypeptide dimer comprising a first Fc polypeptide containing a modification that creates a TfR binding site. Including, The light chain polypeptide sequence of the first antigen-binding site is identical to the light chain polypeptide sequence of the second antigen-binding site.

[0097] In other embodiments, the anti-HER2 antibody is (a) a first antigen-binding site against human HER2 subdomain II; (b) a second antigen-binding site against human HER2 subdomain IV; and (c) a modified Fc polypeptide dimer comprising a first Fc polypeptide containing a modification that creates a TfR binding site. Including, The light chain polypeptide sequence of the first antigen-binding site is identical to the light chain polypeptide sequence of the second antigen-binding site.

[0098] In some embodiments, the first Fc polypeptide comprises a modified CH3 domain that comprises a TfR binding site. In certain embodiments, the modified CH3 domain comprises substitutions at the set of amino acid positions described herein that create the TfR binding site.

[0099] In some embodiments, the first Fc polypeptide and the second Fc polypeptide each comprise a modification that promotes heterodimerization. For example, according to EU numbering, the first Fc polypeptide may comprise a substitution of T366W, and the second Fc polypeptide may comprise a substitution of T366S, L368A, and Y407V. In another example, according to EU numbering, the first Fc polypeptide may comprise a substitution of T366S, L368A, and Y407V, and the second Fc polypeptide may comprise a substitution of T366W. Furthermore, the first Fc polypeptide and / or the second Fc polypeptide may independently comprise a modification that reduces TfR-mediated effector function, i.e., a modification that reduces effector function upon TfR binding. For example, a modification that reduces TfR-mediated effector function is, according to EU numbering, (i) the substitutions L234A and L235A, or (ii) the substitutions L234A and L235A, and the substitution P329G or P329S.

[0100] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., capable of enhancing effector function upon HER2 binding.

[0101] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises substitutions of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a S239D and I332E substitution and the second Fc polypeptide comprises a S239D and I332E substitution. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a S239D and I332E substitution. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a S239D substitution. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a I332E substitution.In some embodiments, the first Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E.

[0102] In certain embodiments, the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering. In certain embodiments, the first Fc polypeptide comprises a I332E substitution and a serine at position 239 and the second Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, according to EU numbering.

[0103] In certain embodiments, the first Fc polypeptide comprises an S239D and an I332E substitution and the second Fc polypeptide comprises an S239D substitution, according to EU numbering. In certain embodiments, the first Fc polypeptide comprises an S239D and an I332E substitution and the second Fc polypeptide comprises an S239D substitution and an isoleucine at position 332, according to EU numbering.

[0104] In certain embodiments, the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a I332E substitution, according to EU numbering. In certain embodiments, the first Fc polypeptide comprises a S239D substitution and an isoleucine at position 332 and the second Fc polypeptide comprises a I332E substitution and a serine at position 239, according to EU numbering.

[0105] In certain embodiments, the second Fc polypeptide comprises a I332E substitution, according to EU numbering. In certain embodiments, the first Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, and the second Fc polypeptide comprises a I332E substitution and a serine at position 239, according to EU numbering.

[0106] In certain embodiments, the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a S239D and I332E substitution, according to EU numbering. In certain embodiments, the first Fc polypeptide comprises a S239D substitution and an isoleucine at position 332 and the second Fc polypeptide comprises a S239D and I332E substitution, according to EU numbering.

[0107] In certain embodiments, the first Fc polypeptide comprises a I332E substitution, according to EU numbering. In certain embodiments, the first Fc polypeptide comprises a I332E substitution and a serine at position 239, and the second Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, according to EU numbering.

[0108] In some embodiments, the first Fc polypeptide comprises a TfR binding site, a T366W substitution, an L234A and an L235A substitution (optionally with a P329G or P329S substitution), and optionally an S239D and / or an I332E substitution, according to EU numbering, and the second Fc polypeptide comprises T366S, L368A, and Y407V substitutions, and optionally an S239D and / or an I332E substitution, according to EU numbering. For example, the first Fc polypeptide may comprise a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of SEQ ID NOs: 74 to 84, 86, and 98, and the second Fc polypeptide may comprise a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of SEQ ID NOs: 71 to 73, 85, and 99 to 100.

[0109] In certain embodiments, the first Fc polypeptide comprises a TfR binding site and comprises a L234A and L235A substitution (optionally comprising a P329G or P329S substitution), and the second Fc polypeptide does not comprise a L234A and L235A substitution (or a P329G or P329S substitution, if present in the first Fc polypeptide). In certain other embodiments, the first Fc polypeptide comprises a TfR binding site and does not comprise a L234A or L325A substitution (or a P329G or P329S substitution, if present in the second Fc polypeptide), and the second Fc polypeptide comprises a L234A and L235A substitution (optionally comprising a P329G or P329S substitution), according to EU numbering.

[0110] In some embodiments, one or both of the Fc polypeptides may have the C-terminal lysine removed (e.g., the Lys residue at position 447 of the Fc polypeptide, according to EU numbering). In some embodiments, removal of the C-terminal lysine of the Fc polypeptide may improve antibody stability.

[0111] In some embodiments, the antigen-binding site of an anti-HER2 antibody against human HER2 subdomain II comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 89; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 90; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 91.

[0112] In certain embodiments, at least one of the following: X1 of SEQ ID NO:89 is not T; X2 of SEQ ID NO:89 is not F; X3 of SEQ ID NO:89 is not T; X1 of SEQ ID NO:90 is not N; X2 of SEQ ID NO:90 is not N; X3 of SEQ ID NO:90 is not S; X4 of SEQ ID NO:90 is not G; X5 of SEQ ID NO:90 is not G; X6 of SEQ ID NO:90 is not Q; X1 of SEQ ID NO:91 is not L; X2 of SEQ ID NO:91 is not G; X3 of SEQ ID NO:91 is not P; and X4 of SEQ ID NO:91 is not S.

[0113] In some embodiments, the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 89 and X1 is N, K, M, or H. In some embodiments, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 90 and X5 is Q. In some embodiments, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 90 and X6 is R, H, or T. In some embodiments, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 91 and X4 is W, F, D, L, or Y. In some embodiments, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 91 and X4 is L.

[0114] In some embodiments, the antigen-binding site of an anti-HER2 antibody against human HER2 subdomain II comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 89; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 90, wherein X5 is Q; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 91, and X4 is L.

[0115] In some embodiments, the antigen-binding site of an anti-HER2 antibody against human HER2 subdomain II comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 49 to 52, or having up to two amino acid substitutions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 49 to 52; (b) a heavy chain CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 6 and 53 to 55, or having up to two amino acid substitutions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 6 and 53 to 55; (c) a heavy chain CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 8 and 56 to 59, or having up to two amino acid substitutions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 8 and 56 to 59.

[0116] In some embodiments, the antigen-binding site of an anti-HER2 antibody against human HER2 subdomain II comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:4, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6; and (c) a heavy chain CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.

[0117] In some embodiments, the antigen-binding site of an anti-HER2 antibody against human HER2 subdomain II comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.

[0118] In some embodiments, the antigen-binding site of an anti-HER2 antibody against human HER2 subdomain II comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:6; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:7.

[0119] In some embodiments, the antigen-binding site of an anti-HER2 antibody against human HER2 subdomain II comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:5; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:8.

[0120] In some embodiments, the antigen-binding site of an anti-HER2 antibody against human HER2 subdomain II comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:6; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:8.

[0121] In some embodiments, the antigen-binding site of the anti-HER2 antibody for human HER2 subdomain II comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 to 3 and 60 to 70. In some embodiments, the antigen-binding site of the anti-HER2 antibody for human HER2 subdomain II comprises a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 3 and 60 to 70.

[0122] In some embodiments, the antigen-binding site of an anti-HER2 antibody against human HER2 subdomain IV comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 16; (b) a heavy chain CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 17, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 17; and (c) a heavy chain CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:18, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:18.

[0123] In some embodiments, the antigen-binding site of an anti-HER2 antibody against human HER2 subdomain IV comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18.

[0124] In some embodiments, the antigen-binding site of an anti-HER2 antibody against human HER2 subdomain IV comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 15. In some embodiments, the antigen-binding site of an anti-HER2 antibody against human HER2 subdomain IV comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15.

[0125] In some embodiments, the light chain polypeptide sequences in the first and second antigen binding sites, one for HER2 subdomain II and the other for HER2 subdomain IV, of an anti-HER2 antibody comprise one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a light chain CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:11, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:11; (b) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 12; and (c) a light chain CDR3 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 13 or 14.

[0126] In some embodiments, the light chain polypeptide sequence comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (b) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and (c) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13 or 14.

[0127] In some embodiments, the light chain polypeptide sequence comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (b) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and (c) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0128] In some embodiments, the light chain polypeptide sequence comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of: (a) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (b) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and (c) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14.

[0129] In certain embodiments, the light chain polypeptide sequence comprises a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13 or 14, and optionally further comprises one or more (e.g., one or both) CDRs selected from the group consisting of: a light chain CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 11, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 11; and a light chain CDR2 having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the light chain polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 13 or 14, and optionally further comprises one or more (e.g., one or both) CDRs selected from the group consisting of a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11; and a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12.

[0130] In some embodiments, the light chain polypeptide sequence comprises a light chain variable region comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 9-10. In some embodiments, the light chain polypeptide sequence comprises a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 9-10.

[0131] In some embodiments, the anti-HER2 antibody comprises heavy and light chain CDRs selected from any one of the combinations listed in Table 1, ie, Combos #A-AC.

[0132] In certain embodiments of this antibody, (a) the first heavy chain comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18; (b) the second heavy chain comprises a heavy chain CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 4 and 49 to 52, a heavy chain CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 5 to 6 and 53 to 55, and a heavy chain CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 7 to 8 and 56 to 59; and (c) The light chain comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a light chain CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 13 to 14.

[0133] In certain embodiments of this anti-HER2 antibody, the first antigen-binding site comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15; the second antigen-binding site comprises an amino acid sequence of SEQ ID NO: 1-3 and 60-70; the first Fc polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74-84, 86, and 98; and the light chain polypeptide sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9-10 and 19. In some embodiments, the antibody further comprises a second Fc polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 71-73, 85, and 99-100.

[0134] In certain other embodiments of the anti-HER2 antibody, the first antigen-binding site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 60-70; the second antigen-binding site comprises the amino acid sequence of SEQ ID NO: 15; the first Fc polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-84, 86, and 98; and the light chain polypeptide sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-10 and 19. In some embodiments, the antibody further comprises a second Fc polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-73, 85, and 99-100.

[0135] In some embodiments, the anti-HER2 antibody comprises a first heavy chain for binding to human HER2 subdomain II or IV, a second heavy chain for binding to the other HER2 subdomain, and two identical light chains.

[0136] In certain embodiments, the anti-HER2 antibody comprises a heavy chain and a light chain each having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from any one of the combinations listed in Table 2, i.e., Combo #A-AT.

