HK40110414A - Anti-her2 antibodies and methods of use thereof - Google Patents

Description

Anti-HER2 antibodies and their usage

Cross-references to related applications

This application claims priority to U.S. Provisional Patent Application No. 63/237,104, filed August 25, 2021, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

Background Technology

Currently, the treatment of brain metastases from cancers such as breast cancer presents daunting clinical challenges. The incidence of brain metastases in breast cancer patients is as high as 50%. Clinical data indicate that HER2-positive breast cancer has a tendency to metastasize to the brain. Notably, anti-HER2 therapy has been shown to control extracranial tumors but not intracranial lesions. The main reason these therapies cannot control metastatic lesions (such as brain metastases from HER2-positive breast cancer) is that the therapeutic agents cannot cross the blood-brain barrier (BBB) and enter the brain parenchyma.

Summary of the Invention

In one aspect, this disclosure provides an isolated antibody comprising one or more (e.g., one, two, or all three) complementarity-determining regions (CDRs) selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:89;

(b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:90; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:91,

At least one of the following:

_X1 in SEQ ID NO:89 is not T;

_X2 in SEQ ID NO:89 is not F;

_X3 in SEQ ID NO:89 is not T;

In SEQ ID NO:90, _X1 is not N;

In SEQ ID NO:90, _X2 is not N;

_X3 in SEQ ID NO:90 is not S;

X ₄ in SEQ ID NO:90 is not G;

X ₅ in SEQ ID NO:90 is not G;

X ₆ in SEQ ID NO:90 is not Q;

In SEQ ID NO:91, _X1 is not L;

X ₂ in SEQ ID NO:91 is not G;

_X3 in SEQ ID NO:91 is not P; and

_X4 in SEQ ID NO:91 is not S.

In some embodiments, heavy chain CDR1 comprises the amino acid sequence SEQ ID NO:89, wherein _X1 is N, K, M, or H. In some embodiments, heavy chain CDR2 comprises the amino acid sequence SEQ ID NO:90, wherein _X5 is Q. In some embodiments, heavy chain CDR2 comprises the amino acid sequence SEQ ID NO:90, wherein _X6 is R, H, or T. In some embodiments, heavy chain CDR3 comprises the amino acid sequence SEQ ID NO:91, wherein _X4 is W, F, D, L, or Y. In some embodiments, heavy chain CDR3 comprises the amino acid sequence SEQ ID NO:91, wherein _X4 is L.

In some embodiments, the antibody comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:89;

(b) A heavy chain CDR2 comprising the amino acid sequence SEQ ID NO:90, wherein _X5 is Q; and

(c) A heavy chain CDR3 containing the amino acid sequence SEQ ID NO:91, wherein X ₄ is L.

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 with an amino acid sequence selected from the group consisting of SEQ ID NO:4 and 49-52, or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO:4 and 49-52;

(b) A heavy chain CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 5-6 and 53-55, or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 5-6 and 53-55; 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 with an amino acid sequence selected from the group consisting of SEQ ID NO: 7-8 and 56-59, or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 7-8 and 56-59.

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 with the amino acid sequence SEQ ID NO:4 or having at most two amino acid substitutions relative to the amino acid sequence 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 with the amino acid sequence SEQ ID NO:5 or SEQ ID NO:6, or having at most two amino acid substitutions relative to the amino acid sequence 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 with the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8, or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8.

In some embodiments, the antibody comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4;

(b) Heavy chain CDR2 containing the amino acid sequence SEQ ID NO:5 or SEQ ID NO:6; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8.

In some embodiments, the antibody comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4;

(b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:6; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:7.

In some embodiments, the antibody comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4;

(b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:5; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:8.

In some embodiments, the antibody comprises one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4;

(b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:6; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:8.

In some embodiments, the antibody comprises a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any of SEQ ID NO:1-3. In some embodiments, the antibody comprises a heavy chain variable region containing an amino acid sequence of any of SEQ ID NO:1-3.

In one related aspect, this disclosure provides an isolated antibody heavy chain comprising one or more (e.g., one, two, or all three) of the CDRs described above. In some embodiments, the antibody heavy chain comprises a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any of SEQ ID NO:1-3. In some embodiments, the antibody heavy chain comprises a heavy chain variable region containing an amino acid sequence of any of SEQ ID NO:1-3.

In another aspect, this disclosure provides an isolated antibody comprising:

(a) A light chain CDR3 containing the amino acid sequence SEQ ID NO:13 or 14.

In some implementations, the antibody also includes one or more (e.g., one or two) 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 with the amino acid sequence SEQ ID NO:11 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:11; and

(c) A light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:12.

In some implementations, the antibody also includes one or more (e.g., one or two) CDRs selected from the group consisting of:

(b) The light chain CDR1 containing the amino acid sequence SEQ ID NO:11; and

(c) The light chain CDR2 containing the amino acid sequence SEQ ID NO:12.

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

In some embodiments, the antibody comprises a light chain variable region containing an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any of SEQ ID NO: 9-10. In some embodiments, the antibody comprises a light chain variable region containing an amino acid sequence of any of SEQ ID NO: 9-10.

In one related aspect, this disclosure provides an isolated antibody light chain comprising one or more (e.g., one, two, or all three) of the CDRs described above. 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 with any of SEQ ID NO: 9-10. In some embodiments, the antibody light chain comprises a light chain variable region comprising an amino acid sequence of any of SEQ ID NO: 9-10.

In another aspect, this disclosure provides an isolated antibody comprising an antigen-binding site including:

(a) A heavy chain CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:4 and 49-52, or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO:4 and 49-52;

(b) A heavy chain CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 5-6 and 53-55, or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 5-6 and 53-55; 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 with an amino acid sequence selected from the group consisting of SEQ ID NO: 7-8 and 56-59, or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 7-8 and 56-59;

(d) A light chain CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with the amino acid sequence SEQ ID NO:11 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:11;

(e) A light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:12; and

(f) A light chain CDR3 containing the amino acid sequence SEQ ID NO:13 or 14.

In some implementations, the antigen binding site includes:

(a) A heavy chain CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with the amino acid sequence SEQ ID NO:4 or having at most two amino acid substitutions relative to the amino acid sequence 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 with the amino acid sequence SEQ ID NO:5 or SEQ ID NO:6, or having at most two amino acid substitutions relative to the amino acid sequence 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 with the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8 or having at most two amino acid substitutions relative to the amino acid sequence 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 with the amino acid sequence SEQ ID NO:11 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:11;

(e) A light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:12; and

(f) A light chain CDR3 containing the amino acid sequence SEQ ID NO:13 or 14.

In some embodiments, the 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 with any of SEQ ID NO: 1-3, 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 with any of SEQ ID NO: 9-10. In some embodiments, the antigen-binding site comprises a heavy chain variable region comprising an amino acid sequence of any of SEQ ID NO: 1-3, and a light chain variable region comprising an amino acid sequence of any of SEQ ID NO: 9-10.

In some implementations, the antibody also includes a second antigen-binding site comprising one or more CDRs selected from the group consisting of:

(a) A heavy chain CDR1 containing the amino acid sequence SEQ ID NO:16 or having at most two amino acid substitutions relative to the amino acid sequence 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 with the amino acid sequence SEQ ID NO:17 or having at most two amino acid substitutions relative to the amino acid sequence 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 with the amino acid sequence SEQ ID NO:18 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:18.

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

In some implementations, the second antigen binding site further includes 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 with the amino acid sequence SEQ ID NO:11 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:11;

(b) A light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:12; and

(c) A light chain CDR3 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:13 or 14.

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 with any of SEQ ID NO: 9-10. In some embodiments, the second antigen binding site comprises a heavy chain variable region comprising a sequence of any of SEQ ID NO: 9-10.

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

In one related aspect, this disclosure provides an isolated antibody comprising a heavy chain and a light chain selected from combinations listed in Table 2.

In another aspect, this disclosure provides an isolated antibody comprising:

(a) The first antigen-binding site of subdomain IV of human epidermal growth factor receptor 2 (HER2);

(b) The second antigen-binding site of human HER2 subdomain II; and

(c) A modified Fc polypeptide dimer comprising a modified first Fc polypeptide containing a TfR binding site.

The light chain polypeptide sequence in the first antigen binding site is the same as the light chain polypeptide sequence in the second antigen binding site.

In one related aspect, this disclosure provides an isolated antibody comprising:

(a) The first antigen-binding site of human HER2 subdomain II;

(b) The second antigen-binding site of human HER2 subdomain IV; and

(c) A modified Fc polypeptide dimer comprising a modified first Fc polypeptide containing a TfR binding site.

The light chain polypeptide sequence in the first antigen binding site is the same as the light chain polypeptide sequence in the second antigen binding site.

In some embodiments, the first Fc polypeptide includes a modified CH3 domain containing a TfR binding site. In some embodiments, the modified CH3 domain is derived from the CH3 domain of human IgG1, IgG2, IgG3, or IgG4.

In some embodiments, according to EU designations, the modified CH3 domain comprises one, two, three, four, five, six, seven, eight, nine, ten, or eleven substitutions at positions of amino acids comprising 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421. In some embodiments, according to EU designations, 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; and an aliphatic amino acid, Gly, Ser, Thr, or A at position 389. sn; Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr at position 390; acidic amino acids, Ala, Ser, Leu, Thr, Pro, Ile, or His at position 413; Glu, Ser, Asp, Gly, Thr, Pro, Gln, or Arg at position 415; Thr, Arg, Asn, or acidic amino acids at position 416; and/or aromatic amino acids, His, or Lys at position 421.

In some implementations, a first Fc polypeptide containing a modification that generates a TfR binding site binds to the top domain of the TfR.

In some embodiments, the first Fc polypeptide and the second Fc polypeptide each contain modifications that promote heterodimerization. In some embodiments, according to EU designations, the first Fc polypeptide contains a T366W substitution, and the second Fc polypeptide contains T366S, L368A, and Y407V substitutions. In other embodiments, according to EU designations, the first Fc polypeptide contains T366S, L368A, and Y407V substitutions, and the second Fc polypeptide contains a T366W substitution.

In some embodiments, the first Fc polypeptide and/or the second Fc polypeptide independently comprises modifications that reduce TfR-mediated effector function. In some embodiments, according to EU designations, the modifications that reduce effector function are L234A and L235A substitutions. In some embodiments, the first Fc polypeptide specifically binds to TfR and comprises L234A and L235A substitutions. In some embodiments, according to EU designations, the first Fc polypeptide also comprises P329G or P329S substitutions. In some embodiments, according to EU designations, the second Fc polypeptide comprises Leu at positions 234 and 235 and proline at position 329. In other embodiments, the second Fc polypeptide specifically binds to TfR and comprises L234A and L235A substitutions. In some embodiments, according to EU designations, the second Fc polypeptide also comprises P329G or P329S substitutions. In some embodiments, according to EU designations, the first Fc polypeptide comprises Leu at positions 234 and 235 and proline at position 329.

In some implementations, the hinge region or a portion thereof is connected to the N-terminus of the first Fc polypeptide and/or the second Fc polypeptide.

In some embodiments, the first Fc polypeptide and/or the second Fc polypeptide independently comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with a sequence selected from the group consisting of SEQ ID NO: 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 with a sequence selected from the group consisting of SEQ ID NO: 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 with a sequence selected from the group consisting of SEQ ID NO: 74-84, 86, and 98.

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

In other embodiments of the antibody, the first antigen-binding site comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3 and 60-70; the second antigen-binding site comprises the amino acid sequence SEQ ID NO: 15; the first Fc polypeptide containing the modification that generates the TfR binding site 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 the amino acid sequence 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 NO: 71-73, 85, and 99-100.

In some embodiments, according to EU designations, the first Fc polypeptide and/or the second Fc polypeptide independently comprises S239D and/or I332E substitutions. In some embodiments, the first Fc polypeptide and/or the second Fc polypeptide independently comprising S239D and/or I332E substitutions can enhance HER2-mediated effector function.

In some embodiments of the antibody:

(a) According to the EU designation, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains an S239D substitution;

(b) According to the EU designation, the first Fc polypeptide contains an I332E substitution and the second Fc polypeptide contains an S239D substitution;

(c) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions and the second Fc polypeptide contains S239D substitution;

(d) According to the EU designation, the second Fc polypeptide contains an S239D substitution;

(e) According to the EU designation, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains an I332E substitution;

(f) According to the EU designation, the first Fc polypeptide contains an I332E substitution and the second Fc polypeptide contains an I332E substitution;

(g) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions and the second Fc polypeptide contains I332E substitutions;

(h) According to the EU designation, the second Fc polypeptide contains an I332E substitution;

(i) According to the EU designation, the first Fc polypeptide contains S239D substitution and the second Fc polypeptide contains S239D and I332E substitution;

(j) According to the EU designation, the first Fc polypeptide contains I332E substitution and the second Fc polypeptide contains S239D and I332E substitution;

(k) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions and the second Fc polypeptide contains S239D and I332E substitutions;

(l) According to the EU designation, the second Fc polypeptide contains S239D and I332E substitutions;

(m) According to the EU designation, the first Fc polypeptide contains an S239D substitution;

(n) According to the EU designation, the first Fc polypeptide contains an I332E substitution; or

(o) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions.

In some embodiments of the antibody:

(a) According to the EU designation, the first Fc polypeptide contains an I332E substitution and the second Fc polypeptide contains an S239D substitution;

(b) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions and the second Fc polypeptide contains S239D substitution;

(c) According to the EU designation, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains an I332E substitution;

(d) According to the EU designation, the second Fc polypeptide contains an I332E substitution;

(e) According to the EU designation, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains both S239D and I332E substitutions; or

(f) According to the EU designation, the first Fc polypeptide contains an I332E substitution.

In the specific implementation scheme of the antibody:

(a) According to the EU designation, the first Fc polypeptide contains an I332E substitution and a serine at position 239, and the second Fc polypeptide contains an S239D substitution and an isoleucine at position 332.

(b) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions, and the second Fc polypeptide contains S239D substitution and isoleucine at position 332;

(c) According to the EU designation, the first Fc polypeptide contains an S239D substitution and an isoleucine at position 332, and the second Fc polypeptide contains an I332E substitution and a serine at position 239.

(d) According to the EU designation, the first Fc polypeptide contains a serine at position 239 and an isoleucine at position 332, and the second Fc polypeptide contains an I332E substitution and a serine at position 239.

(e) According to the EU designation, 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; or

(f) According to the EU designation, the first Fc polypeptide contains an I332E substitution and a serine at position 239, and the second Fc polypeptide contains a serine at position 239 and an isoleucine at position 332.

In some embodiments, the antibody comprises two heavy chains and two light chains. In some embodiments, the antibody comprises heavy and light chains selected from the combinations listed in Table 2. In some embodiments, the first heavy chain comprises _VH and Fc sequences selected from the combinations in Table 3 and the second heavy chain comprises _VH and Fc sequences selected from the combinations in Table 4. In some embodiments, the first heavy chain comprises _VH and Fc sequences selected from the combinations in Table 5 and the second heavy chain comprises _VH and Fc sequences selected from the combinations in Table 6.

In another aspect, this disclosure provides a pharmaceutical composition comprising any of the antibodies described herein and a pharmaceutically acceptable carrier.

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

In another aspect, this disclosure provides a carrier comprising the aforementioned polynucleotides.

In another aspect, this disclosure provides a host cell that contains the polynucleotide or the vector.

In another aspect, this disclosure provides a method for treating a subject with cancer or treating brain metastases of cancer, the method comprising administering to the subject a therapeutically effective amount of the antibody or a pharmaceutical composition thereof described herein.

In some embodiments, the antibody is administered in combination with chemotherapy or radiotherapy. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is HER2-positive cancer.

Attached Figure Description

Figure 1 is a schematic diagram showing an exemplary bispecific antibody having: a first antigen-binding site (“anti-HER2_D4”) of human HER2 subdomain IV and a second antigen-binding site (“anti-HER2_D2”) of human HER2 subdomain II, wherein the first and second antigen-binding sites comprise the same light chain polypeptide; and an Fc polypeptide dimer comprising a first Fc polypeptide having a TfR binding site and a knob mutation and a second Fc polypeptide having a hole mutation.

Figure 2 shows the results of growth inhibition assays on ZR-75-30 cells and the IC50 and maximum growth inhibition % values for different antibodies in Table 12.

Figures 3A and 3B illustrate the in vitro antitumor activity of tumors in two human cell line-derived xenograft models in a single-dose study using the ATV:CLC bispecific antibody. Figure 3A: BT-474; Figure 3B: OE19.

Figures 4A and 4B illustrate the in vitro antitumor activity of tumors in two human cell line-derived xenograft models in a single-dose, low-dose study using the ATV:CLC bispecific antibody. Figure 4A: BT-474; Figure 4B: OE19.

Figures 5A and 5B illustrate the in vitro antitumor activity of tumors in two xenograft models derived from human cell lines during a multi-dose study using the ATV:CLC bispecific antibody. Figure 5A: BT-474; Figure 5B: OE19.

Figure 6 illustrates the brain uptake of the ATV:CLC bispecific antibody.

Figures 7A and 7B illustrate the IHC brain distribution of CLC bispecific antibodies.

Figure 8 illustrates plasma pharmacokinetics in a single-dose study using the ATV:CLC bispecific antibody in cynomolgus monkeys.

Figure 9 illustrates the ADCC of the ATV:CLC bispecific antibody.

Detailed Implementation

I. Introduction

This article describes anti-HER2 bispecific antibodies that utilize a common light chain approach, where two antigen-binding domains pair with the same light chain but still retain individual specificity. Using a common light chain prevents light chain mismatches and thus makes these bispecific antibodies easier to manufacture. In some embodiments, the bispecific antibody comprises a first antigen-binding site of human HER2 subdomain IV and a second antigen-binding site of human HER2 subdomain II, wherein the light chain polypeptide sequence in the first antigen-binding site is identical to the light chain polypeptide sequence in the second antigen-binding site. In another embodiment, the bispecific antibody comprises a first antigen-binding site of human HER2 subdomain II and a second antigen-binding site of human HER2 subdomain IV, wherein the light chain polypeptide sequence in the first antigen-binding site is identical to the light chain polypeptide sequence in the second antigen-binding site.

Furthermore, previous therapies have failed to control brain metastases in HER2-positive breast cancer, primarily because therapeutic agents cannot cross the blood-brain barrier (BBB) and enter the brain parenchyma. Therefore, novel therapeutic agents capable of crossing the BBB and targeting HER2 in the brain parenchyma are needed. We previously described the use of transferrin receptor (TfR) binding as a method to enable BBB delivery across the brain endothelium, as TfR is highly expressed in brain endothelial cells and allows for BBB delivery via receptor-mediated endocytosis. Interestingly, TfR is highly expressed in various cancers, including HER2-positive breast cancer. The mechanism of increased TfR expression in cancer cells may be related to increased tumor cell proliferation and metabolic demands, such as iron uptake. In fact, public microarray datasets have demonstrated the correlation between TfR expression and breast cancer prognosis (Miller et al., Cancer Res. 71:6728, 2011). There are also reports on the use of TfR as a pharmacological target for various types of cancer.

In some embodiments, the anti-HER2 bispecific antibody comprises one or more modified Fc peptides (i.e., Fc peptides that bind to TfR) that specifically bind to a BBB receptor (e.g., TfR). In some embodiments, the anti-HER2 bispecific antibody is capable of trans-BBB transport. In some embodiments, the anti-HER2 bispecific antibody, as described herein, which binds to both HER2 and TfR, may provide additional antitumor benefit when bound to HER2-positive tumor cells that also express high levels of TfR, compared to other therapeutic agents that bind to HER2 alone. Specifically, because these antibodies can bind to both TfR and HER2 simultaneously, this can enhance their potency and/or efficacy.

