Engineered anti-HER2 bispecific proteins

Abstract

In one embodiment, a bispecific protein is provided that has the ability to specifically bind to both subdomain II of human HER2 and subdomain IV of human HER2. In another embodiment, a method of treating cancer or treating brain metastasis of cancer using a bispecific protein that specifically binds to subdomain II and subdomain IV of human HER2 is provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 237,071, filed August 25, 2021. The disclosure of that provisional application is incorporated by reference in its entirety herein for all purposes. [Background technology]

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

[0003] overview In one embodiment, the disclosure provides a protein comprising: (a) a first Fc polypeptide fused at its N-terminus to the Fd portion of a Fab; (b) a second Fc polypeptide N-terminally fused to a single chain variable fragment (scFv), wherein the first and second Fc polypeptides form an Fc dimer; and (c) a light chain polypeptide which pairs with the Fd portion described in (a) to form a Fab; wherein the Fab binds to subdomain II of human HER2 and the scFv binds to subdomain IV of human HER2, or wherein the Fab binds to subdomain IV of human HER2 and the scFv binds to subdomain II of human HER2, and The first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or an I332E substitution according to EU numbering.

[0004] In some embodiments of the protein, the first Fc polypeptide and / or the second Fc polypeptide independently comprising the S239D and / or the I332E substitution are capable of enhancing HER2-mediated effector function.

[0005] In some embodiments of the protein, the first Fc polypeptide or the second Fc polypeptide comprises an S239D and / or I332E substitution according to EU numbering, in other embodiments, the first Fc polypeptide comprises an S239D and / or I332E substitution and the second Fc polypeptide comprises an S239D and / or I332E substitution according to EU numbering.

[0006] In some embodiments of the protein, (a) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (b) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (c) the first Fc polypeptide comprises an S239D and I332E substitution, and the second Fc polypeptide comprises an S239D substitution, according to EU numbering; (d) the second Fc polypeptide comprises the following substitution, according to EU numbering: S239D; (e) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (f) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (g) the first Fc polypeptide comprises an S239D and I332E substitution, and the second Fc polypeptide comprises an I332E substitution, according to EU numbering; (h) the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (i) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; (j) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; (k) the first Fc polypeptide comprises an S239D and an I332E substitution, and the second Fc polypeptide comprises an S239D and an I332E substitution, according to EU numbering; (l) the second Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E; (m) the first Fc polypeptide comprises the following substitution, according to EU numbering: S239D; (n) the first Fc polypeptide comprises a I332E substitution, according to EU numbering; or (o) the first Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E.

[0007] In certain embodiments of the protein, (a) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (b) the first Fc polypeptide comprises an S239D and I332E substitution, and the second Fc polypeptide comprises an S239D substitution, according to EU numbering; (c) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (d) the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (e) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; or (f) the first Fc polypeptide comprises a I332E substitution, according to EU numbering.

[0008] In certain embodiments of the protein, (a) the first Fc polypeptide comprises a I332E substitution and a serine at position 239, and the second Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, according to EU numbering; (b) the first Fc polypeptide comprises an S239D and an I332E substitution, and the second Fc polypeptide comprises an S239D substitution and an isoleucine at position 332, according to EU numbering; (c) the first Fc polypeptide comprises an S239D substitution and an isoleucine at position 332, and the second Fc polypeptide comprises an I332E substitution and a serine at position 239, according to EU numbering; (d) the first Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, and the second Fc polypeptide comprises a I332E substitution and a serine at position 239, according to EU numbering; (e) the first Fc polypeptide comprises an S239D substitution and an isoleucine at position 332, and the second Fc polypeptide comprises an S239D and an I332E substitution, according to EU numbering; or (f) the first Fc polypeptide comprises a I332E substitution and a serine at position 239, and the second Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, according to EU numbering.

[0009] In some embodiments of the protein, the Fab binds to subdomain II of human HER2 and the scFv binds to subdomain IV of human HER2, in other embodiments, the Fab binds to subdomain IV of human HER2 and the scFv binds to subdomain II of human HER2.