[0137] In certain embodiments of this antibody, (a) the first heavy chain comprises SEQ ID NO: 37, the second heavy chain comprises SEQ ID NO: 25, and the light chain comprises SEQ ID NO: 9; (b) the first heavy chain comprises SEQ ID NO:31, the second heavy chain comprises SEQ ID NO:30, and the light chain comprises SEQ ID NO:9; (c) the first heavy chain comprises SEQ ID NO:31, the second heavy chain comprises SEQ ID NO:29, and the light chain comprises SEQ ID NO:9; (d) the first heavy chain comprises SEQ ID NO: 38, the second heavy chain comprises SEQ ID NO: 30, and the light chain comprises SEQ ID NO: 9; (e) the first heavy chain comprises SEQ ID NO: 32, the second heavy chain comprises SEQ ID NO: 30, and the light chain comprises SEQ ID NO: 9; (f) the first heavy chain comprises SEQ ID NO: 32, the second heavy chain comprises SEQ ID NO: 29, and the light chain comprises SEQ ID NO: 9; (g) the first heavy chain comprises SEQ ID NO: 39, the second heavy chain comprises SEQ ID NO: 30, and the light chain comprises SEQ ID NO: 9; (h) the first heavy chain comprises SEQ ID NO: 37, the second heavy chain comprises SEQ ID NO: 26, and the light chain comprises SEQ ID NO: 9; (i) the first heavy chain comprises SEQ ID NO: 31, the second heavy chain comprises SEQ ID NO: 34, and the light chain comprises SEQ ID NO: 9; (j) the first heavy chain comprises SEQ ID NO: 31, the second heavy chain comprises SEQ ID NO: 33, and the light chain comprises SEQ ID NO: 9; (k) the first heavy chain comprises SEQ ID NO: 38, the second heavy chain comprises SEQ ID NO: 34, and the light chain comprises SEQ ID NO: 9; (l) the first heavy chain comprises SEQ ID NO: 32, the second heavy chain comprises SEQ ID NO: 34, and the light chain comprises SEQ ID NO: 9; (m) the first heavy chain comprises SEQ ID NO: 32, the second heavy chain comprises SEQ ID NO: 33, and the light chain comprises SEQ ID NO: 9; (n) the first heavy chain comprises SEQ ID NO: 39, the second heavy chain comprises SEQ ID NO: 34, and the light chain comprises SEQ ID NO: 9; (o) the first heavy chain comprises SEQ ID NO: 37, the second heavy chain comprises SEQ ID NO: 27, and the light chain comprises SEQ ID NO: 9; (p) the first heavy chain comprises SEQ ID NO: 31, the second heavy chain comprises SEQ ID NO: 36, and the light chain comprises SEQ ID NO: 9; (q) the first heavy chain comprises SEQ ID NO: 31, the second heavy chain comprises SEQ ID NO: 35, and the light chain comprises SEQ ID NO: 9; (r) the first heavy chain comprises SEQ ID NO: 38, the second heavy chain comprises SEQ ID NO: 36, and the light chain comprises SEQ ID NO: 9; (s) the first heavy chain comprises SEQ ID NO: 32, the second heavy chain comprises SEQ ID NO: 36, and the light chain comprises SEQ ID NO: 9; (t) the first heavy chain comprises SEQ ID NO: 32, the second heavy chain comprises SEQ ID NO: 35, and the light chain comprises SEQ ID NO: 9; (u) the first heavy chain comprises SEQ ID NO: 39, the second heavy chain comprises SEQ ID NO: 46, and the light chain comprises SEQ ID NO: 9; (v) the first heavy chain comprises SEQ ID NO: 37, the second heavy chain comprises SEQ ID NO: 25, and the light chain comprises SEQ ID NO: 10; (w) the first heavy chain comprises SEQ ID NO: 31, the second heavy chain comprises SEQ ID NO: 30, and the light chain comprises SEQ ID NO: 10; (x) the first heavy chain comprises SEQ ID NO: 31, the second heavy chain comprises SEQ ID NO: 29, and the light chain comprises SEQ ID NO: 10; (y) the first heavy chain comprises SEQ ID NO: 38, the second heavy chain comprises SEQ ID NO: 30, and the light chain comprises SEQ ID NO: 10; (z) the first heavy chain comprises SEQ ID NO: 32, the second heavy chain comprises SEQ ID NO: 30, and the light chain comprises SEQ ID NO: 10; (aa) the first heavy chain comprises SEQ ID NO: 32, the second heavy chain comprises SEQ ID NO: 29, and the light chain comprises SEQ ID NO: 10; (ab) the first heavy chain comprises SEQ ID NO: 39, the second heavy chain comprises SEQ ID NO: 30, and the light chain comprises SEQ ID NO: 10; (ac) the first heavy chain comprises SEQ ID NO: 37, the second heavy chain comprises SEQ ID NO: 26, and the light chain comprises SEQ ID NO: 10; (ad) the first heavy chain comprises SEQ ID NO: 31, the second heavy chain comprises SEQ ID NO: 34, and the light chain comprises SEQ ID NO: 10; (ae) the first heavy chain comprises SEQ ID NO: 31, the second heavy chain comprises SEQ ID NO: 33, and the light chain comprises SEQ ID NO: 10; (af) the first heavy chain comprises SEQ ID NO: 38, the second heavy chain comprises SEQ ID NO: 34, and the light chain comprises SEQ ID NO: 10; (ag) the first heavy chain comprises SEQ ID NO: 32, the second heavy chain comprises SEQ ID NO: 34, and the light chain comprises SEQ ID NO: 10; (ah) the first heavy chain comprises SEQ ID NO: 32, the second heavy chain comprises SEQ ID NO: 33, and the light chain comprises SEQ ID NO: 10; (ai) the first heavy chain comprises SEQ ID NO: 39, the second heavy chain comprises SEQ ID NO: 34, and the light chain comprises SEQ ID NO: 10; (aj) the first heavy chain comprises SEQ ID NO: 37, the second heavy chain comprises SEQ ID NO: 27, and the light chain comprises SEQ ID NO: 10; (ak) the first heavy chain comprises SEQ ID NO: 31, the second heavy chain comprises SEQ ID NO: 36, and the light chain comprises SEQ ID NO: 10; (a1) the first heavy chain comprises SEQ ID NO: 31, the second heavy chain comprises SEQ ID NO: 35, and the light chain comprises SEQ ID NO: 10; (am) the first heavy chain comprises SEQ ID NO: 38, the second heavy chain comprises SEQ ID NO: 36, and the light chain comprises SEQ ID NO: 10; (an) the first heavy chain comprises SEQ ID NO: 32, the second heavy chain comprises SEQ ID NO: 36, and the light chain comprises SEQ ID NO: 10; (ao) the first heavy chain comprises SEQ ID NO: 32, the second heavy chain comprises SEQ ID NO: 35, and the light chain comprises SEQ ID NO: 10; (ap) the first heavy chain comprises SEQ ID NO: 39, the second heavy chain comprises SEQ ID NO: 46, and the light chain comprises SEQ ID NO: 10; (aq) the first heavy chain comprises SEQ ID NO: 20, the second heavy chain comprises SEQ ID NO: 24, and the light chain comprises SEQ ID NO: 19; (ar) the first heavy chain comprises SEQ ID NO:21, the second heavy chain comprises SEQ ID NO:24, and the light chain comprises SEQ ID NO:19; (as) the first heavy chain comprises SEQ ID NO:22, the second heavy chain comprises SEQ ID NO:24, and the light chain comprises SEQ ID NO:19; or (at) the first heavy chain comprises SEQ ID NO:23, the second heavy chain comprises SEQ ID NO:24, and the light chain comprises SEQ ID NO:19.

[0138] In certain embodiments, the anti-HER2 antibody has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from any one of the combinations in Table 3, i.e., Combo #AL. H and a first heavy chain comprising an Fc sequence, each of which has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from any one of the combinations in Table 4, i.e., Combo #AL. H and a second heavy chain comprising an Fc sequence. In any of these heavy chain combinations, the light chain polypeptide sequence has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-10 and 19.

[0139] In certain embodiments of this antibody, (a) the first heavy chain comprises SEQ ID NOs:1 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 86; (b) the first heavy chain comprises SEQ ID NOs:1 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 74; (c) the first heavy chain comprises SEQ ID NOs:1 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 75; (d) the first heavy chain comprises SEQ ID NOs:1 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 76; (e) the first heavy chain comprises SEQ ID NOs:1 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 77; (f) the first heavy chain comprises SEQ ID NOs:1 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 78; (g) the first heavy chain comprises SEQ ID NOs:1 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 79; (h) the first heavy chain comprises SEQ ID NOs:1 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 80; (i) the first heavy chain comprises SEQ ID NOs: 1 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 81; (j) the first heavy chain comprises SEQ ID NOs:1 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 82; (k) the first heavy chain comprises SEQ ID NOs: 1 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 83; or (l) the first heavy chain comprises SEQ ID NOs:1 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 84.

[0140] In certain embodiments of this antibody, (a) the first heavy chain comprises SEQ ID NOs: 2 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 86; (b) the first heavy chain comprises SEQ ID NOs:2 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 74; (c) the first heavy chain comprises SEQ ID NOs:2 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 75; (d) the first heavy chain comprises SEQ ID NOs:2 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 76; (e) the first heavy chain comprises SEQ ID NOs:2 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 77; (f) the first heavy chain comprises SEQ ID NOs:2 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 78; (g) the first heavy chain comprises SEQ ID NOs:2 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 79; (h) the first heavy chain comprises SEQ ID NOs:2 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 80; (i) the first heavy chain comprises SEQ ID NOs: 2 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 81; (j) the first heavy chain comprises SEQ ID NOs:2 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 82; (k) the first heavy chain comprises SEQ ID NOs: 2 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 83; or (l) the first heavy chain comprises SEQ ID NOs: 2 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 84.

[0141] In certain embodiments of this antibody, (a) the first heavy chain comprises SEQ ID NOs: 3 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 86; (b) the first heavy chain comprises SEQ ID NOs: 3 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 74; (c) the first heavy chain comprises SEQ ID NOs: 3 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 75; (d) the first heavy chain comprises SEQ ID NOs:3 and 71, and the second heavy chain comprises SEQ ID NOs:15 and 76; (e) the first heavy chain comprises SEQ ID NOs: 3 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 77; (f) the first heavy chain comprises SEQ ID NOs: 3 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 78; (g) the first heavy chain comprises SEQ ID NOs: 3 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 79; (h) the first heavy chain comprises SEQ ID NOs: 3 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 80; (i) the first heavy chain comprises SEQ ID NOs: 3 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 81; (j) the first heavy chain comprises SEQ ID NOs: 3 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 82; (k) the first heavy chain comprises SEQ ID NOs: 3 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 83; or (l) the first heavy chain comprises SEQ ID NOs: 3 and 71, and the second heavy chain comprises SEQ ID NOs: 15 and 84.

[0142] In certain embodiments of this antibody, (a) the first heavy chain comprises SEQ ID NOs:1 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 86; (b) the first heavy chain comprises SEQ ID NOs:1 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 74; (c) the first heavy chain comprises SEQ ID NOs:1 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 75; (d) the first heavy chain comprises SEQ ID NOs:1 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 76; (e) the first heavy chain comprises SEQ ID NOs:1 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 77; (f) the first heavy chain comprises SEQ ID NOs:1 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 78; (g) the first heavy chain comprises SEQ ID NOs:1 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 79; (h) the first heavy chain comprises SEQ ID NOs:1 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 80; (i) the first heavy chain comprises SEQ ID NOs:1 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 81; (j) the first heavy chain comprises SEQ ID NOs:1 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 82; (k) the first heavy chain comprises SEQ ID NOs: 1 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 83; or (l) the first heavy chain comprises SEQ ID NOs:1 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 84.

[0143] In certain embodiments of this antibody, (a) the first heavy chain comprises SEQ ID NOs: 2 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 86; (b) the first heavy chain comprises SEQ ID NOs:2 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 74; (c) the first heavy chain comprises SEQ ID NOs:2 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 75; (d) the first heavy chain comprises SEQ ID NOs:2 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 76; (e) the first heavy chain comprises SEQ ID NOs:2 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 77; (f) the first heavy chain comprises SEQ ID NOs:2 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 78; (g) the first heavy chain comprises SEQ ID NOs:2 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 79; (h) the first heavy chain comprises SEQ ID NOs:2 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 80; (i) the first heavy chain comprises SEQ ID NOs: 2 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 81; (j) the first heavy chain comprises SEQ ID NOs:2 and 72, and the second heavy chain comprises SEQ ID NOs:15 and 82; (k) the first heavy chain comprises SEQ ID NOs: 2 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 83; or (l) the first heavy chain comprises SEQ ID NOs: 2 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 84.

[0144] In certain embodiments of this antibody, (a) the first heavy chain comprises SEQ ID NOs: 3 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 86; (b) the first heavy chain comprises SEQ ID NOs: 3 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 74; (c) the first heavy chain comprises SEQ ID NOs: 3 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 75; (d) the first heavy chain comprises SEQ ID NOs: 3 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 76; (e) the first heavy chain comprises SEQ ID NOs: 3 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 77; (f) the first heavy chain comprises SEQ ID NOs: 3 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 78; (g) the first heavy chain comprises SEQ ID NOs: 3 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 79; (h) the first heavy chain comprises SEQ ID NOs: 3 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 80; (i) the first heavy chain comprises SEQ ID NOs: 3 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 81; (j) the first heavy chain comprises SEQ ID NOs: 3 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 82; (k) the first heavy chain comprises SEQ ID NOs: 3 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 83; or (l) the first heavy chain comprises SEQ ID NOs: 3 and 72, and the second heavy chain comprises SEQ ID NOs: 15 and 84.

[0145] In certain embodiments of this antibody, (a) the first heavy chain comprises SEQ ID NOs:1 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 86; (b) the first heavy chain comprises SEQ ID NOs:1 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 74; (c) the first heavy chain comprises SEQ ID NOs:1 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 75; (d) the first heavy chain comprises SEQ ID NOs:1 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 76; (e) the first heavy chain comprises SEQ ID NOs:1 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 77; (f) the first heavy chain comprises SEQ ID NOs:1 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 78; (g) the first heavy chain comprises SEQ ID NOs:1 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 79; (h) the first heavy chain comprises SEQ ID NOs:1 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 80; (i) the first heavy chain comprises SEQ ID NOs: 1 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 81; (j) the first heavy chain comprises SEQ ID NOs:1 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 82; (k) the first heavy chain comprises SEQ ID NOs: 1 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 83; or (l) the first heavy chain comprises SEQ ID NOs:1 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 84.

[0146] In certain embodiments of this antibody, (a) the first heavy chain comprises SEQ ID NOs: 2 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 86; (b) the first heavy chain comprises SEQ ID NOs:2 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 74; (c) the first heavy chain comprises SEQ ID NOs:2 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 75; (d) the first heavy chain comprises SEQ ID NOs:2 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 76; (e) the first heavy chain comprises SEQ ID NOs:2 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 77; (f) the first heavy chain comprises SEQ ID NOs:2 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 78; (g) the first heavy chain comprises SEQ ID NOs:2 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 79; (h) the first heavy chain comprises SEQ ID NOs:2 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 80; (i) the first heavy chain comprises SEQ ID NOs: 2 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 81; (j) the first heavy chain comprises SEQ ID NOs:2 and 73, and the second heavy chain comprises SEQ ID NOs:15 and 82; (k) the first heavy chain comprises SEQ ID NOs: 2 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 83; or (l) the first heavy chain comprises SEQ ID NOs: 2 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 84.

[0147] In certain embodiments of this antibody, (a) the first heavy chain comprises SEQ ID NOs: 3 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 86; (b) the first heavy chain comprises SEQ ID NOs: 3 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 74; (c) the first heavy chain comprises SEQ ID NOs: 3 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 75; (d) the first heavy chain comprises SEQ ID NOs: 3 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 76; (e) the first heavy chain comprises SEQ ID NOs: 3 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 77; (f) the first heavy chain comprises SEQ ID NOs: 3 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 78; (g) the first heavy chain comprises SEQ ID NOs: 3 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 79; (h) the first heavy chain comprises SEQ ID NOs: 3 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 80; (i) the first heavy chain comprises SEQ ID NOs: 3 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 81; (j) the first heavy chain comprises SEQ ID NOs: 3 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 82; (k) the first heavy chain comprises SEQ ID NOs: 3 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 83; or (l) the first heavy chain comprises SEQ ID NOs: 3 and 73, and the second heavy chain comprises SEQ ID NOs: 15 and 84.

[0148] In certain embodiments of this antibody, (a) the first heavy chain comprises SEQ ID NOs:1 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 86; (b) the first heavy chain comprises SEQ ID NOs:1 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 74; (c) the first heavy chain comprises SEQ ID NOs:1 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 75; (d) the first heavy chain comprises SEQ ID NOs:1 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 76; (e) the first heavy chain comprises SEQ ID NOs:1 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 77; (f) the first heavy chain comprises SEQ ID NOs:1 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 78; (g) the first heavy chain comprises SEQ ID NOs:1 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 79; (h) the first heavy chain comprises SEQ ID NOs:1 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 80; (i) the first heavy chain comprises SEQ ID NOs:1 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 81; (j) the first heavy chain comprises SEQ ID NOs:1 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 82; (k) the first heavy chain comprises SEQ ID NOs: 1 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 83; or (l) the first heavy chain comprises SEQ ID NOs:1 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 84.

[0149] In certain embodiments of this antibody, (a) the first heavy chain comprises SEQ ID NOs:2 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 86; (b) the first heavy chain comprises SEQ ID NOs: 2 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 74; (c) the first heavy chain comprises SEQ ID NOs: 2 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 75; (d) the first heavy chain comprises SEQ ID NOs:2 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 76; (e) the first heavy chain comprises SEQ ID NOs:2 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 77; (f) the first heavy chain comprises SEQ ID NOs:2 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 78; (g) the first heavy chain comprises SEQ ID NOs:2 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 79; (h) the first heavy chain comprises SEQ ID NOs:2 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 80; (i) the first heavy chain comprises SEQ ID NOs: 2 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 81; (j) the first heavy chain comprises SEQ ID NOs:2 and 85, and the second heavy chain comprises SEQ ID NOs:15 and 82; (k) the first heavy chain comprises SEQ ID NOs: 2 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 83; or (l) the first heavy chain comprises SEQ ID NOs: 2 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 84.