II. Definition

Unless the context clearly indicates otherwise, as used herein, the singular forms “a/an” and “described” include a plural number of indicators. Thus, for example, a reference to “antibody” may optionally include a combination of two or more such molecules, etc.

As used herein, the terms “about” and “approximately” when used to modify quantities specified in numerical values or ranges indicate that the numerical value, as well as reasonable deviations from the value known to those skilled in the art (e.g., ±20%, ±10%, or ±5%), are within the intended meaning of the enumerated value.

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 the tyrosine receptor kinase protein encoded by the human ERBB2 gene, a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family. HER2 amplification or overexpression plays a crucial role in the development and progression of certain aggressive types of cancer, including breast cancer. Non-restrictive examples of human HER2 nucleotide sequences are listed in GenBank references NP_001005862, NP_001289936, NP_001289937, NP_001289938, and NP_004448. Non-restrictive examples of human HER2 peptide sequences are listed in GenBank reference numbers NP_001005862, NP_001276865, NP_001276866, NP_001276867 and NP_004439.

The extracellular domain of HER2, containing approximately 600 amino acids, comprises four subdomains (subdomains I, II, III, and IV). Subdomains I and III form ligand-binding sites. Cysteine-rich subdomains II and IV are involved in receptor homodimerization and heterodimerization. Anti-HER2 antibodies can bind to specific subdomains (e.g., subdomains II and/or subdomain IV).

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

As used herein, the term "antibody" refers to a protein that specifically binds to an antigen via its variable region and has an immunoglobulin fold. 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 classified as κ or λ. Antibodies may contain heavy chains classified as γ, μ, α, δ, or ε, which, respectively, define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE.

An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The N-terminus of each chain defines a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" ( _VL ) and "variable heavy chain" ( _VH ) refer to these light and heavy chains, respectively.

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

The term "complementarity-determining region" or "CDR" refers to the three hypervariable regions in each chain that disrupt the four framework regions established by the variable regions of the light and heavy chains. CDRs are primarily responsible for the binding of antibodies to epitopes of antigens. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are often identified by the chain in which a particular CDR is located. Therefore, _VH CDR3 or CDR-H3 is located in the variable region of the antibody heavy chain in which it is found, while _VL CDR1 or CDR-L1 is CDR1 from the variable region of the antibody light chain in which it is found.

The "frame regions" or "FRs" of different light or heavy chains are relatively conserved within a species. The frame regions of an antibody (i.e., the combined frame regions of constitutive light and heavy chains) are used to locate and align the CDRs in three-dimensional space. Frame sequences can be obtained from public DNA databases containing germline antibody gene sequences or from published references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBASE2" germline variable gene sequence database of human and mouse sequences.

The amino acid sequence of the CDR and frame region can be determined using various well-known definitions in the art, such as Kabat, Chothia, the International ImMunoGeneTics Database (IMGT), AbM, and the observed antigen contact (“Contact”). In some embodiments, the CDR is determined according to the definition of Contact. See MacCallum et al., J. Mol. Biol. 262:732-745, 1996. In some embodiments, the CDR is determined by a combination of the definitions of Kabat, Chothia, and/or Contact CDR.

The term "epitope" refers to a region or area of an antigen that is specifically bound by a molecule (e.g., the CDR of an antibody) and may include a portion of several amino acids, such as 5 or 6 or more (e.g., 20 or more) amino acids or portions of those amino acids. In some cases, epitopes include non-protein components, such as those from carbohydrates, nucleic acids, or lipids. In some cases, epitopes are three-dimensional portions. Thus, for example, when the target is a protein, an epitope may contain continuous amino acids (e.g., a linear epitope) or amino acids from different parts of a protein that are close together through protein folding (e.g., a discontinuous or conformational epitope).

As used herein, the phrase “recognition epitope” for the purposes of this document means that the antibody CDR interacts with or specifically binds to the antigen at the epitope or the antigenic portion containing the epitope.

"Humanized antibodies" are chimeric immunoglobulins derived from non-human sources (e.g., rodents) that contain a minimum amount of sequence derived from non-human immunoglobulins outside the CDR. Generally, humanized antibodies will contain at least one (e.g., two) variable domains, wherein the CDR region substantially corresponds to the CDR region of the non-human immunoglobulin and the frame region substantially corresponds to the frame region of the human immunoglobulin sequence. In some cases, certain frame region residues of the human immunoglobulin may be replaced by corresponding residues from a non-human species to, for example, improve specificity, affinity, and/or serum half-life. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc) (typically the human immunoglobulin sequence). Methods for antibody humanization are known in the art.

"Human antibodies" or "fully human antibodies" are antibodies that possess human heavy and light chain sequences, typically derived from human germline genes. In some embodiments, antibodies are produced by human cells, by non-human animals utilizing a human antibody library (e.g., genetically engineered transgenic mice to express human antibody sequences), or by a phage display platform.

The term "specifically binding" refers to a molecule (e.g., an antibody as described herein) binding to an epitope or target with a greater affinity, stronger binding force, and/or longer duration of binding to that epitope or non-target compound (e.g., a structurally different antigen) compared to its binding to another epitope or non-target compound. In some embodiments, a molecule specifically binding to an epitope or target is a molecule whose binding affinity to said epitope or target is at least 5 times, for example at least 6, 7, 8, 9, 10, 25, 50, 100, 1000, 10,000, or greater than its affinity for other epitopes or non-target compounds. As used herein, the terms “specifically binds to a particular epitope or target,” “specifically binds to a particular epitope or target,” or “is specific to a particular epitope or target” can be expressed, for example, by the fact that the equilibrium dissociation constant _KD of the molecule to which it binds is, 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). Those skilled in the art will recognize that a molecule specifically binds to a target from one species can also specifically bind to an ortholog of said target.

The term “binding affinity” is used herein to refer to the strength of a non-covalent interaction between two molecules, such as an antibody and an antigen as described herein. Therefore, for example, unless otherwise indicated or clearly stated from the context, the term may refer to a 1:1 interaction between an antibody and an antigen. Binding affinity can be quantified by measuring the equilibrium dissociation constant (K_{_d}), which is the dissociation rate constant (k _{_d} , time ^{^-1}) divided by the association rate constant ( _ka , time ^{^-1} M ^{^-1} ). _{K_d} can be determined by measuring the kinetics of complex formation and dissociation, for example using surface plasmon resonance (SPR) methods, such as the Biacore ^™ system; kinetic exclusion assays, such as those using biolayer interference techniques (e.g., using the Octet platform). As used herein, “binding affinity” includes not only formal binding affinity, such as affinity reflecting a 1:1 interaction between an antibody and an antigen, but also apparent affinity for which K_{_d} is calculated to reflect affinity binding.

As used herein, “transferrin receptor” or “TfR” refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is listed 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; bovine, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP_990587.1). The term “transferrin receptor” also covers allelic variants of exemplary reference sequences (e.g., human sequences) encoded by a gene at the transferrin receptor protein 1 chromosomal locus. The full-length transferrin receptor protein comprises 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 a apical domain.

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-sheet structure. An Fc polypeptide contains a constant region sequence comprising at least a CH2 domain and/or a CH3 domain, and may contain at least a portion of a hinge region, but not a variable region.

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

As used in this article, "FcRn" refers to the neonatal Fc receptor. The binding of Fc peptides to FcRn reduces the clearance rate of Fc peptides and prolongs their serum half-life. Human FcRn protein is a heterodimer composed of a protein similar to major histocompatibility (MHC) class I protein with a size of about 50 kDa and a β2-microglobulin with a size of about 15 kDa.

As used herein, "FcRn binding site" refers to the region in an Fc polypeptide that binds to FcRn. In human IgG, FcRn binding sites, as numbered using the EU index, include L251, M252, I253, S254, R255, T256, M428, H433, N434, H435, and Y436. These positions correspond to positions 21 to 26, 198, and 203 to 206 of SEQ ID NO:95.

As used in this article, "natural FcRn binding site" refers to a region in an Fc polypeptide that binds to FcRn and has the same amino acid sequence as the region in a naturally occurring Fc polypeptide that binds to FcRn.

As used herein, the terms "CH3 domain" and "CH2 domain" refer to immunoglobulin constant region domain polypeptides. For the purposes of this application, a CH3 domain polypeptide refers to the amino acid segment numbered from approximately position 341 to position 447 as per the EU numbering scheme, and a CH2 domain polypeptide refers to the amino acid segment numbered from approximately position 231 to position 340 as per the EU numbering scheme, excluding the hinge region sequence. CH2 and CH3 domain polypeptides may also be numbered using the IMGT (ImMunoGeneTics) numbering scheme, where, according to the IMGT Scientific Chart (IMGT website), the CH2 domain is numbered 1-110 and the CH3 domain is numbered 1-107. The CH2 and CH3 domains are part of the immunoglobulin Fc region. The Fc region refers to the amino acid segment numbered from approximately position 231 to position 447 as per the EU numbering scheme, but as used herein, may include at least a portion of the antibody hinge region. An exemplary hinge region sequence is the human IgG1 hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO: 96).

The terms “wild-type,” “natural,” and “naturally existing” used for CH3 or CH2 domains refer to domains that have a sequence that exists in nature.

As used herein, the term "mutant" for mutant polypeptides or mutant polynucleotides may be used interchangeably with "variant." A variant of a given wild-type CH3 or CH2 domain reference sequence may include naturally occurring allelic variants. A "non-naturally occurring" CH3 or CH2 domain refers to a variant or mutant domain not present in naturally occurring cells and generated through genetic modification of the natural CH3 or CH2 domain polynucleotide or polypeptide, for example, using genetic engineering or mutagenesis techniques. A "variant" includes any domain containing at least one amino acid mutation relative to the wild type. Mutations may include substitutions, insertions, and deletions.

The term "isolated" as used with respect to nucleic acids or proteins means that the nucleic acid or protein is substantially free of other cellular components associated with it in its native state. It is preferably in a homogeneous state. Purity and homogeneity are typically determined using analytical chemistry techniques such as electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high-performance liquid chromatography). In some embodiments, the isolated nucleic acid or protein is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure.

The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, and those subsequently modified, such as hydroxyproline, γ-carboxyglutamate, 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 analogues" are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogues have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimics" are compounds whose structure differs from the general chemical structure of amino acids, but whose mode of action is similar to that of naturally occurring amino acids. Amino acids may be referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

The terms “polypeptide” and “peptide” are used interchangeably herein to refer to polymers of amino acid residues that are single-chain amino acids. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or mixtures of L and D amino acids.

As used herein, the term "protein" refers to a polypeptide or a dimer (i.e., two types) or polymer (i.e., three or more types) of a single-chain polypeptide. Single-chain polypeptides of proteins can be linked by covalent bonds (e.g., disulfide bonds) or non-covalent interactions.

The terms “polynucleotide” and “nucleic acid” are used interchangeably to refer to nucleotide chains of any length and include both DNA and RNA. A nucleotide can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base and/or its analogues, or any substrate that can be incorporated into the chain by DNA or RNA polymerase. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogues. Examples of polynucleotides considered herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules containing mixtures of single-stranded and double-stranded DNA and RNA.

The terms "conservative substitution" and "conservative mutation" refer to changes that result in an amino acid being replaced by another amino acid that can be classified as having similar characteristics. Examples of conserved amino acid group categories defined in this way may include: "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); "aromatic group," including Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and "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). Subgroups may also be identified within each group. For example, the charged or polar amino acid group can be subdivided into subgroups including: a "positively charged subgroup" containing Lys, Arg, and His; a "negatively charged subgroup" containing Glu and Asp; and a "polar subgroup" containing Asn and Gln. In another example, the aromatic or cyclic group can be subdivided into subgroups including: a "nitrocyclic subgroup" containing Pro, His, and Trp; and a "phenyl subgroup" containing Phe and Tyr. In yet another further example, the aliphatic group can be subdivided into subgroups such as a "liphatic nonpolar subgroup" containing Val, Leu, Gly, and Ala; and a "liphatic slightly polar subgroup" containing Met, Ser, Thr, and Cys. Examples of conserved mutation categories include amino acid substitutions within the aforementioned subgroups, such as, but not limited to, Lys replacing Arg or vice versa, to retain a positive charge; Glu replacing Asp or vice versa, to retain a negative charge; Ser replacing Thr or vice versa, to retain free -OH; and Gln replacing Asn or vice versa, to retain free _-NH₂ . In some embodiments, hydrophobic amino acids replace naturally occurring hydrophobic amino acids (e.g., in the active site) to maintain hydrophobicity.

In the case of two or more polypeptide sequences, the term "identical" or "identity" percentage refers to the fact that, when compared and aligned within a comparison window or specified region to obtain the maximum correspondence, such as when using sequence comparison algorithms or measured by manual alignment and visual inspection, two or more sequences or subsequences are identical or have a specified percentage (e.g., 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 greater) of the same amino acid residues.

For peptide sequence alignment, a single amino acid sequence is typically used as a reference sequence against which candidate sequences are compared. Various methods available to those skilled in the art can be used for alignment, such as visual alignment or alignment using publicly available software with known algorithms to achieve maximum alignment. Such programs include BLAST, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). Those skilled in the art can determine the parameters used for alignment to achieve maximum alignment. For the purposes of this application, for peptide sequence alignment, the BLASTP algorithm standard protein BLAST, which aligns two protein sequences with preset parameters, is used.

When used in the identification of a given amino acid residue in a polypeptide sequence, the terms "corresponds to," "reference...determined," or "reference...number" specify the position of said residue in the reference sequence when the given amino acid sequence is aligned and compared to a reference sequence to the greatest extent possible. Thus, for example, in optimal alignment with SEQ ID NO:95, when an amino acid residue in the modified Fc polypeptide aligns with an amino acid in SEQ ID NO:95, said residue "corresponds to" the amino acid in SEQ ID NO:95. The polypeptide aligned to the reference sequence does not need to be the same length as the reference sequence.

As may be used interchangeably herein, the terms “subject,” “individual,” and “patient” refer to mammals, including but not limited to humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammal species. In one implementation, the patient is a human.

The terms “treatment” and “treating” are generally used herein to refer to achieving the desired pharmacological and/or physiological effect. “Treatment” can refer to any indicator of success in treating or improving cancer (e.g., HER2-positive and/or metastatic cancer), including any objective or subjective parameters such as relief, remission, improved patient survival, increased survival time or survival rate, reduction of symptoms or increased patient tolerance to the disease, slowing of the rate of deterioration or decline, or improvement of a patient’s physical or mental health. Treatment or improvement of symptoms can be based on objective or subjective parameters. The treatment effect can be compared to individuals or groups of individuals who did not receive the treatment, or to the same patient before or at different times during treatment.

The term "pharmaceuticalally acceptable excipient" refers to an inactive pharmaceutical ingredient that is biologically or pharmacologically suitable for human or animal use, such as, but not limited to, buffers, carriers, or preservatives.

As used herein, a “therapeutic dose” or “therapeutic effective dose” of a molecule (such as an antibody as described herein) is a dose that treats, alleviates, slows, or reduces the severity of a subject’s disease symptoms.

The term "administration" refers to a method of delivering a molecule or composition to the desired site of biological action. These methods include, but are not limited to, local delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, an antibody as described herein is administered intravenously.

III. Anti-HER2 antibodies

In one aspect, an antibody comprising a common light chain polypeptide is provided that binds to subdomains II and IV of human HER2. In some embodiments, one or both of the antibody Fc polypeptides are modified Fc polypeptides (e.g., modified to promote TfR binding and/or enhance the heterodimerization of the Fc polypeptide). A schematic diagram of such a bispecific antibody is shown in Figure 1.

In some implementations, the anti-HER2 antibody comprises:

(a) The first antigen-binding site of human HER2 subdomain IV;

(b) The second antigen-binding site of human HER2 subdomain II; and

(c) A modified Fc polypeptide dimer comprising a modified first Fc polypeptide containing a TfR binding site.

The light chain polypeptide sequence in the first antigen binding site is the same as the light chain polypeptide sequence in the second antigen binding site.

In other embodiments, the anti-HER2 antibody comprises:

(a) The first antigen-binding site of human HER2 subdomain II;

(b) The second antigen-binding site of human HER2 subdomain IV; and

(c) A modified Fc polypeptide dimer comprising a modified first Fc polypeptide containing a TfR binding site.

The light chain polypeptide sequence in the first antigen binding site is the same as the light chain polypeptide sequence in the second antigen binding site.

In some embodiments, the first Fc polypeptide comprises a modified CH3 domain containing a TfR binding site. In some embodiments, the modified CH3 domain comprises substitutions at a set of amino acid positions as described herein that generate a TfR binding site.

In some embodiments, the first Fc peptide and the second Fc peptide each contain modifications that promote heterodimerization. For example, according to EU designations, the first Fc peptide may contain a T366W substitution and the second Fc peptide may contain T366S, L368A, and Y407V substitutions. In another example, according to EU designations, the first Fc peptide may contain T366S, L368A, and Y407V substitutions and the second Fc peptide may contain a T366W substitution. Furthermore, the first Fc peptide and/or the second Fc peptide may independently contain modifications that reduce TfR-mediated effector function, i.e., reduce effector function upon TfR binding. For example, according to EU designations, modifications that reduce TfR-mediated effector function are (i) L234A and L235A substitutions or (ii) L234A and L235A substitutions and P329G or P329S substitutions.

In some embodiments, according to EU designations, the first Fc polypeptide and/or the second Fc polypeptide independently comprises S239D and/or I332E substitutions. In some embodiments, according to EU designations, either the first Fc polypeptide or the second Fc polypeptide comprises S239D and/or I332E substitutions. In some other embodiments, according to EU designations, both the first Fc polypeptide and the second Fc polypeptide comprise S239D and/or I332E substitutions. In a specific embodiment, the first Fc polypeptide and/or the second Fc polypeptide independently comprising S239D and/or I332E substitutions can enhance HER2-mediated effector function, i.e., enhance effector function upon HER2 binding.

In some embodiments, according to EU designations, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains an S239D substitution. In some embodiments, according to EU designations, the first Fc polypeptide contains an I332E substitution and the second Fc polypeptide contains an S239D substitution. In some embodiments, according to EU designations, the first Fc polypeptide contains both S239D and I332E substitutions and the second Fc polypeptide contains an S239D substitution. In some embodiments, according to EU designations, the second Fc polypeptide contains an S239D substitution. In some embodiments, according to EU designations, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains an I332E substitution. In some embodiments, according to EU designations, the first Fc polypeptide contains both S239D and I332E substitutions and the second Fc polypeptide contains an I332E substitution. In some embodiments, according to EU designations, the second Fc polypeptide contains an I332E substitution. In some embodiments, according to EU designations, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains both S239D and I332E substitutions. (This text is repeated four times in the original.)

In some embodiments, according to EU designation, the first Fc polypeptide contains an I332E substitution and the second Fc polypeptide contains an S239D substitution. In a specific embodiment, according to EU designation, the first Fc polypeptide contains an I332E substitution and a serine residue at position 239, and the second Fc polypeptide contains an S239D substitution and an isoleucine residue at position 332.

In some embodiments, according to EU designation, the first Fc polypeptide comprises S239D and I332E substitutions and the second Fc polypeptide comprises an S239D substitution. In a specific embodiment, according to EU designation, the first Fc polypeptide comprises S239D and I332E substitutions and the second Fc polypeptide comprises an S239D substitution and an isoleucine residue at position 332.

In some embodiments, according to EU designation, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains an I332E substitution. In a specific embodiment, according to EU designation, the first Fc polypeptide contains an S239D substitution and an isoleucine at position 332, and the second Fc polypeptide contains an I332E substitution and a serine at position 239.

In some embodiments, according to EU designation, the second Fc polypeptide contains an I332E substitution. In a specific embodiment, according to EU designation, the first Fc polypeptide contains a serine residue at position 239 and an isoleucine residue at position 332, and the second Fc polypeptide contains an I332E substitution and a serine residue at position 239.

In some embodiments, according to EU designation, the first Fc polypeptide comprises an S239D substitution and the second Fc polypeptide comprises S239D and I332E substitutions. In a specific embodiment, according to EU designation, the first Fc polypeptide comprises an S239D substitution and an isoleucine residue at position 332, and the second Fc polypeptide comprises S239D and I332E substitutions.

In some embodiments, according to EU designation, the first Fc polypeptide contains an I332E substitution. In a specific embodiment, according to EU designation, the first Fc polypeptide contains an I332E substitution and a serine residue at position 239, and the second Fc polypeptide contains a serine residue at position 239 and an isoleucine residue at position 332.

In some embodiments, according to EU designations, the first Fc polypeptide comprises a TfR binding site, a T366W substitution, L234A and L235A substitutions (optionally including P329G or P329S substitutions), and optionally S239D and/or I332E substitutions, and according to EU designations, the second Fc polypeptide comprises T366S, L368A and Y407V substitutions and optionally S239D and/or I332E substitutions. For example, the first Fc polypeptide may contain a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with any one of the sequences SEQ ID NO:74-84, 86, and 98, and the second Fc polypeptide may contain a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with any one of the sequences SEQ ID NO:71-73, 85, and 99-100.

In some embodiments, according to EU designations, the first Fc polypeptide contains a TfR binding site and includes L234A and L235A substitutions (optionally including P329G or P329S substitutions), and the second Fc polypeptide does not include L234A or L325A substitutions (or, if present in the first Fc polypeptide, P329G or P329S substitutions). In some other embodiments, according to EU designations, the first Fc polypeptide contains a TfR binding site and does not include L234A or L325A substitutions (or, if present in the second Fc polypeptide, P329G or P329S substitutions), and the second Fc polypeptide contains L234A and L235A substitutions (optionally including P329G or P329S substitutions).

In some embodiments, the C-terminal lysine residue of one or both of the Fc peptides may be removed (e.g., the Lys residue at position 447 of the Fc peptide according to EU designation). In some embodiments, removing the C-terminal lysine residue from the Fc peptide may improve antibody stability.

In some implementations, the antigen-binding site of human HER2 subdomain II in the anti-HER2 antibody contains one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:89;

(b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:90; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:91.

In some implementations, at least one of the following is true: _X1 in SEQ ID NO:89 is not T; _X2 in SEQ ID NO:89 is not F; _X3 in SEQ ID NO:89 is not T; _X1 in SEQ ID NO:90 is not N; _X2 in SEQ ID NO:90 is not N; _X3 in SEQ ID NO:90 is not S; X4 in SEQ ID NO:90 is not G; _X5 in 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 in SEQ ID NO: ₉₁ is not G; _X3 in SEQ ID NO:91 is not P; and _X4 in SEQ ID NO:91 is not S.

In some embodiments, heavy chain CDR1 comprises the amino acid sequence SEQ ID NO:89, wherein _X1 is N, K, M, or H. In some embodiments, heavy chain CDR2 comprises the amino acid sequence SEQ ID NO:90, wherein _X5 is Q. In some embodiments, heavy chain CDR2 comprises the amino acid sequence SEQ ID NO:90, wherein _X6 is R, H, or T. In some embodiments, heavy chain CDR3 comprises the amino acid sequence SEQ ID NO:91, wherein _X4 is W, F, D, L, or Y. In some embodiments, heavy chain CDR3 comprises the amino acid sequence SEQ ID NO:91, wherein _X4 is L.

In some implementations, the antigen-binding site of human HER2 subdomain II in the anti-HER2 antibody contains one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:89;

(b) A heavy chain CDR2 comprising the amino acid sequence SEQ ID NO:90, wherein _X5 is Q; and

(c) A heavy chain CDR3 containing the amino acid sequence SEQ ID NO:91, wherein X ₄ is L.

In some implementations, the antigen-binding site of human HER2 subdomain II in the anti-HER2 antibody contains 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 with an amino acid sequence selected from the group consisting of SEQ ID NO:4 and 49-52, or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO:4 and 49-52;

(b) A heavy chain CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 5-6 and 53-55, or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 5-6 and 53-55; 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 with an amino acid sequence selected from the group consisting of SEQ ID NO: 7-8 and 56-59, or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 7-8 and 56-59.

In some implementations, the antigen-binding site of human HER2 subdomain II in the anti-HER2 antibody contains 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 with the amino acid sequence SEQ ID NO:4 or having at most two amino acid substitutions relative to the amino acid sequence 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 with the amino acid sequence SEQ ID NO:5 or SEQ ID NO:6, or having at most two amino acid substitutions relative to the amino acid sequence 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 with the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8, or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8.

In some implementations, the antigen-binding site of human HER2 subdomain II in the anti-HER2 antibody contains one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4;

(b) Heavy chain CDR2 containing the amino acid sequence SEQ ID NO:5 or SEQ ID NO:6; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8.

In some implementations, the antigen-binding site of human HER2 subdomain II in the anti-HER2 antibody contains one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4;

(b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:6; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:7.

In some implementations, the antigen-binding site of human HER2 subdomain II in the anti-HER2 antibody contains one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4;

(b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:5; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:8.

In some implementations, the antigen-binding site of human HER2 subdomain II in the anti-HER2 antibody contains one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4;

(b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:6; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:8.

In some embodiments, the antigen-binding site of the human HER2 subdomain II in the anti-HER2 antibody comprises a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one of SEQ ID NO: 1-3 and 60-70. In some embodiments, the antigen-binding site of the human HER2 subdomain II in the anti-HER2 antibody comprises a heavy chain variable region containing an amino acid sequence of any one of SEQ ID NO: 1-3 and 60-70.

In some implementations, the antigen-binding site of human HER2 subdomain IV in the anti-HER2 antibody contains one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) A heavy chain CDR1 containing the amino acid sequence SEQ ID NO:16 or having at most two amino acid substitutions relative to the amino acid sequence 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 with the amino acid sequence SEQ ID NO:17 or having at most two amino acid substitutions relative to the amino acid sequence 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 with the amino acid sequence SEQ ID NO:18 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:18.

In some implementations, the antigen-binding site of human HER2 subdomain IV in the anti-HER2 antibody contains one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:16;

(b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:17; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:18.

In some embodiments, the antigen-binding site of the human HER2 subdomain IV in the anti-HER2 antibody includes 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 with SEQ ID NO:15. In some embodiments, the antigen-binding site of the human HER2 subdomain IV in the anti-HER2 antibody includes a heavy chain variable region comprising the sequence SEQ ID NO:15.

In some embodiments, the light chain polypeptide sequences of the first and second antigen-binding sites (i.e., the antigen-binding sites of HER2 subdomain II and HER2 subdomain IV) in the anti-HER2 antibody contain 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 with the amino acid sequence SEQ ID NO:11 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:11;

(b) A light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:12; and

(c) A light chain CDR3 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:13 or 14.

In some implementations, the light chain polypeptide sequence contains one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) The light chain CDR1 containing the amino acid sequence SEQ ID NO:11;

(b) The light chain CDR2 containing the amino acid sequence SEQ ID NO:12; and

(c) A light chain CDR3 containing the amino acid sequence SEQ ID NO:13 or 14.

In some implementations, the light chain polypeptide sequence contains one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) The light chain CDR1 containing the amino acid sequence SEQ ID NO:11;

(b) The light chain CDR2 containing the amino acid sequence SEQ ID NO:12; and

(c) The light chain CDR3 containing the amino acid sequence SEQ ID NO:13.

In some implementations, the light chain polypeptide sequence contains one or more (e.g., one, two, or all three) CDRs selected from the group consisting of:

(a) The light chain CDR1 containing the amino acid sequence SEQ ID NO:11;

(b) The light chain CDR2 containing the amino acid sequence SEQ ID NO:12; and

(c) The light chain CDR3 containing the amino acid sequence SEQ ID NO:14.

In some embodiments, the light chain polypeptide sequence comprises a light chain CDR3 containing the amino acid sequence SEQ ID NO: 13 or 14 and optionally further comprises one or more (e.g., one or two) 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 with the amino acid sequence SEQ ID NO: 11 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO: 11; and a light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO: 12. In a specific embodiment, the light chain polypeptide sequence comprises the amino acid sequence SEQ ID NO: 13 or 14 and optionally further comprises one or more (e.g., one or two) CDRs selected from the group consisting of: a light chain CDR1 containing the amino acid sequence SEQ ID NO: 11; and a light chain CDR2 containing the amino acid sequence SEQ ID NO: 12.

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 with any of SEQ ID NO: 9-10. In some embodiments, the light chain polypeptide sequence comprises a light chain variable region comprising an amino acid sequence of any of SEQ ID NO: 9-10.

In some implementations, the anti-HER2 antibody comprises heavy and light chain CDRs selected from the combinations listed in Table 1 (i.e., any one of combinations #A-AC).

In the specific implementation scheme of the antibody:

(a) The first heavy chain comprises heavy chain CDR1 containing the amino acid sequence SEQ ID NO:16, heavy chain CDR2 containing the amino acid sequence SEQ ID NO:17, and heavy chain CDR3 containing the amino acid sequence SEQ ID NO:18.

(b) The second heavy chain comprises a heavy chain CDR1 containing any one of the amino acid sequences SEQ ID NO:4 and 49-52, a heavy chain CDR2 containing any one of the amino acid sequences SEQ ID NO:5-6 and 53-55, and a heavy chain CDR3 containing any one of the amino acid sequences SEQ ID NO:7-8 and 56-59; and

(c) The light chain comprises light chain CDR1 containing the amino acid sequence SEQ ID NO:11, light chain CDR2 containing the amino acid sequence SEQ ID NO:12, and light chain CDR3 containing any one of the amino acid sequences SEQ ID NO:13-14.

In some embodiments of the 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 amino acid sequences selected from 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.

In some 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 NO: 1-3 and 60-70; the second antigen-binding site comprises the amino acid sequence SEQ ID NO: 15; 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.

In some implementations, the anti-HER2 antibody comprises a first heavy chain that binds to human HER2 subdomain II or IV, a second heavy chain that binds to another HER2 subdomain, and two identical light chains.

In some 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 with an amino acid sequence selected from the combinations listed in Table 2 (i.e., any one of the combinations #A-AT).

In the specific implementation scheme of the antibody:

(a) The first heavy chain contains SEQ ID NO:37, the second heavy chain contains SEQ ID NO:25, and the light chain contains SEQ ID NO:9;

(b) The first heavy chain contains SEQ ID NO:31, the second heavy chain contains SEQ ID NO:30, and the light chain contains SEQ ID NO:9;

(c) The first heavy chain contains SEQ ID NO:31, the second heavy chain contains SEQ ID NO:29, and the light chain contains SEQ ID NO:9;

(d) The first heavy chain contains SEQ ID NO:38, the second heavy chain contains SEQ ID NO:30, and the light chain contains SEQ ID NO:9;

(e) The first heavy chain contains SEQ ID NO:32, the second heavy chain contains SEQ ID NO:30, and the light chain contains SEQ ID NO:9;

(f) The first heavy chain contains SEQ ID NO:32, the second heavy chain contains SEQ ID NO:29, and the light chain contains SEQ ID NO:9;

(g) The first heavy chain contains SEQ ID NO:39, the second heavy chain contains SEQ ID NO:30, and the light chain contains SEQ ID NO:9;

(h) The first heavy chain contains SEQ ID NO:37, the second heavy chain contains SEQ ID NO:26, and the light chain contains SEQ ID NO:9;

(i) The first heavy chain contains SEQ ID NO:31, the second heavy chain contains SEQ ID NO:34, and the light chain contains SEQ ID NO:9;

(j) The first heavy chain contains SEQ ID NO:31, the second heavy chain contains SEQ ID NO:33, and the light chain contains SEQ ID NO:9;

(k) The first heavy chain contains SEQ ID NO:38, the second heavy chain contains SEQ ID NO:34, and the light chain contains SEQ ID NO:9;

(l) The first heavy chain contains SEQ ID NO:32, the second heavy chain contains SEQ ID NO:34, and the light chain contains SEQ ID NO:9;

(m) The first heavy chain contains SEQ ID NO:32, the second heavy chain contains SEQ ID NO:33, and the light chain contains SEQ ID NO:9;

(n) The first heavy chain contains SEQ ID NO:39, the second heavy chain contains SEQ ID NO:34, and the light chain contains SEQ ID NO:9;

(o) The first heavy chain contains SEQ ID NO:37, the second heavy chain contains SEQ ID NO:27, and the light chain contains SEQ ID NO:9;

(p) The first heavy chain contains SEQ ID NO:31, the second heavy chain contains SEQ ID NO:36, and the light chain contains SEQ ID NO:9;

(q) The first heavy chain contains SEQ ID NO:31, the second heavy chain contains SEQ ID NO:35, and the light chain contains SEQ ID NO:9;

(r) The first heavy chain contains SEQ ID NO:38, the second heavy chain contains SEQ ID NO:36, and the light chain contains SEQ ID NO:9;

(s) The first heavy chain contains SEQ ID NO:32, the second heavy chain contains SEQ ID NO:36, and the light chain contains SEQ ID NO:9;

(t) The first heavy chain contains SEQ ID NO:32, the second heavy chain contains SEQ ID NO:35, and the light chain contains SEQ ID NO:9;

(u) The first heavy chain contains SEQ ID NO:39, the second heavy chain contains SEQ ID NO:46, and the light chain contains SEQ ID NO:9;

(v) The first heavy chain contains SEQ ID NO:37, the second heavy chain contains SEQ ID NO:25, and the light chain contains SEQ ID NO:10;

(w) The first heavy chain contains SEQ ID NO:31, the second heavy chain contains SEQ ID NO:30, and the light chain contains SEQ ID NO:10;

(x) The first heavy chain contains SEQ ID NO:31, the second heavy chain contains SEQ ID NO:29, and the light chain contains SEQ ID NO:10;

(y) The first heavy chain contains SEQ ID NO:38, the second heavy chain contains SEQ ID NO:30, and the light chain contains SEQ ID NO:10;

(z) The first heavy chain contains SEQ ID NO:32, the second heavy chain contains SEQ ID NO:30, and the light chain contains SEQ ID NO:10;

(aa) The first heavy chain contains SEQ ID NO:32, the second heavy chain contains SEQ ID NO:29, and the light chain contains SEQ ID NO:10;

(ab) The first heavy chain contains SEQ ID NO:39, the second heavy chain contains SEQ ID NO:30, and the light chain contains SEQ ID NO:10;

(ac) The first heavy chain contains SEQ ID NO:37, the second heavy chain contains SEQ ID NO:26, and the light chain contains SEQ ID NO:10;

(ad) The first heavy chain contains SEQ ID NO:31, the second heavy chain contains SEQ ID NO:34, and the light chain contains SEQ ID NO:10;

(ae) The first heavy chain contains SEQ ID NO:31, the second heavy chain contains SEQ ID NO:33, and the light chain contains SEQ ID NO:10;

(af) The first heavy chain contains SEQ ID NO:38, the second heavy chain contains SEQ ID NO:34, and the light chain contains SEQ ID NO:10;

(ag) The first heavy chain contains SEQ ID NO:32, the second heavy chain contains SEQ ID NO:34, and the light chain contains SEQ ID NO:10;

(ah) The first heavy chain contains SEQ ID NO:32, the second heavy chain contains SEQ ID NO:33, and the light chain contains SEQ ID NO:10;

(ai) The first heavy chain contains SEQ ID NO:39, the second heavy chain contains SEQ ID NO:34, and the light chain contains SEQ ID NO:10;

(aj) The first heavy chain contains SEQ ID NO:37, the second heavy chain contains SEQ ID NO:27, and the light chain contains SEQ ID NO:10;

(ak) The first heavy chain contains SEQ ID NO:31, the second heavy chain contains SEQ ID NO:36, and the light chain contains SEQ ID NO:10;

(al) The first heavy chain contains SEQ ID NO:31, the second heavy chain contains SEQ ID NO:35, and the light chain contains SEQ ID NO:10;

(am) The first heavy chain contains SEQ ID NO:38, the second heavy chain contains SEQ ID NO:36, and the light chain contains SEQ ID NO:10;

(an) The first heavy chain contains SEQ ID NO:32, the second heavy chain contains SEQ ID NO:36, and the light chain contains SEQ ID NO:10;

(ao) The first heavy chain contains SEQ ID NO:32, the second heavy chain contains SEQ ID NO:35, and the light chain contains SEQ ID NO:10;

(ap) The first heavy chain contains SEQ ID NO:39, the second heavy chain contains SEQ ID NO:46, and the light chain contains SEQ ID NO:10;

(aq) The first heavy chain contains SEQ ID NO:20, the second heavy chain contains SEQ ID NO:24, and the light chain contains SEQ ID NO:19;

(ar) The first heavy chain contains SEQ ID NO:21, the second heavy chain contains SEQ ID NO:24, and the light chain contains SEQ ID NO:19;

(as) the first heavy chain contains SEQ ID NO:22, the second heavy chain contains SEQ ID NO:24, and the light chain contains SEQ ID NO:19; or

(at) The first heavy chain contains SEQ ID NO:23, the second heavy chain contains SEQ ID NO:24, and the light chain contains SEQ ID NO:19.

In some embodiments, the anti-HER2 antibody comprises a first heavy chain containing VH and Fc sequences, each having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the combinations selected from Table 3 (i.e., any one of combinations #AL), and a second heavy chain containing _VH and Fc sequences, each having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the combinations selected from _Table 4 (i.e., any one of combinations #AL). 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 with the amino acid sequence selected from the group consisting of SEQ ID NO: 9-10 and 19.

In the specific implementation scheme of the antibody:

(a) The first heavy chain contains SEQ ID NO:1 and 71 and the second heavy chain contains SEQ ID NO:15 and 86;

(b) The first heavy chain contains SEQ ID NO:1 and 71 and the second heavy chain contains SEQ ID NO:15 and 74;

(c) The first heavy chain contains SEQ ID NO:1 and 71 and the second heavy chain contains SEQ ID NO:15 and 75;

(d) The first heavy chain contains SEQ ID NO:1 and 71 and the second heavy chain contains SEQ ID NO:15 and 76;

(e) The first heavy chain contains SEQ ID NO:1 and 71 and the second heavy chain contains SEQ ID NO:15 and 77;

(f) The first heavy chain contains SEQ ID NO:1 and 71 and the second heavy chain contains SEQ ID NO:15 and 78;

(g) The first heavy chain contains SEQ ID NO:1 and 71 and the second heavy chain contains SEQ ID NO:15 and 79;

(h) The first heavy chain contains SEQ ID NO:1 and 71 and the second heavy chain contains SEQ ID NO:15 and 80;

(i) The first heavy chain contains SEQ ID NO:1 and 71 and the second heavy chain contains SEQ ID NO:15 and 81;

(j) The first heavy chain contains SEQ ID NO:1 and 71 and the second heavy chain contains SEQ ID NO:15 and 82;

(k) The first heavy chain contains SEQ ID NO: 1 and 71 and the second heavy chain contains SEQ ID NO: 15 and 83; or

(l) The first heavy chain contains SEQ ID NO:1 and 71 and the second heavy chain contains SEQ ID NO:15 and 84.