[0150] In certain embodiments of this antibody, (a) the first heavy chain comprises SEQ ID NOs: 3 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 86; (b) the first heavy chain comprises SEQ ID NOs: 3 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 74; (c) the first heavy chain comprises SEQ ID NOs: 3 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 75; (d) the first heavy chain comprises SEQ ID NOs: 3 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 76; (e) the first heavy chain comprises SEQ ID NOs: 3 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 77; (f) the first heavy chain comprises SEQ ID NOs: 3 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 78; (g) the first heavy chain comprises SEQ ID NOs: 3 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 79; (h) the first heavy chain comprises SEQ ID NOs: 3 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 80; (i) the first heavy chain comprises SEQ ID NOs: 3 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 81; (j) the first heavy chain comprises SEQ ID NOs: 3 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 82; (k) the first heavy chain comprises SEQ ID NOs: 3 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 83; or (l) the first heavy chain comprises SEQ ID NOs: 3 and 85, and the second heavy chain comprises SEQ ID NOs: 15 and 84.

[0151] In certain embodiments, the anti-HER2 antibody has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from any one of the combinations in Table 5, i.e., Combos #A-AJ, V, VV ...V, VVVVVV, VVVVVV, VVVVVV, VVVVVV, VVVVVV, VVVVVV, VVVVVV, VVVVVVV, VVVVVVV, VVVVVVV, VVVVVVV, VVVVVVVV, VVVVVVVV, VVVVVVVVV, VVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVV Hand a first heavy chain comprising an Fc sequence, each of which has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from any one of the combinations in Table 6, i.e., Combo #AD. H and a second heavy chain comprising an Fc sequence. In any of these heavy chain combinations, the light chain polypeptide sequence has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-10 and 19.

[0152] IV. Fc Polypeptides and Modifications Thereof In some aspects, any of the antibodies described herein comprises an Fc polypeptide dimer, where one or both Fc polypeptides of the dimer comprise an amino acid modification compared to a wild-type Fc polypeptide. In some embodiments, the amino acid modification in the Fc polypeptide (e.g., modified Fc polypeptide) can provide binding of the Fc polypeptide dimer to a BBB receptor (e.g., TfR), promote heterodimerization of the two Fc polypeptides of the dimer, modulate effector function, extend serum half-life, affect glycosylation, and / or reduce immunogenicity in humans. In some embodiments, the Fc polypeptides present in the antibody independently have at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a corresponding wild-type Fc polypeptide (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide). Examples and descriptions of modified Fc polypeptides (e.g., TfR-binding Fc polypeptides) can be found, for example, in International Patent Publication No. WO2018 / 152326, which is incorporated by reference in its entirety.

[0153] Fc Polypeptide Modifications for BBB Receptor Binding Provided herein is an anti-HER2 antibody that can be transported across the BBB. Such a protein comprises a modified Fc polypeptide that binds to a BBB receptor. BBB receptors are expressed in the BBB endothelium, as well as other cell and tissue types. In some embodiments, the BBB receptor is TfR. A modified Fc polypeptide that binds to TfR is also referred to as having a TfR binding site.

[0154] The amino acid residues designated in the various Fc modifications, including those introduced into modified Fc polypeptides that bind to BBB receptors, e.g., TfR, are numbered herein using EU index numbering. Any Fc polypeptide, e.g., IgG1, IgG2, IgG3, or IgG4 Fc polypeptide, may have modifications, e.g., amino acid substitutions, at one or more positions described herein. In some embodiments, the domain modified for BBB (e.g., TfR) receptor binding activity is a human Ig CH3 domain, e.g., IgG1 CH3 domain. This CH3 domain may be from any IgG subtype, i.e., IgG1, IgG2, IgG3, or IgG4. In the context of IgG1 antibodies, the CH3 domain refers to the segment of amino acids from about position 341 to about position 447, numbered according to the EU numbering scheme.

[0155] In some embodiments, a modified Fc polypeptide that specifically binds to TfR can bind to the apical domain of TfR and bind to TfR without blocking or inhibiting the binding of transferrin to TfR. In some embodiments, the binding of transferrin to TfR is not substantially inhibited. In some embodiments, the binding of transferrin to TfR is inhibited by less than about 50% (e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%).

[0156] In some embodiments, a BBB (e.g., TfR) receptor binding Fc polypeptide present in an antibody described herein comprises one or more, at least one, two, or three substitutions, and in some embodiments at least four, five, six, seven, eight, nine, or ten substitutions at amino acid positions including 266, 267, 268, 269, 270, 271, 295, 297, 298, and 299 according to the EU numbering scheme. In some embodiments, a BBB (e.g., TfR) receptor binding Fc polypeptide present in an antibody described herein comprises at least one, two, or three substitutions; and in some embodiments at least four, five, six, seven, eight, or nine substitutions at amino acid positions including 274, 276, 283, 285, 286, 287, 288, 289, and 290 according to the EU numbering scheme. In some embodiments, a BBB (e.g., TfR) receptor binding Fc polypeptide present in an antibody described herein comprises at least one, two, or three substitutions, and in some embodiments, at least 4, 5, 6, 7, 8, 9, or 10 substitutions at amino acid positions including 268, 269, 270, 271, 272, 292, 293, 294, 296, and 300 according to the EU numbering scheme. In some embodiments, a BBB (e.g., TfR) receptor binding Fc polypeptide present in an antibody described herein comprises at least one, two, or three substitutions, and in some embodiments, at least 4, 5, 6, 7, 8, or 9 substitutions at amino acid positions including 272, 274, 276, 322, 324, 326, 329, 330, and 331 according to the EU numbering scheme. In some embodiments, a BBB (e.g., TfR) receptor binding Fc polypeptide present in an antibody described herein comprises at least one, two, or three substitutions, and in some embodiments at least four, five, six, or seven substitutions at amino acid positions including 345, 346, 347, 349, 437, 438, 439, and 440, according to the EU numbering scheme.

[0157] In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide present in the antibodies described herein comprises at least one, two, or three substitutions, and in some embodiments at least four, five, six, seven, eight, or nine substitutions at amino acid positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 according to the EU numbering scheme.

[0158] In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide comprises at least one position having a substitution compared to SEQ ID NO:95 as follows: Leu, Tyr, Met, or Val at position 384, Leu, Thr, His, or Pro at position 386, Val, Pro, or an acidic amino acid at position 387, an aromatic amino acid such as Trp or Gly (e.g., Trp) at position 388, Val, Ser, or Ala at position 389, an acidic amino acid, Ala, Ser, Leu, Thr, or Pro at position 413, a Thr or an acidic amino acid at position 416, or Trp, Tyr, His, or Phe at position 421. In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide may comprise conservative substitutions of particular amino acids at one or more positions of this set, e.g., amino acids of the same charge class, hydrophobic class, side chain ring structure class (e.g., aromatic amino acids), or size class, and / or polar or non-polar class. Thus, for example, Ile can be present at positions 384, 386, and / or 413. In some embodiments, one, two, or each of the acidic amino acids at positions 387, 413, and 416 is Glu. In other embodiments, one, two, or each of the acidic amino acids at positions 387, 413, and 416 is Asp. In some embodiments, two, three, four, five, six, seven, or all eight of positions 384, 386, 387, 388, 389, 413, 416, and 421 have an amino acid substitution as defined in this paragraph.

[0159] In some embodiments, an Fc polypeptide having modifications at amino acid positions 384, 386, 387, 388, 389, 390, 413, 416, and / or 421 includes a native Asn at position 390. In some embodiments, the Fc polypeptide includes a Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, or Asp at position 390. In some embodiments, the Fc polypeptide further includes one, two, three, or four substitutions at positions including 380, 391, 392, and 415. In some embodiments, a Trp, Tyr, Leu, or Gln may be present at position 380. In some embodiments, a Ser, Thr, Gln, or Phe may be present at position 391. In some embodiments, a Gln, Phe, or His may be present at position 392. In some embodiments, a Glu may be present at position 415.

[0160] In certain embodiments, the Fc polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions selected from the following: Trp, Leu or Glu at position 380, Tyr or Phe at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser, Ala, Val or Asn at position 389, Ser or Asn at position 390, Thr or Ser at position 413, Glu or Ser at position 415, Glu at position 416, and / or Phe at position 421. In some embodiments, the Fc polypeptide comprises all eleven of the following positions: Trp, Leu or Glu at position 380, Tyr or Phe at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser, Ala, Val or Asn at position 389, Ser or Asn at position 390, Thr or Ser at position 413, Glu or Ser at position 415, Glu at position 416, and / or Phe at position 421.

[0161] In certain embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide comprises Leu or Met at position 384; Leu, His, or Pro at position 386; Val at position 387; Trp at position 388; Val or Ala at position 389; Pro at position 413; Thr at position 416; and / or Trp at position 421. In some embodiments, the Fc polypeptide further comprises Ser, Thr, Gln, or Phe at position 391. In some embodiments, the Fc polypeptide further comprises Trp, Tyr, Leu, or Gln at position 380 and / or Gln, Phe, or His at position 392. In some embodiments, Trp is present at position 380 and / or Gln is present at position 392. In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide does not have a Trp at position 380.

[0162] In other embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide comprises a Tyr at position 384, a Thr at position 386, a Glu or Val at position 387, a Trp at position 388, a Ser at position 389, a Ser or Thr at position 413, a Glu at position 416, and / or a Phe at position 421. In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide comprises a naturally occurring Asn at position 390. In certain embodiments, the Fc polypeptide further comprises a Trp, Tyr, Leu, or Gln at position 380 and / or a Glu at position 415. In some embodiments, the Fc polypeptide further comprises a Trp at position 380 and / or a Glu at position 415.

[0163] In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide comprises one or more of the following substitutions: Trp at position 380, Thr at position 386, Trp at position 388, Val at position 389, Ser or Thr at position 413, Glu at position 415, and / or Phe at position 421.

[0164] In a further embodiment, the BBB (e.g., TfR) receptor binding Fc polypeptide further comprises one, two or three positions selected from the following: position 414 is Lys, Arg, Gly, or Pro, position 424 is Ser, Thr, Glu, or Lys, and position 426 is Ser, Trp, or Gly.

[0165] In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide has the sequence of SEQ ID NO: 97. In some embodiments of the antibodies described herein, one of the two Fc polypeptides of the Fc polypeptide dimer may be a BBB (e.g., TfR) receptor binding Fc polypeptide having the sequence of SEQ ID NO: 97, while the other Fc polypeptide of the Fc polypeptide dimer may have the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO: 95). In other embodiments of the antibodies described herein, both Fc polypeptides of the Fc polypeptide dimer may be BBB (e.g., TfR) receptor binding Fc polypeptides having the sequence of SEQ ID NO: 97.

[0166] In some embodiments of the antibodies described herein, the first Fc polypeptide and / or the second Fc polypeptide independently comprise a Tyr at position 384, a Thr at position 386, a Glu at position 387, a Trp at position 388, a Ser at position 389, a Ser at position 413, a Glu at position 415, a Glu at position 416, and a Phe at position 421, according to EU numbering, and a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence selected from SEQ ID NOs: 74-84, 86, and 97-101.

[0167] In some embodiments of the antibodies described herein, one of the two Fc polypeptides of the Fc polypeptide dimer may be a BBB (e.g., TfR) receptor binding Fc polypeptide comprising a sequence having at least 90% identity to the sequence of SEQ ID NO: 97, comprising Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser at position 389, Ser at position 413, Glu at position 415, Glu at position 416, and Phe at position 421, according to EU numbering, while the other Fc polypeptide of the Fc polypeptide dimer may have the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO: 95).

[0168] In some embodiments of the antibodies described herein, the first Fc polypeptide and / or the second Fc polypeptide independently comprise a Tyr at position 384, a Thr at position 386, a Glu at position 387, a Trp at position 388, an Ala at position 389, a Thr at position 413, a Glu at position 415, a Glu at position 416, and a Phe at position 421, according to EU numbering, and a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence selected from SEQ ID NOs: 101-105.

[0169] In some embodiments of the antibodies described herein, one of the two Fc polypeptides of the Fc polypeptide dimer may be a BBB (e.g., TfR) receptor binding Fc polypeptide comprising a sequence having at least 90% identity to the sequence of SEQ ID NO: 101, comprising Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ala at position 389, Thr at position 413, Glu at position 415, Glu at position 416, and Phe at position 421, according to EU numbering, while the other Fc polypeptide of the Fc polypeptide dimer may have the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO: 95).

[0170] In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide has the sequence of SEQ ID NO: 101. In some embodiments of the antibodies described herein, one of the two Fc polypeptides of the Fc polypeptide dimer may be a BBB (e.g., TfR) receptor binding Fc polypeptide having the sequence of SEQ ID NO: 101, while the other Fc polypeptide of the Fc polypeptide dimer may have the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO: 95). In other embodiments of the antibodies described herein, both Fc polypeptides of the Fc polypeptide dimer may be BBB (e.g., TfR) receptor binding Fc polypeptides having the sequence of SEQ ID NO: 101.

[0171] In some embodiments, the BBB (eg, TfR) receptor binding Fc polypeptide comprises the following substitutions (according to EU numbering) listed in Table A below.

[0172] [Table A] TIFF2024534118000003.tif207159

[0173] Fc Polypeptide Modification for Heterodimerization In some embodiments, the Fc polypeptide present in any antibody described herein comprises knob and hole mutations to promote heterodimer formation and prevent homodimer formation. Generally, the modifications introduce a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and thus prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain at the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity of equal or similar size to the protrusion is formed at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine). In some embodiments, such additional mutations are at positions in the Fc polypeptide that do not adversely affect binding of the polypeptide to a BBB receptor, e.g., TfR.

[0174] In one exemplary embodiment of the knob-and-hole approach for dimerization, one of the Fc polypeptides present in the antibody contains a tryptophan instead of the native threonine at position 366 (numbered according to the EU numbering scheme). The other Fc polypeptide of the dimer has a valine instead of the native tyrosine at position 407 (numbered according to the EU numbering scheme). The other Fc polypeptide may further contain substitutions, in which the native threonine at position 366 (numbered according to the EU numbering scheme) is replaced with a serine and the native leucine at position 368 (numbered according to the EU numbering scheme) is replaced with an alanine. Thus, one of the Fc polypeptides of the antibodies described herein has a T366W knob mutation and the other Fc polypeptide has a Y407V mutation, which is usually accompanied by T366S and L368A hole mutations.