In the specific implementation scheme of the antibody:

(a) The first heavy chain contains SEQ ID NO:2 and 71 and the second heavy chain contains SEQ ID NO:15 and 86;

(b) The first heavy chain contains SEQ ID NO:2 and 71 and the second heavy chain contains SEQ ID NO:15 and 74;

(c) The first heavy chain contains SEQ ID NO:2 and 71 and the second heavy chain contains SEQ ID NO:15 and 75;

(d) The first heavy chain contains SEQ ID NO:2 and 71 and the second heavy chain contains SEQ ID NO:15 and 76;

(e) The first heavy chain contains SEQ ID NO:2 and 71 and the second heavy chain contains SEQ ID NO:15 and 77;

(f) The first heavy chain contains SEQ ID NO:2 and 71 and the second heavy chain contains SEQ ID NO:15 and 78;

(g) The first heavy chain contains SEQ ID NO:2 and 71 and the second heavy chain contains SEQ ID NO:15 and 79;

(h) The first heavy chain contains SEQ ID NO:2 and 71 and the second heavy chain contains SEQ ID NO:15 and 80;

(i) The first heavy chain contains SEQ ID NO:2 and 71 and the second heavy chain contains SEQ ID NO:15 and 81;

(j) The first heavy chain contains SEQ ID NO:2 and 71 and the second heavy chain contains SEQ ID NO:15 and 82;

(k) The first heavy chain contains SEQ ID NO:2 and 71 and the second heavy chain contains SEQ ID NO:15 and 83; or

(l) The first heavy chain contains SEQ ID NO:2 and 71 and the second heavy chain contains SEQ ID NO:15 and 84.

In the specific implementation scheme of the antibody:

(a) The first heavy chain contains SEQ ID NO:3 and 71 and the second heavy chain contains SEQ ID NO:15 and 86;

(b) The first heavy chain contains SEQ ID NO:3 and 71 and the second heavy chain contains SEQ ID NO:15 and 74;

(c) The first heavy chain contains SEQ ID NO:3 and 71 and the second heavy chain contains SEQ ID NO:15 and 75;

(d) The first heavy chain contains SEQ ID NO:3 and 71 and the second heavy chain contains SEQ ID NO:15 and 76;

(e) The first heavy chain contains SEQ ID NO:3 and 71 and the second heavy chain contains SEQ ID NO:15 and 77;

(f) The first heavy chain contains SEQ ID NO:3 and 71 and the second heavy chain contains SEQ ID NO:15 and 78;

(g) The first heavy chain contains SEQ ID NO:3 and 71 and the second heavy chain contains SEQ ID NO:15 and 79;

(h) The first heavy chain contains SEQ ID NO:3 and 71 and the second heavy chain contains SEQ ID NO:15 and 80;

(i) The first heavy chain contains SEQ ID NO:3 and 71 and the second heavy chain contains SEQ ID NO:15 and 81;

(j) The first heavy chain contains SEQ ID NO:3 and 71 and the second heavy chain contains SEQ ID NO:15 and 82;

(k) The first heavy chain contains SEQ ID NO:3 and 71 and the second heavy chain contains SEQ ID NO:15 and 83; or

(l) The first heavy chain contains SEQ ID NO:3 and 71 and the second heavy chain contains SEQ ID NO:15 and 84.

In the specific implementation scheme of the antibody:

(a) The first heavy chain contains SEQ ID NO:1 and 72 and the second heavy chain contains SEQ ID NO:15 and 86;

(b) The first heavy chain contains SEQ ID NO:1 and 72 and the second heavy chain contains SEQ ID NO:15 and 74;

(c) The first heavy chain contains SEQ ID NO:1 and 72 and the second heavy chain contains SEQ ID NO:15 and 75;

(d) The first heavy chain contains SEQ ID NO:1 and 72 and the second heavy chain contains SEQ ID NO:15 and 76;

(e) The first heavy chain contains SEQ ID NO:1 and 72 and the second heavy chain contains SEQ ID NO:15 and 77;

(f) The first heavy chain contains SEQ ID NO:1 and 72 and the second heavy chain contains SEQ ID NO:15 and 78;

(g) The first heavy chain contains SEQ ID NO:1 and 72 and the second heavy chain contains SEQ ID NO:15 and 79;

(h) The first heavy chain contains SEQ ID NO:1 and 72 and the second heavy chain contains SEQ ID NO:15 and 80;

(i) The first heavy chain contains SEQ ID NO:1 and 72 and the second heavy chain contains SEQ ID NO:15 and 81;

(j) The first heavy chain contains SEQ ID NO:1 and 72 and the second heavy chain contains SEQ ID NO:15 and 82;

(k) The first heavy chain contains SEQ ID NO: 1 and 72 and the second heavy chain contains SEQ ID NO: 15 and 83; or

(l) The first heavy chain contains SEQ ID NO:1 and 72 and the second heavy chain contains SEQ ID NO:15 and 84.

In the specific implementation scheme of the antibody:

(a) The first heavy chain contains SEQ ID NO:2 and 72 and the second heavy chain contains SEQ ID NO:15 and 86;

(b) The first heavy chain contains SEQ ID NO:2 and 72 and the second heavy chain contains SEQ ID NO:15 and 74;

(c) The first heavy chain contains SEQ ID NO:2 and 72 and the second heavy chain contains SEQ ID NO:15 and 75;

(d) The first heavy chain contains SEQ ID NO:2 and 72 and the second heavy chain contains SEQ ID NO:15 and 76;

(e) The first heavy chain contains SEQ ID NO:2 and 72 and the second heavy chain contains SEQ ID NO:15 and 77;

(f) The first heavy chain contains SEQ ID NO:2 and 72 and the second heavy chain contains SEQ ID NO:15 and 78;

(g) The first heavy chain contains SEQ ID NO:2 and 72 and the second heavy chain contains SEQ ID NO:15 and 79;

(h) The first heavy chain contains SEQ ID NO:2 and 72 and the second heavy chain contains SEQ ID NO:15 and 80;

(i) The first heavy chain contains SEQ ID NO:2 and 72 and the second heavy chain contains SEQ ID NO:15 and 81;

(j) The first heavy chain contains SEQ ID NO:2 and 72 and the second heavy chain contains SEQ ID NO:15 and 82;

(k) The first heavy chain contains SEQ ID NO:2 and 72 and the second heavy chain contains SEQ ID NO:15 and 83; or

(l) The first heavy chain contains SEQ ID NO:2 and 72 and the second heavy chain contains SEQ ID NO:15 and 84.

In the specific implementation scheme of the antibody:

(a) The first heavy chain contains SEQ ID NO:3 and 72 and the second heavy chain contains SEQ ID NO:15 and 86;

(b) The first heavy chain contains SEQ ID NO:3 and 72 and the second heavy chain contains SEQ ID NO:15 and 74;

(c) The first heavy chain contains SEQ ID NO:3 and 72 and the second heavy chain contains SEQ ID NO:15 and 75;

(d) The first heavy chain contains SEQ ID NO:3 and 72 and the second heavy chain contains SEQ ID NO:15 and 76;

(e) The first heavy chain contains SEQ ID NO:3 and 72 and the second heavy chain contains SEQ ID NO:15 and 77;

(f) The first heavy chain contains SEQ ID NO:3 and 72 and the second heavy chain contains SEQ ID NO:15 and 78;

(g) The first heavy chain contains SEQ ID NO:3 and 72 and the second heavy chain contains SEQ ID NO:15 and 79;

(h) The first heavy chain contains SEQ ID NO:3 and 72 and the second heavy chain contains SEQ ID NO:15 and 80;

(i) The first heavy chain contains SEQ ID NO:3 and 72 and the second heavy chain contains SEQ ID NO:15 and 81;

(j) The first heavy chain contains SEQ ID NO:3 and 72 and the second heavy chain contains SEQ ID NO:15 and 82;

(k) The first heavy chain contains SEQ ID NO:3 and 72 and the second heavy chain contains SEQ ID NO:15 and 83; or

(l) The first heavy chain contains SEQ ID NO:3 and 72 and the second heavy chain contains SEQ ID NO:15 and 84.

In the specific implementation scheme of the antibody:

(a) The first heavy chain contains SEQ ID NO:1 and 73 and the second heavy chain contains SEQ ID NO:15 and 86;

(b) The first heavy chain contains SEQ ID NO:1 and 73 and the second heavy chain contains SEQ ID NO:15 and 74;

(c) The first heavy chain contains SEQ ID NO:1 and 73 and the second heavy chain contains SEQ ID NO:15 and 75;

(d) The first heavy chain contains SEQ ID NO:1 and 73 and the second heavy chain contains SEQ ID NO:15 and 76;

(e) The first heavy chain contains SEQ ID NO:1 and 73 and the second heavy chain contains SEQ ID NO:15 and 77;

(f) The first heavy chain contains SEQ ID NO:1 and 73 and the second heavy chain contains SEQ ID NO:15 and 78;

(g) The first heavy chain contains SEQ ID NO:1 and 73 and the second heavy chain contains SEQ ID NO:15 and 79;

(h) The first heavy chain contains SEQ ID NO:1 and 73 and the second heavy chain contains SEQ ID NO:15 and 80;

(i) The first heavy chain contains SEQ ID NO:1 and 73 and the second heavy chain contains SEQ ID NO:15 and 81;

(j) The first heavy chain contains SEQ ID NO:1 and 73 and the second heavy chain contains SEQ ID NO:15 and 82;

(k) The first heavy chain contains SEQ ID NO: 1 and 73 and the second heavy chain contains SEQ ID NO: 15 and 83; or

(l) The first heavy chain contains SEQ ID NO:1 and 73 and the second heavy chain contains SEQ ID NO:15 and 84.

In the specific implementation scheme of the antibody:

(a) The first heavy chain contains SEQ ID NO:2 and 73 and the second heavy chain contains SEQ ID NO:15 and 86;

(b) The first heavy chain contains SEQ ID NO:2 and 73 and the second heavy chain contains SEQ ID NO:15 and 74;

(c) The first heavy chain contains SEQ ID NO:2 and 73 and the second heavy chain contains SEQ ID NO:15 and 75;

(d) The first heavy chain contains SEQ ID NO:2 and 73 and the second heavy chain contains SEQ ID NO:15 and 76;

(e) The first heavy chain contains SEQ ID NO:2 and 73 and the second heavy chain contains SEQ ID NO:15 and 77;

(f) The first heavy chain contains SEQ ID NO:2 and 73 and the second heavy chain contains SEQ ID NO:15 and 78;

(g) The first heavy chain contains SEQ ID NO:2 and 73 and the second heavy chain contains SEQ ID NO:15 and 79;

(h) The first heavy chain contains SEQ ID NO:2 and 73 and the second heavy chain contains SEQ ID NO:15 and 80;

(i) The first heavy chain contains SEQ ID NO:2 and 73 and the second heavy chain contains SEQ ID NO:15 and 81;

(j) The first heavy chain contains SEQ ID NO:2 and 73 and the second heavy chain contains SEQ ID NO:15 and 82;

(k) The first heavy chain contains SEQ ID NO:2 and 73 and the second heavy chain contains SEQ ID NO:15 and 83; or

(l) The first heavy chain contains SEQ ID NO:2 and 73 and the second heavy chain contains SEQ ID NO:15 and 84.

In the specific implementation scheme of the antibody:

(a) The first heavy chain contains SEQ ID NO:3 and 73 and the second heavy chain contains SEQ ID NO:15 and 86;

(b) The first heavy chain contains SEQ ID NO:3 and 73 and the second heavy chain contains SEQ ID NO:15 and 74;

(c) The first heavy chain contains SEQ ID NO:3 and 73 and the second heavy chain contains SEQ ID NO:15 and 75;

(d) The first heavy chain contains SEQ ID NO:3 and 73 and the second heavy chain contains SEQ ID NO:15 and 76;

(e) The first heavy chain contains SEQ ID NO:3 and 73 and the second heavy chain contains SEQ ID NO:15 and 77;

(f) The first heavy chain contains SEQ ID NO:3 and 73 and the second heavy chain contains SEQ ID NO:15 and 78;

(g) The first heavy chain contains SEQ ID NO:3 and 73 and the second heavy chain contains SEQ ID NO:15 and 79;

(h) The first heavy chain contains SEQ ID NO:3 and 73 and the second heavy chain contains SEQ ID NO:15 and 80;

(i) The first heavy chain contains SEQ ID NO:3 and 73 and the second heavy chain contains SEQ ID NO:15 and 81;

(j) The first heavy chain contains SEQ ID NO:3 and 73 and the second heavy chain contains SEQ ID NO:15 and 82;

(k) The first heavy chain contains SEQ ID NO:3 and 73 and the second heavy chain contains SEQ ID NO:15 and 83; or

(l) The first heavy chain contains SEQ ID NO:3 and 73 and the second heavy chain contains SEQ ID NO:15 and 84.

In the specific implementation scheme of the antibody:

(a) The first heavy chain contains SEQ ID NO:1 and 85 and the second heavy chain contains SEQ ID NO:15 and 86;

(b) The first heavy chain contains SEQ ID NO:1 and 85 and the second heavy chain contains SEQ ID NO:15 and 74;

(c) The first heavy chain contains SEQ ID NO:1 and 85 and the second heavy chain contains SEQ ID NO:15 and 75;

(d) The first heavy chain contains SEQ ID NO:1 and 85 and the second heavy chain contains SEQ ID NO:15 and 76;

(e) The first heavy chain contains SEQ ID NO:1 and 85 and the second heavy chain contains SEQ ID NO:15 and 77;

(f) The first heavy chain contains SEQ ID NO:1 and 85 and the second heavy chain contains SEQ ID NO:15 and 78;

(g) The first heavy chain contains SEQ ID NO:1 and 85 and the second heavy chain contains SEQ ID NO:15 and 79;

(h) The first heavy chain contains SEQ ID NO:1 and 85 and the second heavy chain contains SEQ ID NO:15 and 80;

(i) The first heavy chain contains SEQ ID NO:1 and 85 and the second heavy chain contains SEQ ID NO:15 and 81;

(j) The first heavy chain contains SEQ ID NO:1 and 85 and the second heavy chain contains SEQ ID NO:15 and 82;

(k) The first heavy chain contains SEQ ID NO: 1 and 85 and the second heavy chain contains SEQ ID NO: 15 and 83; or

(l) The first heavy chain contains SEQ ID NO:1 and 85 and the second heavy chain contains SEQ ID NO:15 and 84.

In the specific implementation scheme of the antibody:

(a) The first heavy chain contains SEQ ID NO:2 and 85 and the second heavy chain contains SEQ ID NO:15 and 86;

(b) The first heavy chain contains SEQ ID NO:2 and 85 and the second heavy chain contains SEQ ID NO:15 and 74;

(c) The first heavy chain contains SEQ ID NO:2 and 85 and the second heavy chain contains SEQ ID NO:15 and 75;

(d) The first heavy chain contains SEQ ID NO:2 and 85 and the second heavy chain contains SEQ ID NO:15 and 76;

(e) The first heavy chain contains SEQ ID NO:2 and 85 and the second heavy chain contains SEQ ID NO:15 and 77;

(f) The first heavy chain contains SEQ ID NO:2 and 85 and the second heavy chain contains SEQ ID NO:15 and 78;

(g) The first heavy chain contains SEQ ID NO:2 and 85 and the second heavy chain contains SEQ ID NO:15 and 79;

(h) The first heavy chain contains SEQ ID NO:2 and 85 and the second heavy chain contains SEQ ID NO:15 and 80;

(i) The first heavy chain contains SEQ ID NO:2 and 85 and the second heavy chain contains SEQ ID NO:15 and 81;

(j) The first heavy chain contains SEQ ID NO:2 and 85 and the second heavy chain contains SEQ ID NO:15 and 82;

(k) The first heavy chain contains SEQ ID NO:2 and 85 and the second heavy chain contains SEQ ID NO:15 and 83; or

(l) The first heavy chain contains SEQ ID NO:2 and 85 and the second heavy chain contains SEQ ID NO:15 and 84.

In the specific implementation scheme of the antibody:

(a) The first heavy chain contains SEQ ID NO:3 and 85 and the second heavy chain contains SEQ ID NO:15 and 86;

(b) The first heavy chain contains SEQ ID NO:3 and 85 and the second heavy chain contains SEQ ID NO:15 and 74;

(c) The first heavy chain contains SEQ ID NO:3 and 85 and the second heavy chain contains SEQ ID NO:15 and 75;

(d) The first heavy chain contains SEQ ID NO:3 and 85 and the second heavy chain contains SEQ ID NO:15 and 76;

(e) The first heavy chain contains SEQ ID NO:3 and 85 and the second heavy chain contains SEQ ID NO:15 and 77;

(f) The first heavy chain contains SEQ ID NO:3 and 85 and the second heavy chain contains SEQ ID NO:15 and 78;

(g) The first heavy chain contains SEQ ID NO:3 and 85 and the second heavy chain contains SEQ ID NO:15 and 79;

(h) The first heavy chain contains SEQ ID NO:3 and 85 and the second heavy chain contains SEQ ID NO:15 and 80;

(i) The first heavy chain contains SEQ ID NO:3 and 85 and the second heavy chain contains SEQ ID NO:15 and 81;

(j) The first heavy chain contains SEQ ID NO:3 and 85 and the second heavy chain contains SEQ ID NO:15 and 82;

(k) The first heavy chain contains SEQ ID NO:3 and 85 and the second heavy chain contains SEQ ID NO:15 and 83; or

(l) The first heavy chain contains SEQ ID NO:3 and 85 and the second heavy chain contains SEQ ID NO:15 and 84.

In some other embodiments, the anti-HER2 antibody comprises a first heavy chain containing VH and Fc sequences, each having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the combinations selected from Table 5 (i.e., any one of combinations #A-AJ), and a second heavy chain containing _VH and Fc sequences, each having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the combinations selected from _Table 6 (i.e., any one of combinations #AD). 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 with the amino acid sequence selected from the group consisting of SEQ ID NO: 9-10 and 19.

IV. FC peptides and their modifications

In some aspects, any antibody described herein comprises an Fc polypeptide dimer, wherein one or both Fc polypeptides in the dimer contain amino acid modifications relative to the wild-type Fc polypeptide. In some embodiments, the amino acid modifications in the Fc polypeptide (e.g., modified Fc polypeptides) can lead to the binding of the Fc polypeptide dimer to a BBB receptor (e.g., TfR), promote heterodimerization of the two Fc polypeptides in the dimer, modulate effector function, prolong serum half-life, affect glycosylation, and/or reduce human immunogenicity. In some embodiments, the Fc polypeptide present in the antibody independently has at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the corresponding wild-type Fc polypeptide (e.g., 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. WO 2018/152326, which is incorporated herein by reference in its entirety.

Fc peptide modification for BBB receptor binding

This article provides an anti-HER2 antibody capable of transport across the BBB. This protein comprises a modified Fc polypeptide that binds to the BBB receptor. The BBB receptor is expressed on the BBB endothelium as well as in other cell and tissue types. In some embodiments, the BBB receptor is a TfR. The modified Fc polypeptide that binds to the TfR is also referred to as having a TfR binding site.