[0175] In some embodiments, one or both of the Fc polypeptides present in an antibody described herein may also be engineered to include other modifications for heterodimerization, such as electrostatic manipulation or hydrophobic patch modifications of contact residues within the naturally charged CH3-CH3 interface.

[0176] For example, in some embodiments, the antibodies described herein may comprise an Fc polypeptide dimer having one Fc polypeptide with a knob mutation of T366W and at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the sequence of SEQ ID NO: 107 and another Fc polypeptide with hole mutations of T366S, L368A, and Y407V and at least 90% identity to the sequence of SEQ ID NO: 85. In certain embodiments, one or both Fc polypeptides of the Fc polypeptide dimer may be a TfR-binding Fc polypeptide. In certain embodiments, the antibodies described herein may comprise an Fc polypeptide dimer having (i) a first Fc polypeptide with a sequence of SEQ ID NO: 85, and (ii) a second Fc polypeptide with a sequence of SEQ ID NO: 98. In certain embodiments, the antibodies described herein may comprise an Fc polypeptide dimer having (i) a first Fc polypeptide having the sequence of SEQ ID NO: 85, and (ii) a second Fc polypeptide having the sequence of SEQ ID NO: 102.

[0177] Fc Polypeptide Modifications to Modulate Effector Function In some embodiments, one or both of the Fc polypeptides present in any of the antibodies described herein may contain modifications that reduce TfR-mediated effector function upon TfR binding, i.e., reduce the ability to induce a particular biological function after binding to an Fc receptor expressed on an effector cell that mediates the effector function. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Effector functions may vary depending on the class of antibody. For example, naturally occurring human IgG1 and IgG3 antibodies can induce ADCC and CDC activity upon binding to the appropriate Fc receptor present on immune system cells, and naturally occurring human IgG1, IgG2, IgG3, and IgG4 can induce ADCP function upon binding to the appropriate Fc receptor present on immune cells.

[0178] In some embodiments, one or both Fc polypeptides present in an antibody described herein may contain a modification that reduces or eliminates TfR-mediated effector function. Exemplary Fc polypeptide mutations that reduce TfR-mediated effector function include, but are not limited to, substitutions in the CH2 domain, e.g., at positions 234 and 235 according to the EU numbering scheme. For example, in some embodiments, one or both Fc polypeptides may contain alanine residues at positions 234 and 235. Thus, one or both Fc polypeptides may have L234A and L235A (also referred to herein as "LALA") substitutions.

[0179] Further Fc polypeptide mutations that modulate effector function include, but are not limited to, the following: position 329 may have a mutation in which proline is substituted with glycine, alanine, serine, or arginine, or an amino acid residue large enough to disrupt the Fc / Fcγ receptor interface formed between proline 329 of Fc and tryptophan residues Trp87 and Trp110 of FcγRIII. Further exemplary substitutions include S228P, E233P, L235E, N297A, N297D, and P331S according to the EU numbering scheme. Multiple substitutions may also be present. For example, according to the EU numbering scheme, L234A and L235A in the Fc region of human IgG1, L234A, L235A, and P329G in the Fc region of human IgG1, S228P and L235E in the Fc region of human IgG4, L234A and G237A in the Fc region of human IgG1, L234A, L235A, and G237A in the Fc region of human IgG1, V234A and G237A in the Fc region of human IgG2, L235A, G237A, and E318A in the Fc region of human IgG4, and S228P and L236E in the Fc region of human IgG4. In some embodiments, one or both Fc polypeptides may have one or more amino acid substitutions that modulate ADCC, for example substitutions at positions 298, 333, and / or 334 according to the EU numbering scheme. In some embodiments, one or both Fc polypeptides may have the following substitutions according to the EU numbering scheme: L234A, L235A, and P329G or P329S.

[0180] In some embodiments, one or both of the Fc polypeptides present in the antibody described herein may contain modifications that can enhance HER2-mediated effector function upon HER2 binding, i.e., enhance the ability to induce a specific biological function after binding to an Fc receptor expressed on an effector cell that mediates the effector function. Examples of antibody effector functions are described above. Exemplary Fc polypeptide mutations that can enhance HER2-mediated effector function include, but are not limited to, substitutions in the CH2 domain, for example, at positions 239 and / or 332 according to the EU numbering scheme. For example, in some embodiments, one or both Fc polypeptides may contain an aspartic acid at position 239 and / or a glutamic acid at position 332. Thus, one or both Fc polypeptides may have the following substitutions according to EU numbering: S239D and / or I332E.

[0181] "cis-LALA" configuration In some embodiments of any of the antibodies described herein, only one of the two Fc polypeptides of the antibody (but not both Fc polypeptides) is modified to reduce TfR-mediated effector function upon TfR binding. The other Fc polypeptide does not contain a TfR binding site or a modification that reduces effector function. An Fc polypeptide dimer in an antibody in which only one of the two Fc polypeptides contains both a TfR binding site and a modification that reduces FcγR binding upon binding to TfR (e.g., a LALA substitution), and the other Fc polypeptide does not contain a TfR binding site or any modification that reduces FcγR binding, is said to have a cis-LALA configuration.

[0182] For example, in some embodiments, the antibodies described herein may comprise an Fc polypeptide dimer having this cis-LALA configuration, which has (i) a first Fc polypeptide having a sequence of SEQ ID NO: 86, which has both a TfR binding site and a LALA substitution, as well as a knob modification, and (ii) a second Fc polypeptide having only the hole modification, which has at least 90% identity to the sequence of SEQ ID NO: 85. In some embodiments, the antibodies described herein may comprise an Fc polypeptide dimer having this cis-LALA configuration, which has (i) a first Fc polypeptide having a sequence of SEQ ID NO: 103, which has both a TfR binding site and a LALA substitution, as well as a knob modification, and (ii) a second Fc polypeptide having only the hole modification, which has at least 90% identity to the sequence of SEQ ID NO: 85.

[0183] In certain embodiments, the antibodies described herein may comprise an Fc polypeptide dimer having this cis-LALA configuration, which comprises (i) a first Fc polypeptide comprising a sequence comprising: Ala at position 234, Ala at position 235, Trp at position 366, Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser at position 389, Ser at position 413, Glu at position 415, Glu at position 416, and Phe at position 421 according to EU numbering, and having at least 90% identity to the sequence of SEQ ID NO: 86; and (ii) a second Fc polypeptide comprising: Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering, and having at least 90% identity to the sequence of SEQ ID NO: 85.

[0184] In certain embodiments, the antibodies described herein may comprise an Fc polypeptide dimer having this cis-LALA configuration, which comprises (i) a first Fc polypeptide comprising a sequence comprising Ser at position 366, Ala at position 368, and Val at position 407, according to EU numbering, and having at least 90% identity to the sequence of SEQ ID NO: 85, and (ii) a second Fc polypeptide comprising a sequence comprising Ala at position 234, Ala at position 235, Trp at position 366, Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser at position 389, Ser at position 413, Glu at position 415, Glu at position 416, and Phe at position 421, according to EU numbering, and having at least 90% identity to the sequence of SEQ ID NO: 86.

[0185] In certain embodiments, the antibodies described herein may comprise an Fc polypeptide dimer having this cis-LALA configuration, which comprises (i) a first Fc polypeptide comprising a sequence comprising Ala at position 234, Ala at position 235, Trp at position 366, Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ala at position 389, Thr at position 413, Glu at position 415, Glu at position 416, and Phe at position 421 according to EU numbering, and having at least 90% identity to the sequence of SEQ ID NO: 103, and (ii) a second Fc polypeptide comprising a sequence comprising Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering, and having at least 90% identity to the sequence of SEQ ID NO: 85.

[0186] In certain embodiments, the antibodies described herein may comprise an Fc polypeptide dimer having this cis-LALA configuration, which comprises (i) a first Fc polypeptide comprising a sequence comprising Ser at position 366, Ala at position 368, and Val at position 407, according to EU numbering, and having at least 90% identity to the sequence of SEQ ID NO: 85, and (ii) a second Fc polypeptide comprising a sequence comprising Ala at position 234, Ala at position 235, Trp at position 366, Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ala at position 389, Thr at position 413, Glu at position 415, Glu at position 416, and Phe at position 421, according to EU numbering, and having at least 90% identity to the sequence of SEQ ID NO: 103.

[0187] Fc Polypeptide Modifications to Extend Serum Half-Life In some embodiments, modifications to extend serum half-life may be introduced into any of the antibodies described herein. For example, in some embodiments, one or both Fc polypeptides present in an antibody described herein may contain a tyrosine at position 252, a threonine at position 254, and a glutamic acid at position 256, numbered according to the EU numbering scheme. Thus, one or both Fc polypeptides may have the following substitutions: M252Y, S254T, and T256E. Alternatively, one or both Fc polypeptides may have the following substitutions: M428L and N434S, numbered according to the EU numbering scheme. Alternatively, one or both Fc polypeptides may have the following substitutions: N434S or N434A.

[0188] Fc polypeptide with C-terminal lysine residue removed In some embodiments of the antibodies described herein, one or both of the Fc polypeptides may have their C-terminal lysine removed (e.g., the Lys residue at position 447 of the Fc polypeptide according to EU numbering). This C-terminal lysine residue is highly conserved in immunoglobulins across many species and may be completely or partially removed by the cellular machinery during protein production. In some embodiments, removal of the C-terminal lysine of the Fc polypeptide may improve the stability of the antibody.

[0189] V. Antibody Preparation Many techniques known in the art may be used for the preparation of the antibodies described herein. In some embodiments, genes encoding the heavy and light chains of the antibody of interest may be cloned from cells, such as hybridomas. Gene libraries encoding the heavy and light chains of monoclonal antibodies may also be generated from hybridomas or plasma cells. Alternatively, phage or yeast display technology may be used to identify antibodies and Fab fragments that specifically bind to a selected antigen.

[0190] The antibody may be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is a mammalian cell expression system, such as a hybridoma or CHO cell expression system. Many such systems are widely available from commercial vendors. In some embodiments, the polynucleotides encoding the polypeptides comprising the antibody may be expressed using a single vector, for example, in a dicistronic expression unit or under the control of different promoters. In other embodiments, the polynucleotides encoding the polypeptides comprising the antibody may be expressed using separate vectors.

[0191] In some aspects, the disclosure provides isolated nucleic acids comprising a nucleic acid sequence encoding any of the polypeptides, including the antibodies, described herein, vectors comprising such nucleic acids, and host cells into which the nucleic acids have been introduced for use in replicating the nucleic acids and / or expressing the antibodies.

[0192] In some embodiments, a polynucleotide (e.g., an isolated polynucleotide) comprises a nucleotide sequence that encodes a polypeptide comprising an antibody disclosed herein (e.g., those described in Section III, above). In some embodiments, the polynucleotide comprises a nucleotide sequence that encodes one or more amino acid sequences (e.g., heavy chain sequences, light chain sequences and / or Fc polypeptide sequences) disclosed in the Informal Sequence Listing below. In some embodiments, the polynucleotide comprises a nucleotide sequence that encodes an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to a sequence disclosed in the Informal Sequence Listing below. In some embodiments, the polynucleotides described herein are operably linked to a heterologous nucleic acid, e.g., a heterologous promoter.

[0193] Suitable vectors containing a polynucleotide encoding an antibody of the present disclosure, or a fragment thereof, include cloning vectors and expression vectors. The cloning vector selected may vary depending on the host cell to be used, but useful cloning vectors generally have the ability to replicate autonomously, may have a single target for a specific restriction endonuclease, and / or may have a marker gene that can be used to select clones containing the vector. Examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors, such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.

[0194] Expression vector is generally a replicable polynucleotide construct that contains the nucleic acid of the present disclosure.This expression vector can replicate in host cell as episome or as integral part of chromosomal DNA.Suitable expression vector includes but is not limited to plasmid, virus vector, including adenovirus, adeno-associated virus, retrovirus, and any other vector.

[0195] Suitable host cells for cloning or expressing the polynucleotide or vector described herein include prokaryotic or eukaryotic cells. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell. In some embodiments, the host cell is a human cell, such as a human embryonic kidney (HEK) cell.

[0196] In another aspect, methods of making the antibodies described herein are provided. In some embodiments, the methods include culturing a host cell described herein (e.g., a host cell expressing a polynucleotide or vector described herein) under conditions suitable for expression of the antibody. In some embodiments, the antibody is then recovered from the host cell (or host cell culture medium). In some embodiments, the antibody is purified, for example, by chromatography.

[0197] VI. Treatment method In some aspects, provided herein is a method for treating cancer (e.g., HER2-positive cancer) or treating brain metastasis of cancer (e.g., HER2-positive cancer) in a subject by administering to the subject a therapeutically effective amount of any antibody or pharmaceutical composition thereof described herein. Also provided herein is a method of transcytosis of an antibody variable region capable of binding to HER2 (e.g., human HER2) or an antigen-binding fragment thereof across an endothelium. In some embodiments, the method comprises contacting the endothelium with a composition comprising an antibody described herein. In some embodiments, the endothelium is the blood-brain barrier (BBB).

[0198] Non-limiting examples of HER2-positive cancers that may be treated according to the methods provided herein include HER2-positive breast cancer, ovarian cancer, bladder cancer, salivary gland cancer, endometrial cancer, pancreatic cancer, and non-small cell lung cancer (NSCLC), as well as HER2-positive gastric and / or esophagogastric junction adenocarcinoma. In some embodiments, the HER2-positive cancer is HER2-positive breast cancer. In some embodiments, the HER2-positive cancer is HER2-positive gastric and / or esophagogastric junction adenocarcinoma. In some embodiments, the HER2-positive cancer is metastatic cancer.

[0199] In yet another aspect, provided herein are methods for treating metastasis of cancer (e.g., HER2-positive cancer). In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an antibody described herein. In some embodiments, the metastasis is a brain metastasis of a HER2-positive cancer as described above. In some embodiments, the metastasis is a brain metastasis of a HER2-positive breast cancer. In some embodiments, the metastasis is a brain metastasis of a HER2-positive gastric adenocarcinoma and / or a HER2-positive gastroesophageal junction adenocarcinoma.

[0200] In some embodiments, the therapeutic benefit may include reducing or slowing tumor growth, reducing tumor size (e.g., volume), reducing tumor cell viability, reducing the number of metastatic lesions, improving one or more signs or symptoms of cancer (e.g., HER2-positive cancer), and / or increasing patient survival. In some embodiments, tumor cell viability, tumor growth, tumor size, and / or the number of metastatic lesions is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.

[0201] In some embodiments, the antibody antagonizes HER2 activity, hi some embodiments, HER2 activity is inhibited (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more).