The amino acid residues specified in various Fc modifications, including those introduced into modified Fc peptides that bind to BBB receptors (e.g., TfR), are numbered herein using EU index numbers. Any Fc peptide (e.g., IgG1, IgG2, IgG3, or IgG4 Fc peptide) may have modifications (e.g., amino acid substitutions) at one or more positions as described herein. In some embodiments, the domain modified for activity binding to BBB receptors (e.g., TfR) is a human IgCH3 domain, such as the IgG1 CH3 domain. The CH3 domain may have any IgG subtype, i.e., derived from IgG1, IgG2, IgG3, or IgG4. In the case of IgG1 antibodies, the CH3 domain refers to the amino acid segment from approximately position 341 to approximately position 447 as numbered according to the EU numbering scheme.

In some embodiments, the modified Fc peptide that specifically binds to TfR binds to the apical domain of TfR and can bind to TfR without blocking or otherwise inhibiting the binding of transferrin to TfR. In some embodiments, the binding of transferrin to TfR is substantially uninhibited. In some embodiments, the binding of transferrin to TfR is inhibited by less than about 50% (e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%).

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

In some embodiments, the Fc polypeptide present in the antibody described herein that binds to a BBB receptor (e.g., TfR) contains at least one, two, or three substitutions according to the EU numbering scheme; 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.

In some embodiments, the Fc polypeptide that binds to a BBB receptor (e.g., TfR) contains at least one position having a substitution relative 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; Thr or an acidic amino acid at position 416; or Trp, Tyr, His, or Phe at position 421. In some embodiments, the Fc polypeptide binding to a BBB receptor (e.g., TfR) may contain conserved substitutions of specified amino acids at one or more positions in the set, such as amino acids in groups of the same charge, hydrophobic groups, side-chain ring structures (e.g., aromatic amino acids), or size groups and/or polar or nonpolar groups. Thus, for example, Ile may be present at positions 384, 386, and/or 413. In some embodiments, the acidic amino acid at one, two, or each of positions 387, 413, and 416 is Glu. In other embodiments, the acidic amino acid at one, two, or each of positions 387, 413, and 416 is Asp. In some embodiments, two, three, four, five, six, seven, or all eight of positions 384, 386, 387, 388, 389, 413, 416, and 421 have amino acid substitutions as specified in this paragraph.

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

In some embodiments, the Fc polypeptide comprises two, three, four, five, six, seven, eight, nine, or ten molecules selected from 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. 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.

In some embodiments, the Fc peptide binding to a BBB receptor (e.g., TfR) includes 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 peptide also includes Ser, Thr, Gln, or Phe at position 391. In some embodiments, the Fc peptide also includes 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 Fc peptide binding to a BBB receptor (e.g., TfR) does not have Trp at position 380.

In other embodiments, the Fc peptide binding to a BBB receptor (e.g., TfR) comprises Tyr at position 384; Thr at position 386; Glu or Val at position 387; Trp at position 388; Ser at position 389; Ser or Thr at position 413; Glu at position 416; and/or Phe at position 421. In some embodiments, the Fc peptide binding to a BBB receptor (e.g., TfR) comprises native Asn at position 390. In some embodiments, the Fc peptide also comprises Trp, Tyr, Leu, or Gln at position 380; and/or Glu at position 415. In some embodiments, the Fc peptide also comprises Trp at position 380 and/or Glu at position 415.

In some embodiments, the Fc peptide that binds to a BBB receptor (e.g., TfR) 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.

In other embodiments, the Fc peptide that binds to a BBB receptor (e.g., TfR) 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.

In some embodiments, the Fc polypeptide binding to a BBB receptor (e.g., TfR) has the sequence SEQ ID NO:97. In some embodiments of the antibody described herein, one of the two Fc polypeptides in the Fc polypeptide dimer may be an Fc polypeptide binding to a BBB receptor (e.g., TfR) having the sequence SEQ ID NO:97, while the other Fc polypeptide in 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 antibody described herein, both Fc polypeptides in the Fc polypeptide dimer may be Fc polypeptides binding to a BBB receptor (e.g., TfR) having the sequence SEQ ID NO:97.

In some embodiments of the antibody described herein, the first Fc polypeptide and/or the second Fc polypeptide independently comprise 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, and a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with a sequence selected from SEQ ID NO: 74-84, 86, and 97-101.

In some embodiments of the antibody described herein, one of the two Fc polypeptides in the Fc polypeptide dimer may be an Fc polypeptide that binds to a BBB receptor (e.g., TfR) and comprises 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, and a sequence having at least 90% identity with sequence SEQ ID NO:97, while the other Fc polypeptide in the Fc polypeptide dimer may have the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO:95).

In some embodiments of the antibody described herein, the first Fc polypeptide and/or the second Fc polypeptide independently comprise 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, and a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with a sequence selected from SEQ ID NO: 101-105.

In some embodiments of the antibody described herein, one of the two Fc polypeptides in the Fc polypeptide dimer may be an Fc polypeptide that binds to a BBB receptor (e.g., TfR) and comprises 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, and a sequence having at least 90% identity with sequence SEQ ID NO:101, while the other Fc polypeptide in the Fc polypeptide dimer may have the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO:95).

In some embodiments, the Fc polypeptide binding to a BBB receptor (e.g., TfR) has the sequence SEQ ID NO: 101. In some embodiments of the antibody described herein, one of the two Fc polypeptides in the Fc polypeptide dimer may be an Fc polypeptide binding to a BBB receptor (e.g., TfR) having the sequence SEQ ID NO: 101, while the other Fc polypeptide in 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 antibody described herein, both Fc polypeptides in the Fc polypeptide dimer may be Fc polypeptides binding to a BBB receptor (e.g., TfR) having the sequence SEQ ID NO: 101.

In some implementations, the Fc peptide that binds to the BBB receptor (e.g., TfR) comprises the following substitutions (according to EU numbers) listed in Table A below:

Table A

Fc peptide modification for heterodimerization

In some embodiments, the Fc peptide present in any of the antibodies described herein includes club and mortar mutations to promote heterodimer formation and inhibit homodimer formation. Typically, the modification introduces a protrusion (“club”) at the interface of the first peptide and a corresponding cavity (“mortar”) at the interface of the second peptide, thereby allowing the protrusion to be localized within the cavity to promote heterodimer formation and thus inhibit homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first peptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity of the same or similar size as the protrusion is created at the interface of the second peptide by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). In some embodiments, the location of such other mutations in the Fc peptide does not negatively affect the binding of the peptide to a BBB receptor (e.g., TfR).

In an exemplary embodiment of the pestle and mortar method for dimerization, position 366 (according to the EU numbering scheme) of one Fc polypeptide in the antibody contains tryptophan instead of native threonine. Another Fc polypeptide in the dimer has valine instead of native tyrosine at position 407 (according to the EU numbering scheme). The other Fc polypeptide may also contain substitutions, wherein the native threonine at position 366 (according to the EU numbering scheme) is replaced by serine and the native leucine at position 368 (according to the EU numbering scheme) is replaced by alanine. Thus, one Fc polypeptide of the antibody described herein has the T366W pestle mutation and the other Fc polypeptide has the Y407V mutation, which is typically accompanied by the T366S and L368A mortar mutations.

In some implementations, one or both Fc peptides present in the antibody described herein may also be engineered to contain other modifications for heterodimerization, such as electrostatic engineering of contact residues within the CH3-CH3 interface that serve as naturally charged or hydrophobic patches.

For example, in some embodiments, the antibody described herein may contain an Fc polypeptide dimer, one Fc polypeptide of which has a T366W mutation and at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with sequence SEQ ID NO:107, and the other Fc polypeptide has T366S, L368A, and Y407V mutations and at least 90% identity with sequence SEQ ID NO:85. In some embodiments, one or both Fc polypeptides of the Fc polypeptide dimer may be TfR-binding Fc polypeptides. In a specific embodiment, the antibody described herein may contain an Fc polypeptide dimer having (i) a first Fc polypeptide having sequence SEQ ID NO:85, and (ii) a second Fc polypeptide having sequence SEQ ID NO:98. In a specific implementation, the antibody described herein may contain an Fc polypeptide dimer having (i) a first Fc polypeptide having the sequence SEQ ID NO: 85 and (ii) a second Fc polypeptide having the sequence SEQ ID NO: 102.

Fc peptide modification for regulating effector function

In some embodiments, one or both Fc peptides present in any of the antibodies described herein may include modifications that reduce TfR-mediated effector function upon TfR binding, i.e., a reduced ability to induce certain biological functions upon binding to an Fc receptor expressed on effector cells mediating 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 function may vary depending on the antibody class. For example, natural human IgG1 and IgG3 antibodies may induce ADCC and CDC activity upon binding to appropriate Fc receptors present on immune system cells; and natural human IgG1, IgG2, IgG3, and IgG4 may induce ADCP function upon binding to appropriate Fc receptors present on immune cells.

In some embodiments, one or both Fc peptides present in the antibodies described herein may contain modifications that reduce or eliminate TfR-mediated effector function. Exemplary Fc peptide mutations that reduce TfR-mediated effector function include, but are not limited to, substitutions in the CH2 domain, for example, substitutions at positions 234 and 235 according to the EU numbering scheme. For example, in some embodiments, one or both Fc peptides may contain alanine residues at positions 234 and 235. Thus, one or both Fc peptides may have L234A and L235A (also referred to herein as “LALA”) substitutions.

Other Fc polypeptide mutations that regulate effector function include, but are not limited to, the following: Position 329 may be mutated, wherein proline is replaced by glycine, alanine, serine, or arginine, or an amino acid residue large enough to disrupt the Fc/Fcγ receptor interface, which is formed between proline 329 of the Fc and tryptophan residues Trp 87 and Trp 110 of the FcγRIII. Other exemplary substitutions according to the EU numbering scheme include S228P, E233P, L235E, N297A, N297D, and P331S. Multiple substitutions may also be present, such as L234A and L235A of the human IgG1 Fc region according to the EU numbering scheme; L234A, L235A and P329G of the human IgG1 Fc region; S228P and L235E of the human IgG4 Fc region; L234A and G237A of the human IgG1 Fc region; L234A, L235A and G237A of the human IgG1 Fc region; V234A and G237A of the human IgG2 Fc region; L235A, G237A and E318A of the human IgG4 Fc region; and S228P and L236E of the human IgG4 Fc region. In some embodiments, one or both Fc peptides may have one or more amino acid substitutions that regulate ADCC, such as substitutions at positions 298, 333 and/or 334 according to the EU numbering scheme. In some implementations, according to the EU numbering scheme, one or both Fc peptides may have L234A, L235A and P329G or P329S substitutions.

In some embodiments, one or both Fc peptides present in the antibodies described herein may contain modifications capable of enhancing HER2-mediated effector function upon HER2 binding, i.e., enhancing the ability to induce certain biological functions upon binding to an Fc receptor expressed on effector cells mediating effector function. Examples of antibody effector function have been described above. Exemplary Fc peptide mutations capable of enhancing HER2-mediated effector function include, but are not limited to, substitutions in the CH2 domain, such as substitutions at positions 239 and/or 332 according to the EU numbering scheme. For example, in some embodiments, one or both Fc peptides may contain aspartic acid at position 239 and/or glutamic acid at position 332. Thus, according to EU numbering, one or both Fc peptides may have S239D and/or I332E substitutions.

"cis-LALA" conformation

In some embodiments of any of the antibodies described herein, only one (not both) of the two Fc peptides in the antibody is modified to reduce TfR-mediated effector function upon TfR binding. The other Fc peptide does not contain a TfR binding site or any modification that reduces effector function. Such an Fc peptide dimer in the antibody is referred to as having a cis-LALA conformation, in which only one of the two Fc peptides contains a TfR binding site and a modification that reduces FcγR binding upon binding to TfR (e.g., LALA substitution), while the other Fc peptide does not contain a TfR binding site or any modification that reduces FcγR binding.

For example, in some embodiments, the antibody described herein may contain an Fc polypeptide dimer having a cis-LALA conformation, the dimer having (i) a first Fc polypeptide having the sequence SEQ ID NO:86, having a TfR binding site and LALA substitution and β-modification, and (ii) a second Fc polypeptide having at least 90% identity with the sequence SEQ ID NO:85, having only β-modification. In some embodiments, the antibody described herein may contain an Fc polypeptide dimer having a cis-LALA conformation, the dimer having (i) a first Fc polypeptide having the sequence SEQ ID NO:103, having a TfR binding site and LALA substitution and β-modification, and (ii) a second Fc polypeptide having at least 90% identity with the sequence SEQ ID NO:85, having only β-modification.

In a specific implementation, the antibody described herein may contain an Fc polypeptide dimer having a cis-LALA conformation, the dimer having (i) a first Fc polypeptide 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, and a sequence having at least 90% identity with sequence SEQ ID NO: 86, and (ii) a second Fc polypeptide comprising Ser at position 366, Ala at position 368, and Val at position 407, and a sequence having at least 90% identity with sequence SEQ ID NO: 85.

In a specific implementation, the antibody described herein may contain an Fc polypeptide dimer having a cis-LALA conformation, the dimer having (i) a first Fc polypeptide comprising Ser at position 366, Ala at position 368, and Val at position 407 according to EU number, and a sequence having at least 90% identity with sequence SEQ ID NO:85, and (ii) a second Fc polypeptide 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, and a sequence having at least 90% identity with sequence SEQ ID NO:86.

In a specific implementation, the antibody described herein may contain an Fc polypeptide dimer having a cis-LALA conformation, the dimer having (i) a first Fc polypeptide 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, and a sequence having at least 90% identity with sequence SEQ ID NO: 103, and (ii) a second Fc polypeptide comprising Ser at position 366, Ala at position 368, and Val at position 407, and a sequence having at least 90% identity with sequence SEQ ID NO: 85.

In a specific implementation, the antibody described herein may contain an Fc polypeptide dimer having a cis-LALA conformation, the dimer having (i) a first Fc polypeptide comprising Ser at position 366, Ala at position 368, and Val at position 407 according to EU number, and a sequence having at least 90% identity with sequence SEQ ID NO: 85, and (ii) a second Fc polypeptide 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, and a sequence having at least 90% identity with sequence SEQ ID NO: 103.

Fc peptide modification for prolonging serum half-life

In some embodiments, modifications to enhance serum half-life may be introduced into any of the antibodies described herein. For example, in some embodiments, one or both Fc peptides present in the antibodies described herein may contain a tyrosine residue at position 252, a threonine residue at position 254, and a glutamic acid residue at position 256, as designated according to the EU numbering scheme. Thus, one or both Fc peptides may have M252Y, S254T, and T256E substitutions. Alternatively, one or both Fc peptides may have M428L and N434S substitutions, as designated according to the EU numbering scheme. Alternatively, one or both Fc peptides may have N434S or N434A substitutions.

Fc peptides with C-terminal lysine residues removed

In some embodiments of the antibodies described herein, the C-terminal lysine residue of one or both of the Fc peptides may be removed (e.g., the Lys residue at position 447 of the Fc peptide according to EU designation). C-terminal lysine residues are highly conserved across many species of immunoglobulins and can be completely or partially removed by cellular machinery during protein production. In some embodiments, removal of the C-terminal lysine residue from the Fc peptide may improve antibody stability.

V. Antibody Preparation

Many techniques known in the art can be used to prepare the antibodies described herein. In some embodiments, the genes encoding the heavy and light chains of the antibody of interest can be cloned from cells (e.g., from hybridomas). Libraries of genes encoding the heavy and light chains of monoclonal antibodies can also be prepared from hybridomas or plasma cells. Alternatively, phage or yeast display techniques can be used to identify antibodies and Fab fragments that specifically bind to selected antigens.

Many expression systems, including prokaryotic and eukaryotic expression systems, can be used to generate antibodies. 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, a single vector (e.g., in a bicistronic expression unit) or under the control of different promoters can be used to express polynucleotides encoding polypeptides that constitute antibodies. In other embodiments, a separate vector can be used to express polynucleotides encoding polypeptides that constitute antibodies.

In some aspects, this disclosure provides: isolated nucleic acids comprising a nucleic acid sequence encoding any polypeptide constituting an antibody as described herein; a vector comprising such nucleic acids; and a host cell in which said nucleic acids are introduced for replicating said nucleic acids and/or expressing antibodies.

In some embodiments, the polynucleotide (e.g., isolated polynucleotide) comprises a nucleotide sequence encoding a polypeptide constituting an antibody as disclosed herein (e.g., as described in Section III above). In some embodiments, the polynucleotide comprises a nucleotide sequence encoding one or more amino acid sequences (e.g., heavy chain, light chain, and/or Fc polypeptide sequences) disclosed in the informal sequence listing below. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding 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) with respect to a sequence disclosed in the informal sequence listing below. In some embodiments, the polynucleotide as described herein is operatively linked to a heterologous nucleic acid, such as a heterologous promoter.

Suitable vectors containing polynucleotides encoding antibodies or fragments thereof disclosed herein include cloning vectors and expression vectors. While the chosen cloning vector may vary depending on the host cell intended for use, available cloning vectors are generally self-replicating, may have a single target of a specific restriction endonuclease, and/or carry genes that can be used to select clones containing said vectors. 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, Stragene, and Invitrogen.

Expression vectors are typically replicable polynucleotide constructs containing the nucleic acids disclosed herein. Expression vectors can replicate in host cells as episomes or as part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors (including adenoviruses, adeno-associated viruses, and retroviruses), and any other vectors.

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

In another aspect, methods for preparing antibodies as described herein are provided. In some embodiments, the method includes culturing host cells as described herein (e.g., host cells expressing polynucleotides or vectors as described herein) under conditions suitable for expressing the antibody. In some embodiments, the antibody is subsequently recovered from the host cells (or host cell culture medium). In some embodiments, the antibody is purified, for example by chromatography.

VI. Treatment methods

In some aspects, this document provides methods for treating a subject's cancer (e.g., HER2-positive cancer) or brain metastases of such cancer (e.g., HER2-positive cancer) by administering a therapeutically effective amount of the antibody or a pharmaceutical composition thereof described herein to the subject. This document also provides methods for transendothelial endocytosis transport of a variable region of an antibody or an antigen-binding fragment thereof capable of binding to HER2 (e.g., human HER2). In some embodiments, the method includes contacting the endothelium with a composition comprising the antibody described herein. In some embodiments, the endothelium is the blood-brain barrier (BBB).

Non-limiting examples of HER2-positive cancers treatable 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 adenocarcinoma and/or HER2-positive gastroesophageal 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 adenocarcinoma and/or HER2-positive gastroesophageal junction adenocarcinoma. In some embodiments, the HER2-positive cancer is metastatic cancer.

In other aspects, this document provides methods for treating metastases of cancer, such as HER2-positive cancer. In some embodiments, the method includes administering a therapeutically effective amount of the antibody described herein to the subject. In some embodiments, the metastasis is a brain metastasis of the HER2-positive cancer described above. In some embodiments, the metastasis is a brain metastasis of HER2-positive breast cancer. In some embodiments, the metastasis is a brain metastasis of HER2-positive gastric adenocarcinoma and/or HER2-positive gastroesophageal junction adenocarcinoma.

In some implementations, the treatment benefits may include reduced or slowed tumor growth, reduced tumor size (e.g., volume), decreased tumor cell viability, reduced number of metastatic lesions, improvement of one or more signs or symptoms of cancer (e.g., HER2-positive cancer), and/or increased patient survival. In some implementations, the reduction in tumor cell viability, tumor growth, tumor size, and/or number of metastatic lesions is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.

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

The routes of administration of the antibodies described herein may include oral, intraperitoneal, percutaneous, subcutaneous, intravenous, intramuscular, intrathecal, inhalation, local, intralesional, rectal, intrabronchial, nasal, mucosal, intestinal, ocular, or ear delivery, or any other method known in the art. In some embodiments, the antibodies are administered orally, intravenously, or intraperitoneally.

VII. Pharmaceutical compositions and medicine boxes

In other respects, pharmaceutical compositions and kits comprising antibodies according to this disclosure are provided.

Pharmaceutical Composition

Guidelines for preparing formulations used in this disclosure are available in many manuals on drug preparation and formulation known to those skilled in the art.

In some embodiments, the pharmaceutical composition comprises an antibody as described herein, and also comprises one or more pharmaceutically acceptable carriers and/or excipients. Pharmaceutically acceptable carriers include any solvent, dispersion medium, or coating that is physiologically compatible and does not interfere with or otherwise inhibit the activity of the active agent.

In some implementations, antibodies may be formulated for parenteral administration via injection. Typically, pharmaceutical compositions intended for in vivo administration are sterile, for example, through heat sterilization, steam sterilization, sterile filtration, or irradiation.

The dosage and desired drug concentration of the pharmaceutical compositions described herein may vary depending on the intended specific use.

medicine box

In some embodiments, a kit for treating cancer (e.g., HER2-positive cancer) is provided, containing the antibody described herein. In some embodiments, the kit also contains one or more additional therapeutic agents. For example, in some embodiments, the kit contains an antibody as described herein and also contains one or more additional therapeutic agents for treating cancer. In some embodiments, the kit also contains instructional material containing instructions (i.e., protocols) for practicing the methods described herein (e.g., instructions for administering the antibody using the kit). While instructional material typically includes written or printed material, it is not limited thereto. This disclosure contemplates any media capable of storing and transmitting such instructions to the end user. Such media includes, but is not limited to, electronic storage media (e.g., disks, magnetic tapes, cassette tapes, chips), optical media (e.g., CD-ROMs), etc. Such media may include the address of an internet site providing such instructional material.

VIII. Examples

The invention will be described in more detail through specific embodiments. The following embodiments are provided for illustrative purposes only and are not intended to limit the invention in any way.

Example 1. Generation of anti-HER2 bispecific antibodies

Expression and purification of recombinant bispecific antibody variants

Expression plasmids consisting of the following components were co-transfected into Expi293 or ExpiCHO cells: (i) a heavy chain polypeptide containing a TfR binding site and a valence (T366W) mutation, (ii) a heavy chain polypeptide containing a valence (T366S/L368A/Y407V) mutation, and (iii) a light chain according to the combination in Table 2. The recombinant bispecific antibody variant was then purified from conditioned medium by loading the supernatant onto a Protein A column (GE Mab Select SuRe). The column was washed with 10 column volumes of PBS (pH 7.4). The protein was eluted with 50 mM sodium citrate (pH 3.0, containing 150 mM NaCl) and immediately neutralized with 200 mM arginine and 137 mM succinate (pH 5.0). The protein was further purified by size exclusion chromatography (GE Superdex 200) using 200 mM arginine and 137 mM succinate (pH 5.0) as the run buffer. The purified protein was confirmed by full-weight LC/MS, and the purity was confirmed to be >95% by SDS-PAGE and analytical HPLC-SEC.

Heavy chain peptides can be further processed during cell culture production, resulting in the removal of C-terminal lysine residues. Therefore, the bispecific antibodies listed in Table 2 may refer to protein molecules containing unprocessed heavy chains (i.e., containing C-terminal lysine residues); protein molecules containing one or more processed heavy chains (i.e., lacking C-terminal lysine residues); or mixtures of protein molecules having processed heavy chains and/or unprocessed heavy chains.

Example 2. Biacore evaluation of anti-HER2 antibody

The binding affinity of the engineered anti-HER2 antibody to the HER2 extracellular domain (ECD) was measured using SPR on a Biacore 8K instrument. Antibodies were captured on a Biacore ^™ Series S CM5 sensor chip immobilized with mouse anti-human Fab (from GE Healthcare's Human Fab Capture Kit), followed by injection of serially 3-fold dilutions of recombinant HER2 ECDs at a rate of 30 μL/min. Each sample was analyzed using a 3-minute association followed by a 10-minute dissociation. After each injection, the sensor chip was regenerated using 50 mM glycine pH 2.0 regeneration buffer. A 1:1 Languir model, simultaneously fitting k _-association and k _{-dissociation} , was used for kinetic analysis.

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

Mature anti-HER2 light chain sequences (SEQ ID NO: 9 and 10) were paired with anti-HER2_D2 heavy chain controls (SEQ ID NO: 92) and anti-HER2_D4 heavy chain controls (SEQ ID NO: 93) for HER2 binding _KD measurements. The results are shown in Table 7.

Table 7

SEQ ID NO:9 and 10 light chains showed HER2 binding when paired with the anti-HER2_D2 and D4 heavy chain controls. SEQ ID NO:9 and 10 light chains showed lower HER2 binding affinity (13 K _D and 14 K _D , respectively) when paired with the anti-HER2_D2 heavy chain control compared to the anti-HER2_D2 light chain control (2.9 K _D ). In contrast, SEQ ID NO:9 and 10 light chains showed higher HER2 binding affinity (1.5 K _D and 1.7 K _D , respectively) when paired with the anti-HER2_D4 heavy chain control compared to the anti-HER2_D4 light chain control (3 K _D ).

Based on structural analysis, thirteen amino acid positions in the CDR H1, H2, or H3 of the anti-HER2_D2 heavy chain control (SEQ ID NO: 92) were selected. The selected residues were randomized to find single-amino acid substitution variants that improved HER2 ECD domain II binding. Antibodies with these single-point mutations were paired with the anti-HER2_D4 light chain control (SEQ ID NO: 87) and expressed in Expi293 cells. Antibodies in the cell culture supernatant were screened for recombinant HER2 ECD binding using SPR. Variants with improved HER2 ECD domain II binding were selected and expressed in Expi293 cells with the light chain SEQ ID NO: 10, and purified for additional SPR binding assessment.

Affinity-matured anti-HER2_D2 heavy chain sequences containing the _VH regions of SEQ ID NO:1-2 and 60-70 were paired with an anti-HER2_D4 light chain control (SEQ ID NO:87) for HER2 binding _KD measurements. The results are shown in Table 8.

Table 8

The V _H region containing SEQ ID NO:1-2 and 60-70 and the affinity-matured antiHER2_D2 heavy chain sequence paired with the antiHER2_D4 light chain control (SEQ ID NO:87) showed HER2 binding and all showed improved HER2 binding compared with the antiHER2_D2 heavy chain control.

The affinity-matured anti-HER2 light chain sequence (SEQ ID NO:10) was paired with the affinity-matured anti-HER2_D2 heavy chain sequence containing the _VH region of SEQ ID NO:1-3 for HER2 binding _KD measurement. The results are shown in Table 9.

Table 9

The SEQ ID NO:10 light chain, paired with a mature anti-HER2_D2 heavy chain sequence containing the _VH region of SEQ ID NO:1-3, exhibited improved HER2 binding affinity (4.2, 6.2, and 2.1 _KD , respectively). As discussed above and shown in Table 7, the HER2 binding affinity of the control light chain, paired with the anti-HER2_D2 heavy chain control, was 2.9 _KD . Therefore, the SEQ ID NO:10 light chain, paired with a mature anti-HER2_D2 heavy chain sequence containing the _VH region of SEQ ID NO:3, showed a higher HER2 binding affinity than the control.

Example 3. In vitro ADCC/ADCP of anti-HER2 bispecific antibody

Based on the combinations in Table 10, the Human ADCC Reporter Gene Bioassay V variant kit (Promega G7018) was used to assess human FcγRIIIa activation, while the Human FcgRIIa ADCP Reporter Gene Bioassay kit (Promega G9995) was used to measure the activation of the human FcγRIIa reporter gene with a bispecific antibody. The kits contain all components described below. Several cell lines with different HER2 and TfR expression levels were tested. Cells of various types, including SKBR3 (ATCC HTB-30), ZR-75-30 (ATCC CRL-1504), BT-474 (ATCC HTB-20), OE-19 (Sigma 96071721), and CHO-KI+human TfR (ChemPartner CRO protocol), were cultured to the exponential phase in RPMI (Liffe Technologies 61870-036) supplemented with 10% FBS (Hyclone bovine serum SH30080.03) and 1% penicillin-streptomycin (Life Technologies 15140-122). Cells were washed twice with PBS and resuspended at 1.0 x ^10⁶ cells/mL in RPMI supplemented with 10% FBS and 1% penicillin/streptomycin. White 96-well high-binding Nunc plates (ThermoFisher) were coated with 25 μL of medium containing 50,000 cells/well.

Antibody titration was prepared in RPMI containing 4% low IgG serum, and 25 μl was added to each well of the plate to condition the cells. The plate was then capped and incubated at 37°C and 5% _CO2 for 30 min. During antibody conditioning, 3.5 mL of medium was preheated to 37°C, and FcγR reporter cells were rapidly thawed in a 37°C water bath without inversion. The cells were then added to preheated medium in 15 mL conical tubes with gentle mixing. After 30 min conditioning, 25 μl of the FcγR reporter cell line was added to each well of each plate and incubated at 37°C and ₅ % CO2 for 6 h (SKBR3, ZR-75-30, and BT-474 for hFcγRIIIa and hFcγRIIa activation) or 16 h (CHO-KI+huTfR for hFcγRIIIa and hFcγRIIa). After incubation, the plates were allowed to acclimatize to room temperature and 75 μL of Bio-Glo luciferase substrate suspension (Promega) was added to each well. The luminescence was measured on a Perkin Elmer Envision reader. The results are shown in Table 11.

Table 10

Table 11

The goal was to develop Fc variants that, compared to control and/or Fc1, did not increase TfR-mediated ADCC and also exhibited comparable levels of HER2-mediated ADCC 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 exhibited comparable levels of TfR-mediated ADCC in TfR-overexpressing CHO cells to control levels. Fc50 and Fc52 showed the highest levels of HER2-mediated ADCC in all tested HER2-overexpressing cell lines without increasing TfR-mediated ADCC activation.

The ADCP levels of the Fc1, Fc41, Fc5, Fc45, Fc42, Fc52, Fc44 and Fc50 variants compared to controls in HER2-overexpressing cell lines (i.e., OE19, ZR-75-30 and SKBR3) are also shown in Table 11.

Example 4. In vitro growth inhibition of anti-HER2 bispecific antibody

Cell viability was determined using a growth inhibition assay after treatment with different antibodies for different durations. Several cell lines with different HER2 and TfR expression levels 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+human TfR (ChemPartner CRO protocol) were cultured to the exponential 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 resuspended at 1.0 x ^10⁵ cells/mL in RPMI supplemented with 10% FBS and 1% penicillin/streptomycin. 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 at 37°C in a 5% _CO2 incubator for 24 hours.

Antibody titration was prepared in RPMI containing 10% FBS serum and 1% penicillin/streptomycin. 65 μl of antibody was added to each well of each plate, which was then capped and incubated at 37°C and 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, followed by _incubation at 37°C and 5% CO2 for another 72 hours.

On day 7, cell growth was measured using 5 μL of WST-1 reagent (Sigma-Aldrich) in 50 μL of growth medium. Plates were incubated for 4 hours in the presence of WST-1 reagent, and absorbance was measured at 440 nm. The percentage of growth inhibition/proliferation was calculated based on A440 nM and normalized to the untreated control.

The results of growth inhibition assays on ZR-75-30 cells by different antibodies, as well as IC50 and maximum growth inhibition % values, are shown in Table 12 and Figure 2. Each of the bispecific antibodies #2, #3, #4, and #5 showed improved EC50 values/efficacy compared to the control. Bispecific antibodies #4 and #5 showed maximum growth inhibition %/efficacy comparable to the control.

Table 12

Example 5. In vivo xenograft studies using ATV:CLC bispecific antibody

The reaction of ATV:CLC bispecific antibody #1 was evaluated using two human HER2+ cell lines in a subcutaneous xenograft model of immunodeficient (NOD/SCID) mice. All molecules were prepared in the same preparation buffer (10 mM sodium acetate, 6% sucrose, pH 5.5) or PBS/saline, except for trastuzumab (Clinical Herceptin) and pertuzumab (Clinical Perjeta), which were purchased and prepared according to their manufacturer's instructions and/or further diluted with PBS or saline.

Table 13. Anti-HER2 Bispecific Molecules

For the BT-474 breast cancer cell line-derived xenograft (CDX) model, BT-474 cells were subcutaneously injected into the axilla of female NSG (NOD scidγ) mice (6-7 weeks old), and treatment began six days post-inoculation when the tumor volume was between 100-200 ^mm³ . Mice were randomly assigned to treatment groups based on mean tumor volume, with n = 11 mice per group. Treatment consisted of either 40 ± 40 mg/kg of trastuzumab and pertuzumab in combination or 80 mg/kg of ATV:CLC bispecific antibody #1 administered via intraperitoneal (IP) injection. Tumor volume was measured three times weekly using calipers.

In the BT-474 xenograft model, which is relatively sensitive to trastuzumab, the ATV:CLC bispecific antibody #1 showed equivalent tumor growth inhibition compared to trastuzumab and pertuzumab after a single dose, with complete tumor regression in the entire treatment group after 21 days (Figure 3A).

For the OE19 gastroesophageal junction CDX model, OE19 cells were subcutaneously injected into the right upper quadrant of female NOD/SCID mice (6-8 weeks old), and treatment began one week post-inoculation when the tumor volume was between 100-200 ^mm³ . Mice were randomly assigned to treatment groups (n=11 per group) based on a matched distribution/stratification method. Treatment consisted of either a combination of 50 ± 50 mg/kg ATV:trastuzumab and ATV:pertuzumab or 100 mg/kg ATV:CLC bispecific antibody #1 administered via intraperitoneal injection. Tumor volume was measured three times weekly using calipers.

In an OE19 xenograft model exhibiting relative resistance to combination therapy with trastuzumab and pertuzumab, ATV:CLC bispecific antibody #1 and the combination of ATV cis-LALA:trastuzumab and ATV cis-LALA:pertuzumab showed significant tumor growth delay compared to the control group (Figure 3B). All groups started with n=11 mice per group. The values on the graph represent the number of animals remaining in the control group after some animals reached the humanitarian endpoint. One animal died without apparent cause on day 17 in the ATV combination group. ATV:trastuzumab and ATV:pertuzumab are trastuzumab and pertuzumab antibodies containing an Fc modification and a cis-LALA mutation binding to TfR (“TV”). These in vivo results are consistent with in vitro growth inhibition data, which indicate that ATV:HER2 (an anti-HER2 molecule with TV) has increased efficacy and maximal effect in the OE19 cell line compared to anti-HER2 molecules lacking TfR-binding Fc modification.

In subsequent lower-dose studies, to investigate the role of TfR binding, ATV:CLC bispecific antibody #1 was compared to a CLC bispecific antibody control, which had the same Fab as ATV:CLC bispecific antibody #1 but lacked TfR-binding Fc modification. For the BT-474 breast cancer CDX model, female NOD/SCID mice (6–8 weeks old) were implanted with estrogen pellets (0.36 mg, 17β-estradiol, 60-day pellets) one day after tumor inoculation. BT-474 cells were then subcutaneously injected into the mammary fat pads, and treatment began eight days post-inoculation when the tumor volume was between 100–200 ^mm³ . Mice were randomly assigned to treatment groups based on a matched distribution/stratification method, with n = 11 mice in each group. Tumor volume was measured twice weekly using calipers.

A single intraperitoneal dose of 20 mg/kg ATV:CLC bispecific antibody #1 showed similar tumor growth delay in a sensitive BT-474 xenograft model as the CLC bispecific antibody control (without TfR binding) (Figure 4A). ATV:CLC bispecific antibody #1 also showed improved responses at an equivalent dose of 20 mg/kg in a more resistant OE19 xenograft model and equivalent antitumor responses at a quarter dose (5 mg/kg) of the CLC bispecific antibody control (Figure 4B).

In another bridging study, ATV:CLC bispecific antibodies #1 and #2 were compared in a multi-dose xenograft study. In the BT-474 model, mice were administered a single dose weekly for 3 weeks via intraperitoneal injection (IP) to each group (n=11) for Q1W. ATV:CLC bispecific antibodies #1 and #2 showed equivalent tumor growth inhibition and delay (Figure 5A). Additionally, in the same groups, tucatinib was administered orally at 50 mg/kg daily for 21 days, but no further improvement was observed with either ATV:CLC bispecific antibody #1 or #2.

Finally, in the multi-dose OE19 xenograft study, the Fc-engineered variant of ATV:CLC bispecific antibody #2, ATV:CLC bispecific antibody #3 (containing additional Fc modifications, such as P329S, I332E, and S239D), was compared with anti-HER2 molecules lacking TfR binding. Mice were administered a single dose via IP for Q2W, i.e., every 2 weeks for 6 weeks. Compared to the combination of trastuzumab and pertuzumab or the CLC bispecific antibody control, ATV:CLC bispecific antibody #3 showed increased tumor growth delay and increased survival (Figure 5B). All groups started with n = 11 mice per group. The values on the graph represent the number of animals remaining in the group after some animals reached the humanitarian endpoint.

Example 6. Brain uptake and distribution of ATV:CLC bispecific antibody

TfR ^mu/hu KI mice (see, for example, International Publication No. WO 2018/152285) were administered a single IV dose of 25 mg/kg CLC bispecific antibody control or ATV:CLC bispecific antibody #3 (n=4/group). Blood was collected at 30 minutes and 6 hours throughout the lifetime, and peripheral blood and fresh frozen brain were collected at 1, 4, 7 and 10 days after administration to assess huIgG concentrations in plasma and brain lysates via ELISA.

Plasma and brain concentrations were measured in TfR ^mu/hu KI mice following a single dose of either the bispecific CLC antibody control or the ATV:CLC bispecific antibody #3. Brain concentrations of ATV:CLC bispecific antibody #3 were approximately 6.5 times higher than those of the CLC bispecific antibody control at 24 h post-administration (Figure 6). This indicates TfR-mediated brain delivery of the ATV molecule. Similarly, in time-course studies, ATV:CLC bispecific antibodies #2, #3, and #7 showed approximately 4–5 times higher brain concentrations at 24 h post-IV administration and up to approximately 2 times higher brain concentrations at 4 days post-IV administration.

In addition, immunohistochemistry was performed on the molecules administered to the brain. One fresh brain hemisphere from each animal was fixed by immersion at 4°C for approximately 24 hours for immunohistochemistry, followed by cryoprotection in sucrose and sectioning on a cryostat. Coronal brain sections (40 μm) from each animal were selected and stained by incubation for three hours at room temperature in blocking buffer (1% BSA + 1x fish gelatin + 0.5% Triton X-100 + 0.01% sodium azide in PBS). The sections were then incubated overnight at 4°C in dilution buffer (1% BSA + 0.3% Triton X-100 + 0.01% sodium azide in PBS) containing primary/secondary antibodies (NeuN, Abcam, ab177487 and donkey anti-hu IgG, Jackson, 709-606-149), washed three times for 15 minutes in PBS with 0.3% Triton X-100, and incubated for three hours in dilution buffer containing secondary antibodies (donkey anti-rabbit, Invitrogen, A21206) and DAPI (5 μg/mL, Invitrogen, D1306), washed three times for 15 minutes in PBS with 0.3% Triton X-100, fixed, and covered with a coverslip using a Prolong glass (Invitrogen, P36984). The slides were imaged using a Leica SP8 confocal microscope at 20X magnification and segmented and visualized using Imaris.