[0202] The route of administration of the antibodies described herein can be oral, intraperitoneal, transdermal, subcutaneous, intravenous, intramuscular, intrathecal, inhalation, topical, intralesional, rectal, intrabronchial, nasal, transmucosal, intestinal, ocular or otic delivery, or any other method known in the art, In some embodiments, the antibodies are administered orally, intravenously, or intraperitoneally.

[0203] VII. Pharmaceutical Compositions and Kits In other aspects, pharmaceutical compositions and kits comprising the antibodies according to the present disclosure are provided.

[0204] Pharmaceutical Compositions Guidance for preparing formulations for use in the present disclosure can be found in many handbooks on pharmaceuticals and formulations known to those of skill in the art.

[0205] In some embodiments, a pharmaceutical composition comprises an antibody described herein and further comprises one or more pharma- ceutically acceptable carriers and / or excipients. A pharma- ceutically acceptable carrier includes any solvent, dispersion medium, or coating that is physiologically compatible with the active agent and does not interfere with or inhibit the activity of the active agent.

[0206] In some embodiments, the antibody can be formulated for parenteral administration by injection. Typically, pharmaceutical compositions to be used for in vivo administration are sterile, e.g., by heat sterilization, steam sterilization, sterile filtration, or irradiation sterilization.

[0207] Dosages and desired drug concentrations of the pharmaceutical compositions described herein may vary depending on the particular use envisioned.

[0208] kit In some embodiments, a kit for use in treating cancer (e.g., HER2-positive cancer) is provided that includes an antibody described herein. In some embodiments, the kit further includes one or more additional therapeutic agents. For example, in some embodiments, the kit includes an antibody described herein and further includes one or more additional therapeutic agents for use in treating cancer. In some embodiments, the kit further includes instructional materials (e.g., instructions for using the kit to administer the antibody) that include directions (i.e., protocols) for carrying out the methods described herein. The instructional materials typically include, but are not limited to, written or printed materials. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, magnetic tapes, magnetic cartridges, magnetic chips), optical media (e.g., CD-ROMs), and the like. Such media may include the address of an internet site that provides such instructional materials. EXAMPLES

[0209] VIII. Examples The present invention will now be described in more detail through specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the present invention in any manner.

[0210] Example 1. Generation of anti-HER2 bispecific antibodies Expression and purification of recombinant bispecific antibody variants Expression plasmids consisting of (i) a heavy chain polypeptide containing the TfR binding site and the knob (T366W) mutation, (ii) a heavy chain polypeptide containing the hole (T366S / L368A / Y407V) mutation, and (iii) a light chain according to the combinations in Table 2 are co-transfected into Expi293 or ExpiCHO cells. Recombinant bispecific antibody variants are then purified from the conditioned medium by loading the supernatant onto a Protein A column (GE Mab Select SuRe). The column is washed with 10 column volumes of PBS, pH 7.4. The protein is eluted with 50 mM sodium citrate, pH 3.0, containing 150 mM NaCl, and immediately neutralized with 200 mM arginine, 137 mM succinate, pH 5.0. The protein is further purified by size exclusion chromatography (GE Superdex200) using 200 mM arginine, 137 mM succinic acid, pH 5.0 as the running buffer. The purified protein is confirmed by intact mass LC / MS and is >95% pure by SDS-PAGE and analytical HPLC-SEC.

[0211] Heavy chain polypeptides may be further processed during cell culture production to remove C-terminal lysine residues. Thus, the bispecific antibodies listed in Table 2 may refer to protein molecules that contain a heavy chain that is unprocessed (i.e., contains a C-terminal lysine residue); protein molecules that contain one or more heavy chains that are processed (i.e., lacking a C-terminal lysine residue); or mixtures of protein molecules having processed and / or unprocessed heavy chains.

[0212] Example 2. Evaluation of anti-HER2 antibodies by Biacore The HER2 extracellular domain (ECD) binding affinity of engineered anti-HER2 antibodies was measured by SPR using a Biacore 8K instrument. Antibodies were captured on a Biacore™ Series S CM5 sensor chip immobilized with mouse anti-human Fab (human Fab capture kit from GE Healthcare), followed by injection of 3-fold serial dilutions of recombinant HER2 ECD at a flow rate of 30 μL / min. Each sample was analyzed using 3 min of binding followed by 10 min of dissociation. After each injection, the sensor chip was regenerated using 50 mM glycine (pH 2.0) regeneration buffer. on and k off A 1:1 Languir model of simultaneous fitting was used for kinetic analysis.

[0213] The consensus sequences of anti-HER2_D4 light chain control (SEQ ID NO: 87) and anti-HER2_D2 light chain control (SEQ ID NO: 94) were analyzed. Structural analysis suggested that the Y residue at position 91 is involved in the structural organization of the CDR loop for efficient HER2 D4 binding, while the H residue at the same position is less involved in HER2 D2 binding. After screening single amino acid substitutions at position 91, the Y and F residues at this position were selected for further testing.

[0214] The affinity matured anti-HER2 light chain sequences (SEQ ID NOs: 9 and 10) were analyzed using HER2 binding K D For the measurements, anti-HER2_D2 heavy chain control (SEQ ID NO: 92) and anti-HER2_D4 heavy chain control (SEQ ID NO: 93) were paired. The results are shown in Table 7.

[0215] [Table 7]

[0216] The light chains of SEQ ID NOs: 9 and 10, when paired with both the anti-HER2_D2 and D4 heavy chain controls, demonstrated HER2 binding. The light chains of SEQ ID NOs: 9 and 10, when paired with the anti-HER2_D2 heavy chain control, demonstrated HER2 binding relative to the anti-HER2_D2 light chain control (2.9K D ) compared with lower HER2 binding affinity (13K, respectively)D and 14K D In contrast, the light chains of SEQ ID NOs: 9 and 10, when paired with the anti-HER2_D4 heavy chain control, showed a similar increase in the expression of the anti-HER2_D4 light chain control (3K D ) compared with higher HER2 binding affinity (1.5K D and 1.7K D ) was shown.

[0217] Thirteen amino acid positions in CDR H1, H2, or H3 of anti-HER2_D2 heavy chain control (SEQ ID NO: 92) were selected based on structural analysis. The selected residues were randomized to find single amino acid substitution variants with improved HER2 ECD domain II binding. Antibodies with these single point mutations were expressed in Expi293 cells in combination with anti-HER2_D4 light chain control (SEQ ID NO: 87), and antibodies in cell culture supernatants were screened for recombinant HER2 ECD binding using SPR. Variants with improved HER2 ECD domain II were selected and expressed in Expi293 cells with the light chain of SEQ ID NO: 10, and purified for further SPR binding evaluation.

[0218] V of SEQ ID NOs: 1 to 2 and 60 to 70 H The affinity matured anti-HER2_D2 heavy chain sequence containing the HER2 binding K D Anti-HER2_D4 light chain control (SEQ ID NO: 87) was paired for measurement. Results are shown in Table 8.

[0219] [Table 8]

[0220] V of SEQ ID NO: 1-2 and 60-70 paired with anti-HER2_D4 light chain control (SEQ ID NO: 87) H Affinity matured anti-HER2_D2 heavy chain sequences containing the region exhibited HER2 binding, and all showed improved HER2 binding compared to the anti-HER2_D2 heavy chain control.

[0221] The affinity matured anti-HER2 light chain sequence (SEQ ID NO: 10) was used as a HER2 binding K D For the measurement, V of SEQ ID NO: 1 to 3 H The results are shown in Table 9.

[0222] [Table 9]

[0223] V of SEQ ID NOs: 1 to 3 H The light chain of SEQ ID NO: 10 paired with the affinity matured anti-HER2_D2 heavy chain sequence containing the region showed improved HER2 binding affinity (4.2, 6.2, and 2.1K, respectively). D As discussed above and shown in Table 7, the anti-HER2_D2 light chain control light chain paired with the anti-HER2_D2 heavy chain control showed a 2.9K D Thus, V of SEQ ID NO:3 had a HER2 binding affinity of 0.01 to 0.01. H The light chain of SEQ ID NO: 10 paired with an affinity matured anti-HER2_D2 heavy chain sequence containing the region binds to HER2 with higher affinity than the control.

[0224] Example 3. In vitro ADCC / ADCP of anti-HER2 bispecific antibodies Human ADCC Reporter Bioassay, V Variant Kit (Promega G7018) was used to assess human FcγRIIIa activation, while Human FcgRIIa ADCP Reporter Bioassay Kit (Promega G9995) was used to measure human FcγRIIa reporter activation of bispecific antibody combinations from Table 10. The kit contains all components as described below. Several cell lines with different expression levels of HER2 and TfR were tested. Cells SKBR3 (ATCC HTB-30), ZR-75-30 (ATCC CRL-1504), BT-474 (ATCC HTB-20), OE-19 (Sigma 96071721), and CHO-KI+HumanTfR (ChemPartner CRO agreement) were cultured to log phase in RPMI (Liffe Technologies 61870-036) supplemented with 10% FBS (Hyclone bovine serum SH30080.03) and 1% penicillin-streptomycin (Life Technologies 15140-122), washed twice with PBS, and cultured at 1.0 × 10 in RPMI supplemented with 10% FBS and 1% penicillin / streptomycin. 6 White 96-well high-binding Nunc plates (ThermoFisher) were coated with 25 μL of medium containing 50,000 cells / well.

[0225] Antibody titrations were prepared in RPMI with 4% low IgG serum and 25 μl per well was added to the plates to opsonize the cells, then covered and incubated for 30 min at 37°C, 5% CO2. During antibody opsonization, 3.5 mL of medium was pre-warmed to 37°C and FcγR reporter cells were quickly thawed in a 37°C water bath without inversion and added to the pre-warmed medium in a 15 mL conical tube with gentle mixing. After 30 min of opsonization, FcγR reporter cell lines were added to each plate at 25 μl per well and incubated for 6 h (SKBR3, ZR-75-30, BT-474 for hFcγRIIIa and hFcγRIIa activation) or 16 h (CHO-KI+huTfR for hFcγRIIIa and hFcγRIIa) at 37°C, 5% CO2. After incubation, plates were acclimated to room temperature, 75 μL per well of Bio-Glo luciferase substrate suspension (Promega) was added, and luminescence was measured on a Perkin Elmer Envision reader. The results are shown in Table 11.

[0226] [Table 10]

[0227] [Table 11] TIFF2024534118000009.tif139160

[0228] The objective was to develop Fc variants that did not increase TfR-mediated ADCC compared to the control and / or Fc1, and had comparable levels of HER2-mediated ADCC compared to the control and / or improved levels of HER2-mediated ADCC compared to Fc1. As shown in Table 11 above, Fc1, Fc41, Fc5, Fc45, Fc42, Fc52, Fc44, Fc50, Fc68, Fc7, Fc8, Fc2, Fc34, and Fc4 all had comparable levels of TfR-mediated ADCC compared to the control in TfR-overexpressing CHO cells. Fc50 and Fc52 showed the highest levels of HER2-mediated ADCC across all HER2-overexpressing cell lines tested, without increasing TfR-mediated ADCC activation.

[0229] Also shown in Table 11 are ADCP levels for Fc1, Fc41, Fc5, Fc45, Fc42, Fc52, Fc44, and Fc50 variants compared to controls across HER2-overexpressing cell lines, namely OE19, ZR-75-30, and SKBR3.

[0230] Example 4. In vitro proliferation inhibition of anti-HER2 bispecific antibodies A proliferation inhibition assay was used to measure the viability of cells after treatment with different antibodies for different periods of time. Several cell lines with different expression levels of HER2 and TfR were tested. Cells SKBR3 (ATCC HTB-30), ZR-75-30 (ATCC CRL-1504), BT-474 (ATCC HTB-20), OE-19 (Sigma 96071721), CHO-KI+HumanTfR (ChemPartner CRO agreement) were cultured to log phase in RPMI (Life Technologies 61870-036) supplemented with 10% FBS (Hyclone Bovine serum SH30080.03) and 1% penicillin-streptomycin (Life Technologies 15140-122). After washing with PBS, cells were cultured at 1.0 × 10 5The cells were resuspended at 10,000 cells / mL. Black poly-D-lysine plates (Corning 354640) were coated with 100 μl of cell culture medium containing 10,000 cells / well. The plates were incubated in a 37° C., 5% CO2 incubator for 24 hours.

[0231] Antibody titrations were prepared in RPMI with 10% FBS serum and 1% penicillin / streptomycin. Antibodies were added to each plate at 65 μl per well, then covered and incubated at 37° C., 5% CO2 for 72 hours (OE-19 cell line only). For BT-474 and ZR-75-30 cell lines, an additional 65 μl of antibody was added after 72 hours and incubated at 37° C., 5% CO2 for an additional 72 hours.

[0232] On day 7, cell proliferation was measured using 5 μL of WST-1 reagent (Sigma Aldrich) in 50 μL of growth medium. The plates were incubated in the presence of WST-1 reagent for 4 hours and the absorbance at 440 nm was measured. Percentage of growth inhibition / proliferation was calculated based on A440nM and normalized to untreated controls.

[0233] The results of the growth inhibition assay on ZR-75-30 cells for the different antibodies in Table 12, as well as IC50 and maximum % growth inhibition values, are shown in Figure 2. Bispecific antibodies #2, #3, #4, and #5 each showed improved EC50 values / efficacy compared to the control. Bispecific antibodies #4 and 5 showed maximum % growth inhibition / efficacy similar to the control.

[0234] [Table 12]

[0235] Example 5. In vivo xenograft studies with ATV:CLC bispecific antibodies Two human HER2+ cell lines were used to evaluate the response of ATV:CLC bispecific antibody #1 in a subcutaneous xenograft model in immunodeficient (NOD / SCID) mice. All molecules were prepared in the same formulation buffer (10 mM sodium acetate, 6% sucrose, pH 5.5) or PBS / saline, except for trastuzumab (Clinical Herceptin) and pertuzumab (Clinical Perjeta), which were either purchased and prepared according to manufacturer's instructions and / or further diluted in PBS or saline.

[0236] [Table 13]

[0237] For the BT-474 breast cancer cell line-derived xenograft (CDX) model, BT-474 cells were injected subcutaneously into the axilla of female NSG (NOD scid gamma) mice (6–7 weeks old) until tumor volumes reached 100–200 mm. 3 Treatment was initiated 6 days after inoculation when tumor volumes were 100-150%. Mice were randomly assigned to treatment groups based on mean tumor volume, with n=11 mice per group. Trastuzumab and pertuzumab combination treatment at 40+40mg / kg or ATV:CLC bispecific antibody #1 at 80mg / kg were administered by intraperitoneal (IP) injection. Tumor volumes were measured 3 times a week using calipers.