Immunohistochemistry of the huIgG backbone of the drug-administered molecule revealed widespread distribution of ATV:CLC bispecific antibody #3 in the normal brain, localizing to diffuse signals within blood vessels and NeuN+ neurons along with the parenchyma (Fig. 7A). In contrast, the CLC bispecific antibody control showed limited entry or distribution in brain tissue (Fig. 7B). This is consistent with the significantly lower brain concentrations observed for non-TV anti-HER2 molecules (i.e., molecules that do not bind to TfR). Similar results (i.e., vascular and neuronal/parenchymal localization) were observed using ATV:CLC bispecific antibodies #2 and #7.

Example 7. Plasma pharmacokinetic analysis of ATV:CLC bispecific antibodies in cynomolgus monkeys.

To assess the effect of Fc modification on systemic clearance, Fc-modified variants were compared. Since TV35.23.4 (i.e., CH3C.35.23.4) lacks cynomolgus cross-reactivity (i.e., it does not bind to cynomolgus TfR), a bispecific HER2 ATV with TV35.21 (see CH3C.35.21 in Table A above) was used instead of TV35.23.4. As shown in Table 14 below, these molecules used the same Fab as the ATV:CLC bispecific antibodies #2, #3, and #7 used in the aforementioned mouse studies.

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

Table 14. Anti-HER2 bispecific molecules used in cynomolgus monkey studies

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

Example 8. In vitro ADCC/ADCP of anti-HER2 bispecific antibody in NK cells.

Cell-based antibody-dependent cytotoxicity (ADCC) assays are used to assess whether differences in the binding affinity of the Fcγ receptor to different Fc mutants affect HER2-mediated tumor cell or TfR-mediated cell killing using isolated human NK cells.

NK cells were isolated from whole blood and used to assess human FcγRIIIa activation. Blood was collected on a Trizma. Cells were isolated according to the RosetteSep human NK cell enrichment protocol (Stemcell 15065). The RosetteSep mixture was added to the blood sample in SepMate tubes and incubated at room temperature for 15 min. 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 Lymphoprep density gradient medium (Stemcell 07801) and centrifuged for 10 min. The enriched cells were then collected and washed twice with PBS. Finally, 20 ng/ml IL-21 was added to the cells and then incubated overnight for use the next day.

Cell lines with different HER2 and TfR expression levels were tested. SKBR3 (ATCC HTB-30) and CHO-KI+human TfR (ChemPartner CRO protocol) cells were cultured to the exponential 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 resuspended at 1.0 x ^10⁶ cells/mL in RPMI supplemented with 10% FBS and 1% penicillin/streptomycin.

Untreated 96-well clear V-shaped plates (Costar 3897) were coated with 25 μL of medium containing 50,000 cells/well. Antibody titration was prepared in RPMI with 10% FBS serum and 25 μL was added to each well to condition the cells. The plates were then capped and incubated at 37°C and 5% _CO2 for 30 min. During antibody conditioning, 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 for cell density. After 30 min conditioning, NK cells were added to each well at 25 μL and incubated for 4 h. After incubation, the plates were allowed to acclimatize to room temperature and rotated at 300 x g for 5 min. 50 μL of the supernatant was transferred to a white 96-well clear plate (Thermo 165306). 50 μL of CytoTox assay reagent was added to each well of the plate containing the supernatant. The plate was capped to protect it from light and incubated at room temperature for 30 minutes. Stop solution was added, and the absorbance signal was measured at 490 nm using a plate reader. The release of LDH in the culture supernatant was measured using a 30-minute coupled enzymatic assay that resulted in the conversion of tetrazolium salt (iodonitrotetrazole violet; INT) to a red formazan product. The amount of color formed was proportional to the number of lysed cells.

Interestingly, in this assay performed on HER2-expressing tumor cells, cis-LALA modification only caused a slight rightward shift in the curve indicating a slight decrease in efficacy, but the maximum cytotoxic effect was observed to be equal to that of non-TV anti-HER2 bispecific antibodies (i.e., CLC bispecific antibody control #2 and trastuzumab) (Figure 9).

No cell killing was observed in TfR-expressing cells with cis-LALA or additional Fc mutations, suggesting that these molecules do not adversely affect TfR-expressing cells.

ADCP reporter gene assays measuring FcgRIIA activation also demonstrated that ATV:CLC bispecific #2 and Fc variants (ATV:CLC bispecific #3 and #7) showed similar receptor activation to each other, greater than trastuzumab and slightly less than the CLC bispecific antibody control #2.

Example 9. FcgR binding assay of ATV:CLC bispecific antibody

The Fcγ receptor binding affinity of engineered anti-HER2 antibodies was measured using a Biacore 8K instrument via SPR (spatial reactivity ratio). Biotinylated recombinant Fcγ receptors were captured on a Biacore ^™ Series SA sensor chip, followed by injection of sequentially 3-fold dilutions of Fc-engineered anti-HER2 antibodies at a rate of 30 μL/min. Each sample was analyzed using five 60-second injections with increasing antibody concentrations, followed by 5-minute dissociation. A 1:1 Languir model simultaneously fitting k-association and k-dissociation was used for kinetic analysis.

Table 15. Fcγ receptor binding affinity of Fc-engineered anti-Her2 antibodies

Increased FcgR affinity was observed in the Fc variants engineered with S239D and I332E (i.e., ATV:CLC bispecific antibodies #5 and #6) compared to ATV:CLC bispecific antibodies #4 and trastuzumab. However, along with the results from the ADCC assay described above, this increase in affinity may only apply when the antibody binds to the Fab target (i.e., HER2).

IX. Exemplary Implementation

Exemplary embodiments provided based on the currently disclosed subject matter include, but are not limited to, the claims and the following embodiments:

1. An isolated antibody comprising one or more complementarity-determining regions (CDRs) selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:89;

(b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:90; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:91,

At least one of the following:

_X1 in SEQ ID NO:89 is not T;

_X2 in SEQ ID NO:89 is not F;

_X3 in SEQ ID NO:89 is not T;

In SEQ ID NO:90, _X1 is not N;

In SEQ ID NO:90, _X2 is not N;

_X3 in SEQ ID NO:90 is not S;

X ₄ in SEQ ID NO:90 is not G;

X ₅ in SEQ ID NO:90 is not G;

X ₆ in SEQ ID NO:90 is not Q;

In SEQ ID NO:91, _X1 is not L;

X ₂ in SEQ ID NO:91 is not G;

_X3 in SEQ ID NO:91 is not P; and

_X4 in SEQ ID NO:91 is not S.

2. The isolated antibody as described in Embodiment 1, wherein the heavy chain CDR1 comprises the amino acid sequence SEQ ID NO:89, wherein _X1 is N, K, M or H.

3. The isolated antibody as described in embodiment 1, wherein the heavy chain CDR2 comprises the amino acid sequence SEQ ID NO:90, wherein X ₅ is Q.

4. The isolated antibody as described in Embodiment 1, wherein the heavy chain CDR2 comprises the amino acid sequence SEQ ID NO:90, wherein _X6 is R, H or T.

5. The isolated antibody as described in Embodiment 1, wherein the heavy chain CDR3 comprises the amino acid sequence SEQ ID NO:91, wherein _X4 is W, F, D, L or Y.

6. The isolated antibody as described in Embodiment 1, wherein the heavy chain CDR3 comprises the amino acid sequence SEQ ID NO:91, wherein X ₄ is L.

7. The isolated antibody as described in Embodiment 1, comprising one or more CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:89;

(b) A heavy chain CDR2 comprising the amino acid sequence SEQ ID NO:90, wherein _X5 is Q; and

(c) A heavy chain CDR3 containing the amino acid sequence SEQ ID NO:91, wherein X ₄ is L.

8. The isolated antibody as described in Embodiment 1, comprising one or more CDRs selected from the group consisting of:

(a) A heavy chain CDR1 having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:4 and 49-52 or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO:4 and 49-52;

(b) A heavy chain CDR2 having at least 90% sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 5-6 and 53-55, or having at most two amino acid substitutions relative to the amino acid sequence selected from the group consisting of SEQ ID NO: 5-6 and 53-55; and

(c) A heavy chain CDR3 having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 7-8 and 56-59 or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 7-8 and 56-59.

9. The isolated antibody as described in embodiment 8, comprising one or more CDRs selected from the group consisting of:

(a) A heavy chain CDR1 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:4 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:4;

(b) A heavy chain CDR2 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:5 or SEQ ID NO:6, or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:5 or SEQ ID NO:6; and

(c) A heavy chain CDR3 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8.

10. The isolated antibody as described in embodiment 9, comprising one or more CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4;

(b) Heavy chain CDR2 containing the amino acid sequence SEQ ID NO:5 or SEQ ID NO:6; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8.

11. The isolated antibody as described in embodiment 10, comprising one or more CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4;

(b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:6; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:7.

12. The isolated antibody as described in embodiment 10, comprising one or more CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4;

(b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:5; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:8.

13. The isolated antibody as described in embodiment 10, comprising one or more CDRs selected from the group consisting of:

(a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4;

(b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:6; and

(c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:8.

14. The isolated antibody as described in embodiment 8, comprising a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 1-3.

15. The isolated antibody as described in embodiment 8, comprising a heavy chain variable region containing an amino acid sequence comprising any one of SEQ ID NO: 1-3.

16. An isolated antibody comprising:

(a) A light chain CDR3 containing the amino acid sequence SEQ ID NO:13 or 14.

17. The isolated antibody as described in embodiment 16, further comprising one or more CDRs selected from the group consisting of:

(b) A light chain CDR1 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:11 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:11; and

(c) A light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:12.

18. The isolated antibody as described in embodiment 16, further comprising one or more CDRs selected from the group consisting of:

(b) The light chain CDR1 containing the amino acid sequence SEQ ID NO:11; and

(c) The light chain CDR2 containing the amino acid sequence SEQ ID NO:12.

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

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

21. The isolated antibody as described in embodiment 17, comprising a light chain variable region containing an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 9-10.

22. The isolated antibody as described in embodiment 17, comprising a light chain variable region comprising an amino acid sequence including any one of SEQ ID NO: 9-10.

23. An isolated antibody comprising an antigen-binding site including:

(a) A heavy chain CDR1 having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:4 and 49-52 or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO:4 and 49-52;

(b) A heavy chain CDR2 having at least 90% sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 5-6 and 53-55, or having at most two amino acid substitutions relative to the amino acid sequence selected from the group consisting of SEQ ID NO: 5-6 and 53-55; and

(c) A heavy chain CDR3 having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 7-8 and 56-59 or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 7-8 and 56-59;

(d) A light chain CDR1 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:11 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:11;

(e) A light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:12; and

(f) A light chain CDR3 containing the amino acid sequence SEQ ID NO:13 or 14.

24. The isolated antibody as described in embodiment 23, wherein the antigen binding site comprises:

(a) A heavy chain CDR1 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:4 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:4;

(b) A heavy chain CDR2 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:5 or SEQ ID NO:6 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:5 or SEQ ID NO:6;

(c) A heavy chain CDR3 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8;

(d) A light chain CDR1 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:11 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:11;

(e) A light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:12; and

(f) A light chain CDR3 containing the amino acid sequence SEQ ID NO:13 or 14.

25. The isolated antibody as described in embodiment 24, wherein the antigen binding site comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 1-3 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 9-10.

26. The isolated antibody as described in embodiment 24, wherein the antigen binding site comprises a heavy chain variable region comprising an amino acid sequence comprising any one of SEQ ID NO: 1-3 and a light chain variable region comprising an amino acid sequence comprising any one of SEQ ID NO: 9-10.

27. The isolated antibody as described in any one of embodiments 23 to 26 further comprises a second antigen-binding site comprising one or more CDRs selected from the group consisting of:

(a) A heavy chain CDR1 containing the amino acid sequence SEQ ID NO:16 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:16;

(b) A heavy chain CDR2 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:17 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:17; and

(c) A heavy chain CDR3 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:18 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:18.

28. The isolated antibody as described in 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 with SEQ ID NO:15.

29. The isolated antibody as described in embodiment 27 or 28, wherein 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% sequence identity with the amino acid sequence SEQ ID NO:11 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:11;

(b) A light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:12; and

(c) A light chain CDR3 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:13 or 14.

30. The isolated antibody as described in 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 with any one of SEQ ID NO: 9-10.

31. The isolated antibody as described in 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.

32. The isolated antibody as described in embodiment 31, comprising heavy chain and light chain CDRs selected from combinations listed in Table 1.

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

34. An isolated antibody comprising:

(a) The first antigen-binding site of subdomain IV of human epidermal growth factor receptor 2 (HER2);

(b) The second antigen-binding site of human HER2 subdomain II; and

(c) A modified Fc polypeptide dimer comprising a modified first Fc polypeptide containing a TfR binding site.

The light chain polypeptide sequence in the first antigen binding site is the same as the light chain polypeptide sequence in the second antigen binding site.

35. The isolated antibody as described in embodiment 34, wherein the first Fc polypeptide comprises a modified CH3 domain including the TfR binding site.

36. The isolated antibody as described in embodiment 35, wherein the modified CH3 domain is derived from the CH3 domain of human IgG1, IgG2, IgG3 or IgG4.

37. The isolated antibody as described in embodiment 35 or 36, wherein, according to EU numbering, the modified CH3 domain comprises one, two, three, four, five, six, seven, eight, nine, ten, or eleven substitutions at a set of amino acid positions comprising 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421.

38. The isolated antibody as described in any one of embodiments 35 to 37, wherein, according to the EU designation, 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; and an aliphatic amino acid, Gln, or Gln at position 389. y, Ser, Thr or Asn; Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg or Thr at position 390; 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 acidic amino acid at position 416; and/or aromatic amino acid, His or Lys at position 421.

39. The isolated antibody as described in any one of embodiments 34 to 38, wherein the first Fc polypeptide, which is modified to generate the TfR binding site, binds to the top domain of the TfR.

40. The isolated antibody as described in any one of embodiments 34 to 39, wherein the first Fc polypeptide and the second Fc polypeptide each contain a modification that promotes heterodimerization.

41. The isolated antibody as described in embodiment 40, wherein, according to the EU designation, the first Fc polypeptide comprises a T366W substitution, and the second Fc polypeptide comprises T366S, L368A, and Y407V substitutions.

42. The isolated antibody as described in embodiment 40, wherein, according to EU designation, the first Fc polypeptide comprises T366S, L368A and Y407V substitutions, and the second Fc polypeptide comprises T366W substitution.

43. The isolated antibody as described in any one of embodiments 34 to 42, wherein the first Fc polypeptide and/or the second Fc polypeptide independently comprise modifications that reduce TfR-mediated effector function.

44. The isolated antibody as described in embodiment 43, wherein, according to EU designation, the modification that reduces effector function is a substitution of L234A and L235A.

45. The isolated antibody as described in embodiment 44, wherein the first Fc polypeptide specifically binds to TfR and comprises L234A and L235A substitutions.

46. The isolated antibody as described in embodiment 45, wherein, according to the EU designation, the first Fc polypeptide further comprises a P329G or P329S substitution.

47. The isolated antibody as described in embodiment 46, wherein, according to the EU designation, the second Fc polypeptide contains Leu at positions 234 and 235 and proline at position 329.

48. The isolated antibody as described in embodiment 44, wherein the second Fc polypeptide specifically binds to TfR and comprises L234A and L235A substitutions.

49. The isolated antibody as described in embodiment 48, wherein, according to the EU designation, the second Fc polypeptide further comprises a P329G or P329S substitution.

50. The isolated antibody as described in embodiment 49, wherein, according to the EU designation, the first Fc polypeptide contains Leu at positions 234 and 235 and proline at position 329.

51. The isolated antibody as described in any one of embodiments 34 to 50, wherein the hinge region or a portion thereof is connected to the N-terminus of the first Fc polypeptide and/or the second Fc polypeptide.

52. The isolated antibody as described in any one of embodiments 34 to 51, wherein the first Fc polypeptide and/or the second Fc polypeptide independently comprises a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 71-86 and 98-100.

53. The isolated antibody as described in embodiment 52, wherein the first Fc polypeptide or the second Fc polypeptide comprises a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 71-73, 85 and 99-100.

54. The isolated antibody as described in embodiment 52, wherein the first Fc polypeptide or the second Fc polypeptide comprises a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 74-84, 86 and 98.

55. The isolated antibody as described in embodiment 34, wherein:

The first antigen binding site contains the amino acid sequence SEQ ID NO:15;

The second antigen binding site comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3 and 60-70;

The first Fc polypeptide containing the modification that generates the TfR binding site 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 the amino acid sequence SEQ ID NO:9 or SEQ ID NO:10.

56. The isolated antibody as described in embodiment 55 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.

57. The isolated antibody as described in any one of embodiments 34 to 56, wherein, according to the EU designation, the first Fc polypeptide and/or the second Fc polypeptide independently comprises S239D and/or I332E substitutions.

58. The isolated antibody as described in embodiment 57, wherein the first Fc polypeptide and/or the second Fc polypeptide, which independently comprises the S239D-substituted and/or the I332E-substituted, can enhance HER2-mediated effector function.

59. The isolated antibody as described in embodiment 57 or 58, wherein:

(a) According to the EU designation, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains an S239D substitution;

(b) According to the EU designation, the first Fc polypeptide contains an I332E substitution and the second Fc polypeptide contains an S239D substitution;

(c) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions and the second Fc polypeptide contains S239D substitution;

(d) According to the EU designation, the second Fc polypeptide contains an S239D substitution;

(e) According to the EU designation, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains an I332E substitution;

(f) According to the EU designation, the first Fc polypeptide contains an I332E substitution and the second Fc polypeptide contains an I332E substitution;

(g) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions and the second Fc polypeptide contains I332E substitutions;

(h) According to the EU designation, the second Fc polypeptide contains an I332E substitution;

(i) According to the EU designation, the first Fc polypeptide contains S239D substitution and the second Fc polypeptide contains S239D and I332E substitution;

(j) According to the EU designation, the first Fc polypeptide contains I332E substitution and the second Fc polypeptide contains S239D and I332E substitution;

(k) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions and the second Fc polypeptide contains S239D and I332E substitutions;

(l) According to the EU designation, the second Fc polypeptide contains S239D and I332E substitutions;

(m) According to the EU designation, the first Fc polypeptide contains an S239D substitution;

(n) According to the EU designation, the first Fc polypeptide contains an I332E substitution; or

(o) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions.

60. The isolated antibody as described in embodiment 59, wherein:

(a) According to the EU designation, the first Fc polypeptide contains an I332E substitution and the second Fc polypeptide contains an S239D substitution;

(b) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions and the second Fc polypeptide contains S239D substitution;

(c) According to the EU designation, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains an I332E substitution;

(d) According to the EU designation, the second Fc polypeptide contains an I332E substitution;

(e) According to the EU designation, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains both S239D and I332E substitutions; or

(f) According to the EU designation, the first Fc polypeptide contains an I332E substitution.

61. The isolated antibody as described in embodiment 60, wherein:

(a) According to the EU designation, the first Fc polypeptide contains an I332E substitution and a serine at position 239, and the second Fc polypeptide contains an S239D substitution and an isoleucine at position 332.