[0238] In the relatively trastuzumab-sensitive BT-474 xenograft model, ATV:CLC bispecific antibody #1 demonstrated comparable tumor growth inhibition compared to trastuzumab and pertuzumab after a single dose, with complete tumor regression in all treatment groups after 21 days (Figure 3A).

[0239] For the OE19 gastroesophageal junction CDX model, female NOD / SCID mice (6–8 weeks old) were injected with OE19 cells subcutaneously in the right upper flank region, and 1 week after inoculation, tumors were grown to 100–200 mm 3Treatment was initiated when tumor volumes were between 0.01 and 0.1. Mice were randomly assigned to treatment groups with n=11 mice per group based on the corresponding distribution / stratification method. ATV:Trastuzumab and ATV:Pertuzumab combination treatments at 50+50 mg / kg or ATV:CLC bispecific antibody #1 at 100 mg / kg were administered by IP injection. Tumor volumes were measured 3 times a week using calipers.

[0240] In the OE19 xenograft model, which shows relative resistance to combination trastuzumab and pertuzumab treatment, both ATV:CLC bispecific antibody #1 and the combination of ATVcis-LALA:trastuzumab and ATVcis-LALA:pertuzumab showed significant tumor growth delay compared to the control group (Figure 3B). All groups start with n=11 mice per group. The numbers on the graphs represent the number of animals remaining in the control group after a subset of animals reached a humane endpoint. On day 17, one animal was found dead in the ATV combo group with no apparent cause. ATV:trastuzumab and ATV:pertuzumab are trastuzumab and pertuzumab antibodies that contain Fc modifications that bind to TfR ("TV") and cis-LALA mutations. These in vivo results are consistent with in vitro growth inhibition data that demonstrated increased potency and increased maximal efficacy of ATV:HER2 (anti-HER2 molecule containing TV) in OE19 cell lines compared with anti-HER2 molecules lacking the TfR-binding Fc modification.

[0241] In a follow-up low-dose study, ATV:CLC bispecific antibody #1 was compared to a CLC bispecific antibody control with an identical Fab as ATV:CLC bispecific antibody #1 but lacking the TfR-binding Fc modification to study the effect of TfR binding. In the BT-474 breast cancer CDX model, female NOD / SCID mice (6-8 weeks old) were implanted with estrogen pellets (0.36 mg, 17B-estradiol, 60-day pellet) 1 day prior to tumor inoculation. BT-474 cells were then injected subcutaneously into the mammary fat pad and 8 days after inoculation, tumors were grown to a volume of 100-200 mm. 3Treatment was initiated when tumor volume reached 0.01 mg / kg / day. Mice were randomly assigned to treatment groups based on corresponding distribution / stratification methods with n=11 mice per group. Tumor volumes were measured twice weekly using calipers.

[0242] A single 20 mg / kg dose of ATV:CLC bispecific antibody #1 administered intraperitoneally demonstrated similar tumor growth delay as the CLC bispecific antibody control (no TfR binding) in the sensitive BT-474 xenograft model (Figure 4A). ATV:CLC bispecific antibody #1 demonstrated improved responses in the better tolerated OE19 xenograft model at an equivalent dose of 20 mg / kg compared to the CLC bispecific antibody control, and demonstrated comparable antitumor responses at a 4-fold lower dose (5 mg / kg) (Figure 4B).

[0243] In further cross-linking studies, ATV:CLC bispecific antibodies #1 and #2 were compared in a multiple dose xenograft study. In the BT-474 model, mice (n=11 per group) were dosed Q1W via IP, i.e., mice were given one dose per week for 3 weeks. ATV:CLC bispecific antibody #1 and ATV:CLC bispecific antibody #2 showed comparable inhibition and delay of tumor growth (Figure 5A). Furthermore, the same groups were given a combination of tucatinib at 50 mg / kg daily orally for 21 days, but no further improvement was observed with ATV:CLC bispecific antibody #1 or #2.

[0244] Finally, Fc-engineered variants of ATV:CLC bispecific antibody #2, ATV:CLC bispecific antibody #3 (containing additional Fc modifications, e.g., P329S, I332E, and S239D) were also compared to an anti-HER2 molecule lacking TfR binding in a multiple-dose OE19 xenograft study. Mice were dosed Q2W via IP, i.e., mice were administered one dose every 2 weeks for 6 weeks. ATV:CLC bispecific antibody #3 showed increased tumor growth delay and increased survival compared to the combination of trastuzumab and pertuzumab, or the CLC bispecific antibody control (Figure 5B). All groups start with n=11 mice per group. Numbers on graphs represent the number of animals remaining in a group after a subset of animals reached a humane endpoint.

[0245] Example 6. Brain uptake and distribution of ATV:CLC bispecific antibodies TfR mu / hu KI mice (see, e.g., International Publication No. WO2018 / 152285) were administered a single 25 mg / kg IV dose of CLC bispecific antibody control or ATV:CLC bispecific antibody #3 (n=4 / group). Antemortem blood was collected at 30 minutes and 6 hours, terminal blood and fresh frozen brains were collected 1, 4, 7, and 10 days post-dosing, and huIgG concentrations in plasma and brain lysates were assessed by ELISA.

[0246] TfR mu / hu Plasma and brain concentrations were measured after a single dose of bispecific CLC bispecific antibody control or ATV:CLC bispecific antibody #3 in KI mice. The brain concentration of ATV:CLC bispecific antibody #3 was approximately 6.5-fold higher 24 h after dosing compared to the CLC bispecific antibody control (Figure 6). This indicates brain delivery of the ATV molecule via the TfR. Similarly, in a time course study, ATV:CLC bispecific antibodies #2, #3, and #7 showed approximately 4-5-fold higher brain concentrations 24 h after IV dosing and up to approximately 2-fold higher brain concentrations after 4 days.

[0247] Furthermore, immunohistochemistry of the molecules administered in the brain was performed. One fresh brain hemisphere per animal was immersion fixed for immunohistochemistry at 4°C for approximately 24 hours, then cryoprotected in sucrose and sectioned with a freezing microtome. Coronal brain sections (40 μm) from each animal were selected and stained by incubating in blocking buffer (1% BSA + 1x fish gelatin + 0.5% Triton X-100 + 0.01% sodium azide in PBS) for 3 hours at room temperature. Sections were then incubated overnight at 4°C in dilution buffer (PBS with 1% BSA + 0.3% Triton X-100 + 0.01% sodium azide) containing primary / secondary antibodies (NeuN, Abcam, ab177487 and donkey anti-huIgG, Jackson, 709-606-149), washed three times for 15 min each with PBS with 0.3% Triton X-100, incubated for 3 h in dilution buffer containing secondary antibodies (donkey anti-rabbit, Invitrogen, A21206) and DAPI (5 μg / mL, Invitrogen, D1306), washed three times for 15 min each with PBS with 0.3% Triton X-100, followed by mounting and coverslipping with Prolong Glass (Invitrogen, P36984). Slides were imaged at 20x magnification using a Leica SP8 confocal microscope and segmentation and visualization was performed using Imaris.

[0248] Immunohistochemistry of the huIgG backbone of the administered molecules revealed that ATV:CLC bispecific antibody #3 was distributed extensively throughout the normal brain, localizing within blood vessels and NeuN+ neurons with diffuse signal in the parenchyma (Figure 7A). In contrast, the CLC bispecific antibody control showed limited penetration or distribution within brain tissue (Figure 7B). This is consistent with the significantly lower brain concentrations observed with non-TFR anti-HER2 molecules, i.e., molecules without TfR binding. Similar results, i.e., blood vessel and neuronal / parenchymal localization, were observed with ATV:CLC bispecific antibody #2 and ATV:CLC bispecific antibody #7.

[0249] Example 7. Plasma PK of ATV:CLC Bispecific Antibody in Cynomolgus Monkeys To assess the impact of Fc modifications on systemic clearance, Fc modified variants were compared. Bispecific HER2 ATVs containing TV35.21 (see CH3C.35.21 in Table A above) were used instead of TV35.23.4 (i.e., CH3C.35.23.4) because TV35.23.4 (i.e., CH3C.35.23.4) has no cynomolgus cross-reactivity, i.e., does not bind to the cynomolgus TfR. As shown in Table 14 below, these molecules use the same Fab as ATV:CLC bispecific antibodies #2, #3, and #7 used in the mouse studies above.

[0250] ATV:CLC bispecific antibodies #4, #5, and #6 were compared to Clinical Herceptin (trastuzumab) and serum concentrations of huIgG were measured at various time points after a single 50 mg / kg intravenous dose in female cynomolgus monkeys (n=3 / group).

[0251] [Table 14]

[0252] All ATV:HER2 molecules showed more rapid clearance from the systemic circulation compared to Herceptin (trastuzumab), as expected due to TfR-mediated clearance (Figure 8).

[0253] Example 8. In vitro ADCC / ADCP of anti-HER2 bispecific antibodies on NK cells A cell-based antibody-dependent cellular cytotoxicity (ADCC) assay was used to evaluate whether differences in Fc gamma receptor binding affinity for different Fc variants affect HER2-mediated tumor cell or TfR-mediated cell killing using isolated human NK cells.

[0254] NK cells were isolated from whole blood and used to evaluate the activation of human FcγRIIIa. Blood was collected with Trizma. Cells were isolated according to the RosetteSep human NK cell enrichment protocol (Stemcell 15065). RosetteSep Cocktail was added to the blood sample in a SepMate tube and left at room temperature for 15 minutes. After incubation, the sample was diluted with an equal volume of PBS (Gibco 10010-0310) and 10% FBS (Hyclone Bovine serum SH30080.03). The diluted sample was then added to density gradient medium Lymphoprep (Stemcell 07801) and centrifuged for 10 minutes. The enriched cells are then collected and washed twice with PBS. Finally, 20ng / ml of IL-21 was added to the cells and then left overnight for use the next day.

[0255] Several cell lines with different expression levels of HER2 and TfR were tested. Cells SKBR3 (ATCC HTB-30) and CHO-KI+HumanTfR (ChemPartner CRO agreement) were cultured to log phase in RPMI (Life Technologies 61870-036) supplemented with 10% FBS (Hyclone bovine serum SH30080.03) and 1% penicillin-streptomycin (Life Technologies 15140-122), washed twice with PBS and cultured at 1.0 × 10 in RPMI supplemented with 10% FBS and 1% penicillin / streptomycin. 6 Resuspended at 100 cells / mL.

[0256] Clear 96-well untreated V-bottom plates (Costar 3897) were coated with 25 μL of medium containing 50,000 cells / well. Antibody titrations were prepared in RPMI with 10% FBS serum and 25 μl per well was added to the plate to opsonize the cells, then covered and incubated for 30 min at 37° C., 5% CO2. During antibody opsonization, NK cells were washed once with medium containing RPMI and 10% FBS. Cells were counted and an E:T ratio of 25:1 was used as the cell density. After 30 min of opsonization, NK cells were added to each plate at 25 μl per well and incubated for 4 h. After incubation, plates were acclimated to room temperature and spun down at 300×g for 5 min. 50 μL of supernatant was transferred to a white 96-well clear bottom plate (Thermo 165306). 50 μl of CytoTox 96® assay reagent was added to each well of the plate containing the supernatant. The plate was covered to protect from light and incubated at room temperature for 30 minutes. Stop solution was added and the absorbance signal was measured at 490 nm in a plate reader. LDH released into the culture supernatant was measured in a 30-minute coupled enzyme assay that converts a tetrazolium salt (iodonitrotetrazolium violet; INT) into a red formazan product. The amount of color formed is proportional to the number of lysed cells.

[0257] Interestingly, in this assay against HER2-expressing tumor cells, the cis-LALA modification resulted in only a slight rightward shift of the curve indicating a slight decrease in potency, although a comparable maximal efficacy of cell killing was observed compared to the non-TV anti-HER2 bispecifics, i.e., CLC bispecific antibody control #2 and trastuzumab (Figure 9).

[0258] No cell killing was observed for TfR-expressing (HER2-) cells carrying cis-LALA or additional Fc mutations, suggesting that these molecules do not adversely affect TfR-expressing cells.

[0259] ADCP reporter assays to measure FcgRIIa activation also showed that ATV:CLC bispecific #2 and the Fc variants (ATV:CLC bispecific #3 and #7) showed similar receptor activation to each other, which was greater than trastuzumab and slightly less than the CLC bispecific antibody control #2.

[0260] Example 9. FcgR binding assay of ATV:CLC bispecific antibodies The Fc gamma receptor binding affinity of engineered anti-HER2 antibodies was measured by SPR using a Biacore 8K instrument. Biotinylated recombinant Fc gamma receptors were captured on a Biacore™ Series SA sensor chip, followed by injection of 3-fold serial dilutions of Fc engineered anti-Her2 antibodies at a flow rate of 30 μL / min. Each sample was analyzed with five 60-second injections of increasing antibody concentrations, followed by a 5-minute dissociation. A 1:1 Languir model of simultaneous fitting of k on and k off was used for kinetic analysis.

[0261] [Table 15]

[0262] Increased FcgR affinity is observed for the S239D and I332E engineered Fc variants, i.e., ATV:CLC bispecific antibody #5 and #6, compared to ATV:CLC bispecific antibody #4 and trastuzumab, but taken together with the results of the ADCC assay above, this increased affinity may only apply when the antibody is bound to the Fab target, i.e., HER2.

[0263] IX. ILLUSTRATIVE EMBODIMENTS Exemplary embodiments provided in accordance with the subject matter of this disclosure include, but are not limited to, the following embodiments and claims.

[0264] 1. The following: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 89; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 90; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 91 An isolated antibody comprising one or more complementarity determining regions (CDRs) selected from the group consisting of: below: X1 of SEQ ID NO:89 is not T; X2 of SEQ ID NO:89 is not F; X3 of SEQ ID NO:89 is not T; X1 in SEQ ID NO:90 is not N; X2 of SEQ ID NO:90 is not N; X3 of SEQ ID NO:90 is not S; X4 of SEQ ID NO:90 is not G; X5 of SEQ ID NO:90 is not G; X6 in SEQ ID NO:90 is not Q; X1 in SEQ ID NO:91 is not L; X2 of SEQ ID NO:91 is not G; X3 of SEQ ID NO:91 is not P; and X4 in SEQ ID NO:91 is not S; The isolated antibody is at least one of:

[0265] 2. The isolated antibody of embodiment 1, wherein said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 89, and X1 is N, K, M, or H.

[0266] 3. The isolated antibody of embodiment 1, wherein the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 90 and X5 is Q.

[0267] 4. The isolated antibody of embodiment 1, wherein the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 90, and X6 is R, H, or T.

[0268] 5. The isolated antibody of embodiment 1, wherein the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 91, and X4 is W, F, D, L, or Y.

[0269] 6. The isolated antibody of embodiment 1, wherein the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 91 and X4 is L.

[0270] 7. Below: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 89; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 90, wherein X5 is Q; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 91, and X4 is L 2. The isolated antibody of embodiment 1, comprising one or more CDRs selected from the group consisting of:

[0271] 8. Below: (a) a heavy chain CDR1 having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 49 to 52, or having up to two amino acid substitutions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 49 to 52; (b) a heavy chain CDR2 having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 6 and 53 to 55, or having up to two amino acid substitutions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 6 and 53 to 55; and (c) a heavy chain CDR3 having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 8 and 56 to 59, or having up to two amino acid substitutions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 8 and 56 to 59; 2. The isolated antibody of embodiment 1, comprising one or more CDRs selected from the group consisting of:

[0272] 9. Below: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6; and (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. 9. The isolated antibody of embodiment 8, comprising one or more CDRs selected from the group consisting of:

[0273] 10. The following: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 10. The isolated antibody of embodiment 9, comprising one or more CDRs selected from the group consisting of:

[0274] 11. The following: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:6; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:7; 11. The isolated antibody of embodiment 10, comprising one or more CDRs selected from the group consisting of:

[0275] 12. The following: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:5; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:8 11. The isolated antibody of embodiment 10, comprising one or more CDRs selected from the group consisting of:

[0276] 13. The following: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:6; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:8 11. The isolated antibody of embodiment 10, comprising one or more CDRs selected from the group consisting of:

[0277] 14. The isolated antibody of embodiment 8, comprising a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1 to 3.

[0278] 15. The isolated antibody of embodiment 8, comprising a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 3.

[0279] 16. The following: (a) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13 or 14; 1. An isolated antibody comprising:

[0280] 17. The following: (b) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:11 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:11; and (c) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 12; 17. The isolated antibody of embodiment 16, further comprising one or more CDRs selected from the group consisting of:

[0281] 18. The following: (b) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11; and (c) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12. 17. The isolated antibody of embodiment 16, further comprising one or more CDRs selected from the group consisting of:

[0282] 19. The isolated antibody of any one of embodiments 16 to 18, wherein the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:13.

[0283] 20. The isolated antibody of any one of embodiments 16 to 18, wherein the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:14.

[0284] 21. The isolated antibody of embodiment 17, comprising a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9-10.

[0285] 22. The isolated antibody of embodiment 17, comprising a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 9 to 10.

[0286] 23. The following: (a) a heavy chain CDR1 having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 49 to 52, or having up to two amino acid substitutions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 49 to 52; (b) a heavy chain CDR2 having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 6 and 53 to 55, or having up to two amino acid substitutions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 6 and 53 to 55; and (c) a heavy chain CDR3 having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 8 and 56 to 59, or having up to two amino acid substitutions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 8 and 56 to 59; (d) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:11 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:11; (e) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 12; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13 or 14. An antigen-binding site comprising 1. An isolated antibody comprising:

[0287] 24. The antigen-binding site comprises: (a) a heavy chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:4; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6; (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8; (d) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:11 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:11; (e) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 12; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13 or 14. 24. The isolated antibody of embodiment 23, comprising:

[0288] 25. The isolated antibody of embodiment 24, wherein the antigen-binding site comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1 to 3, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9 to 10.

[0289] 26. The isolated antibody described in embodiment 24, wherein the antigen-binding site comprises a heavy chain variable region comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 3, and a light chain variable region comprising any one of the amino acid sequences of SEQ ID NOs: 9 to 10.

[0290] 27. The following: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, or having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 16; (b) a heavy chain CDR2 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 17; and (c) a heavy chain CDR3 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18 or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO: 18; 27. The isolated antibody of any one of embodiments 23 to 26, further comprising a second antigen-binding site comprising one or more CDRs selected from the group consisting of:

[0291] 28. The isolated antibody of embodiment 27, wherein the second antigen-binding site comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15.

[0292] 29. The second antigen-binding site comprises: (a) a light chain CDR1 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:11, or having up to two amino acid substitutions to the amino acid sequence of SEQ ID NO:11; (b) a light chain CDR2 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 12; and (c) a light chain CDR3 having up to two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 13 or 14; 29. The isolated antibody of embodiment 27 or 28, further comprising one or more CDRs selected from the group consisting of:

[0293] 30. The isolated antibody of embodiment 29, wherein the second antigen-binding site comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9-10.

[0294] 31. The isolated antibody of embodiment 29 or 30, wherein the first antigen-binding site and the second antigen-binding site comprise the same light chain CDR1, CDR2, and CDR3 sequences.

[0295] 32. The isolated antibody of embodiment 31, comprising heavy chain and light chain CDRs selected from the combinations listed in Table 1.

[0296] 33. An isolated antibody comprising a heavy chain and a light chain selected from the combinations listed in Table 2.

[0297] 34. Below: (a) a first antigen-binding site against human epidermal growth factor receptor 2 (HER2) subdomain IV; (b) a second antigen-binding site against human HER2 subdomain II; and (c) a modified Fc polypeptide dimer comprising a first Fc polypeptide containing a modification that creates a TfR binding site. 1. An isolated antibody comprising: the light chain polypeptide sequence of the first antigen-binding site is identical to the light chain polypeptide sequence of the second antigen-binding site; The isolated antibody.

[0298] 35. The isolated antibody of embodiment 34, wherein the first Fc polypeptide comprises a modified CH3 domain that comprises the TfR binding site.

[0299] 36. The isolated antibody of embodiment 35, wherein the modified CH3 domain is derived from the CH3 domain of human IgG1, IgG2, IgG3, or IgG4.

[0300] 37. The isolated antibody of embodiment 35 or 36, wherein the modified CH3 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions at a set of amino acid positions comprising 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421, according to EU numbering.

[0301] 38. The modified CH3 domain has, according to EU numbering, Glu, Leu, Ser, Val, Trp, Tyr, or Gln at position 380; Leu, Tyr, Phe, Trp, Met, Pro, or Val at position 384; Leu, Thr, His, Pro, Asn, Val, or Phe at position 386; Val, Pro, Ile, or an acidic amino acid at position 387; Trp at position 388; an aliphatic amino acid, Gly, Ser, Thr, or Asn at position 389; Gly, His, 38. The isolated antibody of any one of embodiments 35-37, comprising: Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr; an acidic amino acid, Ala, Ser, Leu, Thr, Pro, Ile, or His, at position 413; Glu, Ser, Asp, Gly, Thr, Pro, Gln, or Arg at position 415; Thr, Arg, Asn, or an acidic amino acid at position 416; and / or an aromatic amino acid, His, or Lys, at position 421.

[0302] 39. The isolated antibody of any one of embodiments 34-38, wherein the first Fc polypeptide containing a modification that creates the TfR binding site binds to the apical domain of TfR.

[0303] 40. The isolated antibody of any one of embodiments 34-39, wherein the first Fc polypeptide and the second Fc polypeptide each comprise a modification that promotes heterodimerization.

[0304] 41. The isolated antibody of embodiment 40, wherein the first Fc polypeptide comprises a T366W substitution and the second Fc polypeptide comprises a T366S, L368A, and Y407V substitution, according to EU numbering.

[0305] 42. The isolated antibody of embodiment 40, wherein the first Fc polypeptide comprises the following substitutions, according to EU numbering: T366S, L368A, and Y407V, and the second Fc polypeptide comprises the following substitution: T366W.

[0306] 43. The isolated antibody of any one of embodiments 34-42, wherein the first Fc polypeptide and / or the second Fc polypeptide independently comprise a modification that reduces TfR-mediated effector function.

[0307] 44. The isolated antibody of embodiment 43, wherein said modification that reduces effector function is a substitution at L234A and L235A according to EU numbering.

[0308] 45. The isolated antibody of embodiment 44, wherein the first Fc polypeptide specifically binds to TfR and comprises the following substitutions: L234A and L235A.

[0309] 46. ​​The isolated antibody of embodiment 45, wherein said first Fc polypeptide further comprises a P329G or P329S substitution according to EU numbering.

[0310] 47. The isolated antibody of embodiment 46, wherein said second Fc polypeptide comprises Leu at positions 234 and 235, and a proline at position 329, according to EU numbering.

[0311] 48. The isolated antibody of embodiment 44, wherein the second Fc polypeptide specifically binds to TfR and comprises the substitutions L234A and L235A.

[0312] 49. The isolated antibody of embodiment 48, wherein said second Fc polypeptide further comprises a P329G or P329S substitution according to EU numbering.

[0313] 50. The isolated antibody of embodiment 49, wherein said first Fc polypeptide comprises Leu at positions 234 and 235, and a proline at position 329, according to EU numbering.

[0314] 51. An isolated antibody according to any one of embodiments 34 to 50, wherein the hinge region or a portion thereof is linked to the N-terminus of the first Fc polypeptide and / or the second Fc polypeptide.

[0315] 52. The isolated antibody of any one of embodiments 34 to 51, wherein the first Fc polypeptide and / or the second Fc polypeptide independently comprise a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 71 to 86 and 98 to 100.

[0316] 53. The isolated antibody of embodiment 52, wherein the first Fc polypeptide or the second Fc polypeptide comprises a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 71-73, 85, and 99-100.

[0317] 54. The isolated antibody of embodiment 52, wherein the first Fc polypeptide or the second Fc polypeptide comprises a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 74-84, 86 and 98.

[0318] 55. the first antigen-binding site comprises the amino acid sequence of SEQ ID NO: 15; the second antigen-binding site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 and 60 to 70; the first Fc polypeptide containing a modification that creates a TfR binding site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-84, 86, and 98; and the light chain polypeptide sequence comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; 35. An isolated antibody according to embodiment 34.

[0319] 56. The isolated antibody of embodiment 55, further comprising a second Fc polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-73, 85, and 99-100.

[0320] 57. The isolated antibody of any one of embodiments 34-56, wherein the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or an I332E substitution according to EU numbering.

[0321] 58. The isolated antibody of embodiment 57, wherein the first Fc polypeptide and / or the second Fc polypeptide independently comprising the S239D and / or I332E substitutions are capable of enhancing HER2-mediated effector function.

[0322] 59.(a) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (b) said first Fc polypeptide comprises a I332E substitution and said second Fc polypeptide comprises a S239D substitution, according to EU numbering; (c) the first Fc polypeptide comprises an S239D and I332E substitution, and the second Fc polypeptide comprises an S239D substitution, according to EU numbering; (d) the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (e) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (f) said first Fc polypeptide comprises a I332E substitution and said second Fc polypeptide comprises a I332E substitution, according to EU numbering; (g) the first Fc polypeptide comprises an S239D and I332E substitution, and the second Fc polypeptide comprises an I332E substitution, according to EU numbering; (h) the second Fc polypeptide comprises a I332E substitution according to EU numbering; (i) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; (j) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; (k) the first Fc polypeptide comprises an S239D and I332E substitution, and the second Fc polypeptide comprises an S239D and I332E substitution, according to EU numbering; (l) the second Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E; (m) the first Fc polypeptide comprises a S239D substitution, according to EU numbering; (n) the first Fc polypeptide comprises a I332E substitution, according to EU numbering; or (o) the first Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E; 59. An isolated antibody according to embodiment 57 or 58.

[0323] 60. (a) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (b) the first Fc polypeptide comprises an S239D and I332E substitution, and the second Fc polypeptide comprises an S239D substitution, according to EU numbering; (c) said first Fc polypeptide comprises a S239D substitution and said second Fc polypeptide comprises a I332E substitution, according to EU numbering; (d) the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (e) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; or (f) the first Fc polypeptide comprises a I332E substitution, according to EU numbering; 60. An isolated antibody according to embodiment 59.

[0324] 61. (a) the first Fc polypeptide comprises a I332E substitution and a serine at position 239, and the second Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, according to EU numbering; (b) the first Fc polypeptide comprises an S239D and an I332E substitution, and the second Fc polypeptide comprises an S239D substitution and an isoleucine at position 332, according to EU numbering; (c) the first Fc polypeptide comprises an S239D substitution and an isoleucine at position 332, and the second Fc polypeptide comprises an I332E substitution and a serine at position 239, according to EU numbering; (d) said first Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, and said second Fc polypeptide comprises a I332E substitution and a serine at position 239, according to EU numbering; (e) the first Fc polypeptide comprises an S239D substitution and an isoleucine at position 332, and the second Fc polypeptide comprises an S239D and I332E substitution, according to EU numbering; or (f) the first Fc polypeptide comprises a I332E substitution and a serine at position 239, and the second Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, according to EU numbering; 61. An isolated antibody according to embodiment 60.

[0325] 62. An isolated antibody according to any one of embodiments 34 to 61, comprising two heavy chains and two light chains.

[0326] 63. The isolated antibody of embodiment 62, comprising a heavy chain and a light chain selected from the combinations listed in Table 2.

[0327] 64. The first heavy chain is selected from the combinations in Table 3. H and the second heavy chain comprises an Fc sequence selected from the combinations in Table 4. H and an Fc sequence.

[0328] 65. The first heavy chain is selected from the combinations in Table 5. H and the second heavy chain comprises an Fc sequence selected from the combinations in Table 6. H and an Fc sequence.

[0329] 66. A pharmaceutical composition comprising an isolated antibody according to any one of embodiments 1 to 65, and a pharma- ceutically acceptable carrier.

[0330] 67. An isolated polynucleotide comprising a nucleotide sequence encoding an isolated antibody according to any one of embodiments 1 to 65.

[0331] 68. A vector comprising a polynucleotide according to embodiment 67.

[0332] 69. A host cell comprising a polynucleotide according to embodiment 67 or a vector according to embodiment 68.

[0333] 70. A method for treating cancer or treating brain metastasis of cancer in a subject, comprising administering to the subject a therapeutically effective amount of an isolated antibody described in any one of embodiments 1 to 65 or a pharmaceutical composition described in embodiment 66.

[0334] 71. The method of embodiment 70, wherein the isolated antibody is administered in combination with chemotherapy or radiation therapy.

[0335] 72. The method of embodiment 70 or 71, wherein the cancer is a metastatic cancer.

[0336] 73. The method of any one of embodiments 70-72, wherein the cancer is breast cancer.

[0337] 74. The method of any one of embodiments 70-73, wherein the cancer is a HER2-positive cancer.

[0338] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light of them will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the scope of the appended claims. The sequences of the sequence accession numbers cited herein are incorporated herein by reference.