(b) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions, and the second Fc polypeptide contains S239D substitution and isoleucine at position 332;

(c) According to the EU designation, the first Fc polypeptide contains an S239D substitution and an isoleucine at position 332, and the second Fc polypeptide contains an I332E substitution and a serine at position 239.

(d) According to the EU designation, the first Fc polypeptide contains a serine at position 239 and an isoleucine at position 332, and the second Fc polypeptide contains an I332E substitution and a serine at position 239.

(e) According to the EU designation, 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; or

(f) According to the EU designation, the first Fc polypeptide contains an I332E substitution and a serine at position 239, and the second Fc polypeptide contains a serine at position 239 and an isoleucine at position 332.

62. The isolated antibody as described in any one of embodiments 34 to 61, comprising two heavy chains and two light chains.

63. The isolated antibody as described in embodiment 62, comprising heavy and light chains selected from combinations listed in Table 2.

64. The isolated antibody as described in embodiment 62, wherein the first heavy chain comprises _VH and Fc sequences selected from combinations in Table 3 and the second heavy chain comprises _VH and Fc sequences selected from combinations in Table 4.

65. The isolated antibody as described in embodiment 62, wherein the first heavy chain comprises _VH and Fc sequences selected from combinations in Table 5 and the second heavy chain comprises _VH and Fc sequences selected from combinations in Table 6.

66. A pharmaceutical composition comprising an isolated antibody as described in any one of embodiments 1 to 65 and a pharmaceutically acceptable carrier.

67. An isolated polynucleotide comprising a nucleotide sequence encoding an isolated antibody as described in any one of embodiments 1 to 65.

68. A vector comprising the polynucleotide as described in embodiment 67.

69. A host cell comprising a polynucleotide as described in embodiment 67 or a vector as described in embodiment 68.

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

71. The method of embodiment 70, wherein the isolated antibody is administered in combination with chemotherapy or radiotherapy.

72. The method as described in embodiment 70 or 71, wherein the cancer is a metastatic cancer.

73. The method as described in any one of embodiments 70 to 72, wherein the cancer is breast cancer.

74. The method as described in any one of embodiments 70 to 73, wherein the cancer is HER2-positive cancer.

It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and various modifications or variations will be proposed by those skilled in the art based thereon, and such modifications or variations should be included within the spirit and scope of this application and the appended claims. The sequences of serial accession numbers referenced herein are hereby incorporated by reference.

Table 1. CDR Combinations

Table 2. Combinations of heavy chains (HC) and light chains (LC)

Table 3. Combinations of HC_D2 V _H and Fc (mortar)

Table 4. Combinations of HC_D4 V _H and Fc (pestle)

combination# <![CDATA[V_H_D4 SEQ ID NO]]> Fc SEQ ID NO A 15 86 B 15 74 C 15 75 D 15 76 E 15 77 F 15 78 G 15 79 H 15 80 I 15 81 J 15 82 K 15 83 L 15 84

Table 5. Combinations of HC_D2 V _H and Fc (pestle)

Table 6. Combinations of HC_D4 V _H and Fc (mortar)

combination# <![CDATA[V_H_D4 SEQ ID NO]]> Fc SEQ ID NO A 15 71 B 15 72 C 15 73 D 15 85

Informal sequence list

Claims (74)

1. An isolated antibody comprising one or more complementarity-determining regions (CDRs) selected from the group consisting of: (a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:89; (b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:90; and (c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:91, At least one of the following: _X1 in SEQ ID NO:89 is not T; _X2 in SEQ ID NO:89 is not F; _X3 in SEQ ID NO:89 is not T; In SEQ ID NO:90, _X1 is not N; In SEQ ID NO:90, _X2 is not N; _X3 in SEQ ID NO:90 is not S; X ₄ in SEQ ID NO:90 is not G; X ₅ in SEQ ID NO:90 is not G; X ₆ in SEQ ID NO:90 is not Q; In SEQ ID NO:91, _X1 is not L; X ₂ in SEQ ID NO:91 is not G; _X3 in SEQ ID NO:91 is not P; and _X4 in SEQ ID NO:91 is not S. 2. The isolated antibody according to claim 1, wherein the heavy chain CDR1 comprises the amino acid sequence SEQ ID NO:89, wherein _X1 is N, K, M or H. 3. The isolated antibody according to claim 1, wherein the heavy chain CDR2 comprises the amino acid sequence SEQ ID NO:90, wherein X ₅ is Q. 4. The isolated antibody according to claim 1, wherein the heavy chain CDR2 comprises the amino acid sequence SEQ ID NO:90, wherein _X6 is R, H or T. 5. The isolated antibody according to claim 1, wherein the heavy chain CDR3 comprises the amino acid sequence SEQ ID NO:91, wherein _X4 is W, F, D, L or Y. 6. The isolated antibody of claim 1, wherein the heavy chain CDR3 comprises the amino acid sequence SEQ ID NO:91, wherein X ₄ is L. 7. The isolated antibody as claimed in claim 1, comprising one or more CDRs selected from the group consisting of: (a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:89; (b) A heavy chain CDR2 comprising the amino acid sequence SEQ ID NO:90, wherein _X5 is Q; and (c) A heavy chain CDR3 containing the amino acid sequence SEQ ID NO:91, wherein X ₄ is L. 8. The isolated antibody as claimed in claim 1, comprising one or more CDRs selected from the group consisting of: (a) A heavy chain CDR1 having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:4 and 49-52 or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO:4 and 49-52; (b) A heavy chain CDR2 having at least 90% sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 5-6 and 53-55, or having at most two amino acid substitutions relative to the amino acid sequence selected from the group consisting of SEQ ID NO: 5-6 and 53-55; and (c) A heavy chain CDR3 having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 7-8 and 56-59 or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 7-8 and 56-59. 9. The isolated antibody of claim 8, comprising one or more CDRs selected from the group consisting of: (a) A heavy chain CDR1 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:4 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:4; (b) A heavy chain CDR2 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:5 or SEQ ID NO:6, or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:5 or SEQ ID NO:6; and (c) A heavy chain CDR3 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8. 10. The isolated antibody of claim 9, comprising one or more CDRs selected from the group consisting of: (a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4; (b) Heavy chain CDR2 containing the amino acid sequence SEQ ID NO:5 or SEQ ID NO:6; and (c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8. 11. The isolated antibody of claim 10, comprising one or more CDRs selected from the group consisting of: (a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4; (b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:6; and (c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:7. 12. The isolated antibody of claim 10, comprising one or more CDRs selected from the group consisting of: (a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4; (b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:5; and (c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:8. 13. The isolated antibody of claim 10, comprising one or more CDRs selected from the group consisting of: (a) Heavy chain CDR1 containing the amino acid sequence SEQ ID NO:4; (b) The heavy chain CDR2 containing the amino acid sequence SEQ ID NO:6; and (c) Heavy chain CDR3 containing the amino acid sequence SEQ ID NO:8. 14. The isolated antibody of claim 8, comprising a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 1-3. 15. The isolated antibody as claimed in claim 8, comprising a heavy chain variable region comprising an amino acid sequence comprising any one of SEQ ID NO: 1-3. 16. An isolated antibody comprising: (a) A light chain CDR3 containing the amino acid sequence SEQ ID NO:13 or 14. 17. The isolated antibody of claim 16, further comprising one or more CDRs selected from the group consisting of: (b) A light chain CDR1 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:11 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:11; and (c) A light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:12. 18. The isolated antibody of claim 16, further comprising one or more CDRs selected from the group consisting of: (b) The light chain CDR1 containing the amino acid sequence SEQ ID NO:11; and (c) The light chain CDR2 containing the amino acid sequence SEQ ID NO:12. 19. The isolated antibody of claim 16, wherein the light chain CDR3 comprises the amino acid sequence SEQ ID NO:13. 20. The isolated antibody of claim 16, wherein the light chain CDR3 comprises the amino acid sequence SEQ ID NO:14. 21. The isolated antibody of claim 17, comprising a light chain variable region comprising an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 9-10. 22. The isolated antibody of claim 17, comprising a light chain variable region comprising an amino acid sequence comprising any one of SEQ ID NO: 9-10. 23. An isolated antibody comprising an antigen-binding site including: (a) A heavy chain CDR1 having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:4 and 49-52 or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO:4 and 49-52; (b) A heavy chain CDR2 having at least 90% sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 5-6 and 53-55, or having at most two amino acid substitutions relative to the amino acid sequence selected from the group consisting of SEQ ID NO: 5-6 and 53-55; and (c) A heavy chain CDR3 having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 7-8 and 56-59 or having at most two amino acid substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 7-8 and 56-59; (d) A light chain CDR1 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:11 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:11; (e) A light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:12; and (f) A light chain CDR3 containing the amino acid sequence SEQ ID NO:13 or 14. 24. The isolated antibody of claim 23, wherein the antigen binding site comprises: (a) A heavy chain CDR1 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:4 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:4; (b) A heavy chain CDR2 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:5 or SEQ ID NO:6 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:5 or SEQ ID NO:6; (c) A heavy chain CDR3 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:7 or SEQ ID NO:8; (d) A light chain CDR1 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:11 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:11; (e) A light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:12; and (f) A light chain CDR3 containing the amino acid sequence SEQ ID NO:13 or 14. 25. The isolated antibody of claim 24, wherein the antigen binding site comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 1-3 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 9-10. 26. The isolated antibody of claim 24, wherein the antigen binding site comprises a heavy chain variable region comprising an amino acid sequence comprising any one of SEQ ID NO: 1-3 and a light chain variable region comprising an amino acid sequence comprising any one of SEQ ID NO: 9-10. 27. The isolated antibody of claim 23, further comprising a second antigen-binding site comprising one or more CDRs selected from the group consisting of: (a) A heavy chain CDR1 containing the amino acid sequence SEQ ID NO:16 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:16; (b) A heavy chain CDR2 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:17 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:17; and (c) A heavy chain CDR3 having at least 90% sequence identity with the amino acid sequence SEQ ID NO:18 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:18. 28. The isolated antibody of claim 27, wherein the second antigen binding site comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO:15. 29. The isolated antibody of claim 27, wherein 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% sequence identity with the amino acid sequence SEQ ID NO:11 or having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:11; (b) A light chain CDR2 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:12; and (c) A light chain CDR3 having at most two amino acid substitutions relative to the amino acid sequence SEQ ID NO:13 or 14. 30. The isolated antibody of claim 29, wherein the second antigen binding site comprises a light chain variable region comprising an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 9-10. 31. The isolated antibody of claim 29, wherein the first antigen binding site and the second antigen binding site comprise the same light chain CDR1, CDR2 and CDR3 sequences. 32. The isolated antibody of claim 31, comprising heavy chain and light chain CDRs selected from the combinations listed in Table 1. 33. An isolated antibody comprising a heavy chain and a light chain selected from combinations listed in Table 2. 34. An isolated antibody comprising: (a) The first antigen-binding site of subdomain IV of human epidermal growth factor receptor 2 (HER2); (b) The second antigen-binding site of human HER2 subdomain II; and (c) A modified Fc polypeptide dimer comprising a modified first Fc polypeptide containing a TfR binding site. The light chain polypeptide sequence in the first antigen binding site is the same as the light chain polypeptide sequence in the second antigen binding site. 35. The isolated antibody of claim 34, wherein the first Fc polypeptide comprises a modified CH3 domain including the TfR binding site. 36. The isolated antibody of claim 35, wherein the modified CH3 domain is derived from the CH3 domain of human IgG1, IgG2, IgG3 or IgG4. 37. The isolated antibody of claim 35, wherein, according to EU designation, the modified CH3 domain comprises one, two, three, four, five, six, seven, eight, nine, ten, or eleven substitutions at positions of one of the following amino acids: 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421. 38. The isolated antibody of claim 35, wherein, according to EU designation, 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; and an aliphatic amino acid, Gly, or Se at position 389. r, Thr, or Asn; Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr at position 390; acidic amino acids, Ala, Ser, Leu, Thr, Pro, Ile, or His at position 413; Glu, Ser, Asp, Gly, Thr, Pro, Gln, or Arg at position 415; Thr, Arg, Asn, or acidic amino acids at position 416; and/or aromatic amino acids, His, or Lys at position 421. 39. The isolated antibody of claim 34, wherein the first Fc polypeptide containing the modification that generates the TfR binding site binds to the top domain of the TfR. 40. The isolated antibody of claim 34, wherein the first Fc polypeptide and the second Fc polypeptide each contain a modification that promotes heterodimerization. 41. The isolated antibody of claim 40, wherein, according to the EU designation, the first Fc polypeptide comprises a T366W substitution, and the second Fc polypeptide comprises T366S, L368A, and Y407V substitutions. 42. The isolated antibody of claim 40, wherein, according to the EU designation, the first Fc polypeptide comprises T366S, L368A and Y407V substitutions, and the second Fc polypeptide comprises T366W substitution. 43. The isolated antibody of claim 34, wherein the first Fc polypeptide and/or the second Fc polypeptide independently comprise modifications that reduce TfR-mediated effector function. 44. The isolated antibody as claimed in claim 43, wherein, according to EU designation, the modification reducing effector function is L234A and L235A substitution. 45. The isolated antibody of claim 44, wherein the first Fc polypeptide specifically binds to TfR and comprises L234A and L235A substitutions. 46. The isolated antibody of claim 45, wherein, according to the EU designation, the first Fc polypeptide further comprises a P329G or P329S substitution. 47. The isolated antibody of claim 46, wherein, according to the EU designation, the second Fc polypeptide contains Leu at positions 234 and 235 and proline at position 329. 48. The isolated antibody of claim 44, wherein the second Fc polypeptide specifically binds to TfR and comprises L234A and L235A substitutions. 49. The isolated antibody of claim 48, wherein, according to the EU designation, the second Fc polypeptide further comprises a P329G or P329S substitution. 50. The isolated antibody of claim 49, wherein, according to the EU designation, the first Fc polypeptide contains Leu at positions 234 and 235 and proline at position 329. 51. The isolated antibody of claim 34, wherein the hinge region or a portion thereof is connected to the N-terminus of the first Fc polypeptide and/or the second Fc polypeptide. 52. The isolated antibody of claim 34, wherein the first Fc polypeptide and/or the second Fc polypeptide independently comprises a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 71-86 and 98-100. 53. The isolated antibody of claim 52, wherein the first Fc polypeptide or the second Fc polypeptide comprises a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 71-73, 85 and 99-100. 54. The isolated antibody of claim 52, wherein the first Fc polypeptide or the second Fc polypeptide comprises a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 74-84, 86 and 98. 55. The isolated antibody as described in claim 34, wherein: The first antigen binding site contains the amino acid sequence SEQ ID NO:15; The second antigen binding site comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3 and 60-70; The first Fc polypeptide containing the modification that generates the TfR binding site 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 the amino acid sequence SEQ ID NO:9 or SEQ ID NO:10. 56. The isolated antibody of claim 55, further comprising a second Fc polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 71-73, 85 and 99-100. 57. The isolated antibody of claim 34, wherein, according to the EU designation, the first Fc polypeptide and/or the second Fc polypeptide independently comprise S239D and/or I332E substitutions. 58. The isolated antibody of claim 57, wherein the first Fc polypeptide and/or the second Fc polypeptide, which independently comprises the S239D-substituted and/or the I332E-substituted, can enhance HER2-mediated effector function. 59. The isolated antibody as described in claim 57, wherein: (a) According to the EU designation, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains an S239D substitution; (b) According to the EU designation, the first Fc polypeptide contains an I332E substitution and the second Fc polypeptide contains an S239D substitution; (c) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions and the second Fc polypeptide contains S239D substitution; (d) According to the EU designation, the second Fc polypeptide contains an S239D substitution; (e) According to the EU designation, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains an I332E substitution; (f) According to the EU designation, the first Fc polypeptide contains an I332E substitution and the second Fc polypeptide contains an I332E substitution; (g) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions and the second Fc polypeptide contains I332E substitutions; (h) According to the EU designation, the second Fc polypeptide contains an I332E substitution; (i) According to the EU designation, the first Fc polypeptide contains S239D substitution and the second Fc polypeptide contains S239D and I332E substitution; (j) According to the EU designation, the first Fc polypeptide contains I332E substitution and the second Fc polypeptide contains S239D and I332E substitution; (k) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions and the second Fc polypeptide contains S239D and I332E substitutions; (l) According to the EU designation, the second Fc polypeptide contains S239D and I332E substitutions; (m) According to the EU designation, the first Fc polypeptide contains an S239D substitution; (n) According to the EU designation, the first Fc polypeptide contains an I332E substitution; or (o) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions. 60. The isolated antibody as described in claim 59, wherein: (a) According to the EU designation, the first Fc polypeptide contains an I332E substitution and the second Fc polypeptide contains an S239D substitution; (b) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions and the second Fc polypeptide contains S239D substitution; (c) According to the EU designation, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains an I332E substitution; (d) According to the EU designation, the second Fc polypeptide contains an I332E substitution; (e) According to the EU designation, the first Fc polypeptide contains an S239D substitution and the second Fc polypeptide contains both S239D and I332E substitutions; or (f) According to the EU designation, the first Fc polypeptide contains an I332E substitution. 61. The isolated antibody as described in claim 60, wherein: (a) According to the EU designation, the first Fc polypeptide contains an I332E substitution and a serine at position 239, and the second Fc polypeptide contains an S239D substitution and an isoleucine at position 332. (b) According to the EU designation, the first Fc polypeptide contains S239D and I332E substitutions, and the second Fc polypeptide contains S239D substitution and isoleucine at position 332; (c) According to the EU designation, the first Fc polypeptide contains an S239D substitution and an isoleucine at position 332, and the second Fc polypeptide contains an I332E substitution and a serine at position 239. (d) According to the EU designation, the first Fc polypeptide contains a serine at position 239 and an isoleucine at position 332, and the second Fc polypeptide contains an I332E substitution and a serine at position 239. (e) According to the EU designation, 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; or (f) According to the EU designation, the first Fc polypeptide contains an I332E substitution and a serine at position 239, and the second Fc polypeptide contains a serine at position 239 and an isoleucine at position 332. 62. The isolated antibody as described in claim 34, comprising two heavy chains and two light chains. 63. The isolated antibody as claimed in claim 62, comprising heavy and light chains selected from combinations listed in Table 2. 64. The isolated antibody of claim 62, wherein the first heavy chain comprises _VH and Fc sequences selected from combinations in Table 3 and the second heavy chain comprises _VH and Fc sequences selected from combinations in Table 4. 65. The isolated antibody of claim 62, wherein the first heavy chain comprises _VH and Fc sequences selected from combinations in Table 5 and the second heavy chain comprises _VH and Fc sequences selected from combinations in Table 6. 66. A pharmaceutical composition comprising the isolated antibody as described in claim 1 and a pharmaceutically acceptable carrier. 67. An isolated polynucleotide comprising a nucleotide sequence encoding the isolated antibody as claimed in claim 1. 68. A vector comprising the polynucleotide as described in claim 67. 69. A host cell comprising the polynucleotide of claim 67 or the vector of claim 68. 70. A method for treating a subject with cancer or treating brain metastases of cancer, the method comprising administering to the subject a therapeutically effective amount of the isolated antibody as claimed in claim 1 or the pharmaceutical composition as claimed in claim 66. 71. The method of claim 70, wherein the isolated antibody is administered in combination with chemotherapy or radiotherapy. 72. The method of claim 70, wherein the cancer is a metastatic cancer. 73. The method of claim 70, wherein the cancer is breast cancer. 74. The method of claim 70, wherein the cancer is a HER2-positive cancer.