[0339] [Table 1]

[0340] [Table 2] TIFF2024534118000016.tif70145

[0341] [Table 3]

[0342] [Table 4]

[0343]

Table 5

[0344]

Table 6

[0345] TIFF2024534118000021.tif185165TIFF2024534118000022.tif158165TIFF2024534118000023.tif137165TIFF2024534118000024.tif137165TIFF2024534118000025.tif137165TIFF2024534118000026.tif137165TIFF2024534118000027.tif137165TIFF2024534118000028.tif137165TIFF2024534118000029.tif137165TIFF2024534118000030.tif137165TIFF2024534118000031.tif137165TIFF2024534118000032.tif137165TIFF2024534118000033.tif137165TIFF2024534118000034.tif137165TIFF2024534118000035.tif137165TIFF2024534118000036.tif197165TIFF2024534118000037.tif204165TIFF2024534118000038.tif205165TIFF2024534118000039.tif195165TIFF2024534118000040.tif195165TIFF2024534118000041.tif198165TIFF2024534118000042.tif201165TIFF2024534118000043.tif195165TIFF2024534118000044.tif91165

Claims

1. below: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 89; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 90; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 91; (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14 or 13.

1. An isolated antibody comprising: below: X of SEQ ID NO: 89 １ is not T; X of SEQ ID NO: 89 ２ is not F; X of SEQ ID NO: 89 ３ is not T; X of SEQ ID NO: 90 １ is not N; X of SEQ ID NO: 90 ２ is not N; X of SEQ ID NO: 90 ３ is not S; X of SEQ ID NO: 90 ４ is not G; X of SEQ ID NO: 90 ５ is not G; X of SEQ ID NO: 90 ６ is not Q; X of SEQ ID NO: 91 １ is not L; X of SEQ ID NO: 91 ２ is not G; X of SEQ ID NO: 91 ３ is not P; and X of SEQ ID NO: 91 ４ is not S, At least one of The isolated antibody.

2. (i) X of SEQ ID NO: 89 １ is T, N, K, M, or H; (ii) X 5 of SEQ ID NO: 90 is Q; (iii) X 6 of SEQ ID NO: 90 is Q, R, H, or T; (iv) X 4 in SEQ ID NO: 91 is L, W, F, D, or Y; (v) X 4 of SEQ ID NO: 91 is L; (vi) X 5 of SEQ ID NO: 90 is Q, and X 4 of SEQ ID NO: 91 is L; (vii) a heavy chain CDR1 comprises the amino acid sequence of any one of SEQ ID NOs: 4 and 49-52, a heavy chain CDR2 comprises the amino acid sequence of any one of SEQ ID NOs: 6, 5, and 53-55, and a heavy chain CDR3 comprises the amino acid sequence of any one of SEQ ID NOs: 8, 7, and 56-59; or (viii) the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 5, and the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 7; The isolated antibody of claim 1.

3. below: (i) (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (ii) (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (iii) (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7; or (iv) (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7; 2. The isolated antibody of claim 1, comprising: (i) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3, 2, and 1; or (ii) a heavy chain variable region comprising any one of the amino acid sequences of SEQ ID NOs: 3, 2, and 1; 3. The isolated antibody of claim 2, comprising:

5. below: (a) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (b) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and (c) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14 or 13.

1. An isolated antibody comprising a light chain comprising: (a) a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10 or 9; or (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10 or 9; 6. The isolated antibody of claim 5, comprising:

7. below: (a) a heavy chain CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 4 and 49-52, a heavy chain CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 6, 5, and 53-55, and a heavy chain CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 8, 7, and 56-59; or (b) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6 or 5, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8 or 7; a first antigen-binding site comprising:

6. The isolated antibody of claim 5, comprising:

8. the antigen-binding site is A heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 3, 2, and 1, or an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3, 2, and 1.

8. The isolated antibody of claim 7, comprising:

9. below: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16; (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17; and (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:

18. and further comprising a second antigen-binding site comprising: the first antigen-binding site and the second antigen-binding site each comprise a light chain; The isolated antibody of claim 7.

10. 10. The isolated antibody of claim 9, wherein the second antigen-binding site comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

15.

11. The following:

6. The isolated antibody of claim 5, comprising heavy chain CDRs and light chain CDRs having sequences selected from the combination: (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 36, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (b) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 31, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 36, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (c) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 38, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 36, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (d) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 37, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 27, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (e) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 31, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 35, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (f) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 35, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (g) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 39, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 46, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (h) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 37, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 25, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 31, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 30, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (j) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 31, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 29, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (k) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 38, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 30, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (l) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 30, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (m) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 29, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (n) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 39, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 30, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (o) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 37, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 26, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (p) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 31, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 34, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (q) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 31, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 33, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (r) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 38, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 34, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (s) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 34, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (t) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 33, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (u) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 39, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 34, and two light chains each comprising the amino acid sequence of SEQ ID NO: 10; (v) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 37, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 25, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (w) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 31, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 30, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (x) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 31, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 29, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (y) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 38, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 30, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (z) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 30, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (aa) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 29, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (ab) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 39, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 30, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (ac) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 37, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 26, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (ad) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 31, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 34, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (ae) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 31, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 33, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (af) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 38, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 34, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (ag) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 34, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (ah) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 33, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (ai) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 39, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 34, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (aj) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 37, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 27, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (ak) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 31, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 36, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (a1) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 31, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 35, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (am) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 38, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 36, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (an) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 36, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; (ao) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 32, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 35, and two light chains each comprising the amino acid sequence of SEQ ID NO: 9; or (ap) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 39, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 46, and two light chains each comprising the amino acid sequence of SEQ ID NO:

9.

7. The isolated antibody of claim 6, comprising:

13. below: (a) a first antigen-binding site against human epidermal growth factor receptor 2 (HER2) subdomain IV, comprising a first Fd domain and a first light chain; (b) a second antigen-binding site for human HER2 subdomain II, comprising a second Fd domain and a second light chain; and (c) a modified Fc polypeptide dimer comprising a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc polypeptide and / or the second Fc polypeptide contains a modification that creates a TfR binding site.

1. An isolated antibody comprising: The first light chain is identical to the second light chain, and the first light chain and the second light chain each comprise the light chain of claim 5. The isolated antibody.

14. the first Fc polypeptide and / or the second Fc polypeptide comprises a modified CH3 domain comprising the TfR-binding site; the modified CH3 domain is derived from a CH3 domain of human IgG1, IgG2, IgG3, or IgG4; and the modified CH3 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions at a set of amino acid positions comprising: 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 according to EU numbering; 14. The isolated antibody of claim 13.

15. The modified CH3 domain may contain, according to EU numbering, Glu, Leu, Ser, Val, Trp, Tyr, or Gln at position 380; Leu, Tyr, Phe, Trp, Met, Pro, or Val at position 384; Leu, Thr, His, Pro, Asn, Val, or Phe at position 386; Val, Pro, lie, or an acidic amino acid at position 387; Trp at position 388; an aliphatic amino acid, Gly, Ser, Thr, or Asn at position 389; Gly at position 390, 15. The isolated antibody of claim 14, comprising: His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr; an acidic amino acid, Ala, Ser, Leu, Thr, Pro, Ile, or His, at position 413; Glu, Ser, Asp, Gly, Thr, Pro, Gln, or Arg at position 415; Thr, Arg, Asn, or an acidic amino acid at position 416; and / or an aromatic amino acid, His, or Lys, at position 421.

16. 14. The isolated antibody of claim 13, wherein the first Fc polypeptide containing a modification that creates the TfR binding site binds to the apical domain of TfR. (a) the first Fc polypeptide and the second Fc polypeptide each comprise a modification that promotes heterodimerization; and / or (b) the first Fc polypeptide and / or the second Fc polypeptide independently comprise a modification that reduces a TfR-mediated effector function; 14. The isolated antibody of claim 13. (a) the first Fc polypeptide comprises a T366W substitution and the second Fc polypeptide comprises T366S, L368A, and Y407V substitutions according to EU numbering; or (b) said first Fc polypeptide comprises the following substitutions, according to EU numbering: T366S, L368A, and Y407V, and said second Fc polypeptide comprises the following substitution: T366W; 18. The isolated antibody of claim 17. (a) the first Fc polypeptide specifically binds to TfR and comprises the substitutions L234A and L235A according to EU numbering. (b) the first Fc polypeptide specifically binds to TfR and comprises substitutions of L234A and L235A, and the second Fc polypeptide comprises Leu at positions 234 and 235, according to EU numbering; (c) the first Fc polypeptide specifically binds to TfR and comprises substitutions of L234A and L235A, and further comprises substitutions of P329G or P329S, according to EU numbering; (d) the first Fc polypeptide specifically binds to TfR and comprises substitutions of L234A and L235A, and further comprises substitutions of P329G or P329S, and the second Fc polypeptide comprises Leu at positions 234 and 235, and proline at position 329, according to EU numbering. (e) the second Fc polypeptide specifically binds to TfR and comprises the following substitutions, according to EU numbering: L234A and L235A; (f) the second Fc polypeptide specifically binds to TfR and comprises substitutions of L234A and L235A, and the first Fc polypeptide comprises Leu at positions 234 and 235, according to EU numbering; (g) the second Fc polypeptide specifically binds to TfR and comprises substitutions of L234A and L235A, and further comprises substitutions of P329G or P329S, according to EU numbering; or (h) the second Fc polypeptide specifically binds to TfR and comprises substitutions of L234A and L235A, and further comprises substitutions of P329G or P329S, and the first Fc polypeptide comprises Leu at positions 234 and 235 and proline at position 329, according to EU numbering.

18. The isolated antibody of claim 17.

20. 14. The isolated antibody of claim 13, wherein the hinge region or a portion thereof is linked to the N-terminus of the first Fc polypeptide and / or the second Fc polypeptide. (a) the first Fc polypeptide and / or the second Fc polypeptide independently comprise a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 71-86 and 98-100; (b) the first Fc polypeptide comprises a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 71-73, 85, and 99-100, and the second Fc polypeptide comprises a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 74-84, 86, and 98; or (c) the first Fc polypeptide comprises a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 74-84, 86, and 98, and the second Fc polypeptide comprises a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 71-73, 85, and 99-100; 14. The isolated antibody of claim 13.

22. (a) the first antigen-binding site comprises the amino acid sequence of SEQ ID NO: 15; (b) the second antigen-binding site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 2, 1, and 60-70; (c) the first Fc polypeptide contains a modification that creates the TfR binding site and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-84, 86, and 98, and the second Fc polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-73, 85, and 99-100, or the second Fc polypeptide contains a modification that creates the TfR binding site and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-84, 86, and 98, and the first Fc polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-73, 85, and 99-100; and (d) the first light chain polypeptide and the second light chain polypeptide each comprise the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 9; 14. The isolated antibody of claim 13.

23. (a) the first Fc polypeptide and / or the second Fc polypeptide independently comprise a S239D and / or I332E substitution according to EU numbering; (b) said first Fc polypeptide comprises a S239D substitution and said second Fc polypeptide comprises a S239D substitution, according to EU numbering; (c) said first Fc polypeptide comprises a I332E substitution and said second Fc polypeptide comprises a S239D substitution, according to EU numbering; (d) said first Fc polypeptide comprises a S239D and I332E substitution, and said second Fc polypeptide comprises a S239D substitution, according to EU numbering; (e) said second Fc polypeptide comprises a S239D substitution, according to EU numbering; (f) said first Fc polypeptide comprises a S239D substitution and said second Fc polypeptide comprises a I332E substitution, according to EU numbering; (g) said first Fc polypeptide comprises a I332E substitution and said second Fc polypeptide comprises a I332E substitution, according to EU numbering; (h) said first Fc polypeptide comprises a S239D and I332E substitution, and said second Fc polypeptide comprises a I332E substitution, according to EU numbering; (i) the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (j) said first Fc polypeptide comprises a S239D substitution and said second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; (k) said first Fc polypeptide comprises a I332E substitution and said second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; (l) said first Fc polypeptide comprises a S239D and I332E substitution, and said second Fc polypeptide comprises a S239D and I332E substitution, according to EU numbering; (m) said second Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E; (n) said first Fc polypeptide comprises a S239D substitution, according to EU numbering; (o) said first Fc polypeptide comprises a I332E substitution according to EU numbering; (p) the first Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E; (q) said first Fc polypeptide comprises a I332E substitution and a serine at position 239, and said second Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, according to EU numbering; (r) said first Fc polypeptide comprises a S239D and I332E substitution, and said second Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, according to EU numbering; (s) said first Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, and said second Fc polypeptide comprises a I332E substitution and a serine at position 239, according to EU numbering; (t) said first Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, and said second Fc polypeptide comprises a I332E substitution and a serine at position 239, according to EU numbering; (u) said first Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, and said second Fc polypeptide comprises a S239D and I332E substitution, according to EU numbering; or (v) said first Fc polypeptide comprises a I332E substitution and a serine at position 239, and said second Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, according to EU numbering; 14. The isolated antibody of claim 13. (a)(i) the first heavy chain comprises a V having the amino acid sequence of SEQ ID NO: 3, 2, or 1; Ｈ and an Fc polypeptide having the amino acid sequence of any one of SEQ ID NOs: 71, 72, 73, and 85; (ii) the second heavy chain has the amino acid sequence of SEQ ID NO: 15 Ｈ and an Fc polypeptide having the amino acid sequence of any one of SEQ ID NOs: 74-84 and 86; or (b)(i) the first heavy chain comprises a V H having the amino acid sequence of amino acid sequence 1, 2, or 3 and an Fc polypeptide having the amino acid sequence of any one of SEQ ID NOs: 74-84 and 86; and (ii) the second heavy chain comprises a VH having the amino acid sequence of SEQ ID NO: 15 and an Fc polypeptide having the amino acid sequence of any one of SEQ ID NOs: 71, 72, 73, and 85; 14. The isolated antibody of claim 13.

25. A pharmaceutical composition comprising the isolated antibody of any one of claims 1 to 24 and a pharmaceutically acceptable carrier.

26. An isolated polynucleotide or vector comprising a nucleotide sequence encoding the isolated antibody of any one of claims 1 to 24.

27. 27. A host cell comprising the polynucleotide or vector of claim 26.

28. 26. The pharmaceutical composition of claim 25, for use in treating cancer or treating brain metastasis of cancer in a subject.

29. 29. The pharmaceutical composition for use as described in claim 28, wherein said isolated antibody or pharmaceutical composition is formulated for use in combination with chemotherapy or radiation therapy.

30. 29. The pharmaceutical composition for use as described in claim 28, wherein the cancer is metastatic cancer, breast cancer, or HER2-positive cancer.

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