[0010] In some embodiments of the protein, the second Fc polypeptide is fused to the scFv via a first linker. The first linker is 1 to 20 amino acids in length, for example, GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 116, (GGSG) 5 ), GGGGS (SEQ ID NO: 117, G 4 S), GGGGSGGGGS (SEQ ID NO: 118, (G 4 S) 2 ), GGGGSGGGGSGGGGS (SEQ ID NO: 119, (G 4 S) 3 ), GGGGSGGGGSGGGG (SEQ ID NO: 120, (G 4 S) 2 -G 4 ), GGGGSGGGGSGG (SEQ ID NO: 121), GGGGSGGGGGS (SEQ ID NO: 122), and GGGGGSGGGGGSGGGGS (SEQ ID NO: 123).

[0011] In some embodiments of the protein, the scFv comprises a V L Area and V H The second linker may be 1 to 20 amino acids long, for example, GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 116, (GGSG) 5 ), GGGGS (SEQ ID NO: 117, G 4 S), GGGGSGGGGS (SEQ ID NO: 118, (G 4 S) 2 ), GGGGSGGGGSGGGGS (SEQ ID NO: 119, (G 4 S) 3 ), GGGGSGGGGSGGGG (SEQ ID NO: 120, (G 4 S) 2 -G 4 ), GGGGSGGGGSGG (SEQ ID NO: 121), GGGGSGGGGGS (SEQ ID NO: 122), and GGGGGSGGGGGSGGGGS (SEQ ID NO: 123).

[0012] In some embodiments of the protein, the first Fc polypeptide and / or the second Fc polypeptide specifically binds to the transferrin receptor (TfR), e.g., comprises any of the sequence modifications described herein that create a TfR binding site. In some embodiments, the first Fc polypeptide and the second Fc polypeptide each comprise a modification that promotes heterodimerization. In certain embodiments, the first Fc polypeptide comprises a T366W substitution according to EU numbering, and the second Fc polypeptide comprises a T366S, L368A, and Y407V substitution. In other embodiments, the first Fc polypeptide comprises a T366S, L368A, and Y407V substitution according to EU numbering, and the second Fc polypeptide comprises a T366W substitution.

[0013] In some embodiments of the protein, the first Fc polypeptide and / or the second Fc polypeptide independently comprise a modification that reduces TfR-mediated effector function. In certain embodiments, the modification that reduces effector function is a substitution of L234A and L235A according to EU numbering. As an example, the first Fc polypeptide may specifically bind to TfR and comprise a substitution of L234A and L235A according to EU numbering, the first Fc polypeptide may further comprise a substitution of P329G or P329S, and the second Fc polypeptide may comprise Leu at positions 234 and 235 and Proline at position 329. As another example, the second Fc polypeptide may specifically bind to TfR and comprise substitutions L234A and L235A according to EU numbering, the second Fc polypeptide may further comprise substitutions P329G or P329S, and the first Fc polypeptide may comprise Leu at positions 234 and 235 and Proline at position 329.

[0014] In some embodiments of the protein, the hinge region or a portion thereof is linked to the N-terminus of the first Fc polypeptide and / or the second Fc polypeptide.

[0015] In some embodiments of the protein, the first Fc polypeptide and / or the second Fc polypeptide independently comprise a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence selected from the group consisting of SEQ ID NOs: 131-149 and 183-196. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence selected from SEQ ID NOs: 133 and 183-185. In certain other embodiments, the first Fc polypeptide or the second Fc polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence selected from SEQ ID NOs: 137 and 186-196.

[0016] In some embodiments of the protein, the first Fc polypeptide and / or the second Fc polypeptide independently comprise a Tyr at position 384, a Thr at position 386, a Glu at position 387, a Trp at position 388, a Ser at position 389, a Ser at position 413, a Glu at position 415, a Glu at position 416, and a Phe at position 421 according to EU numbering, and a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence selected from SEQ ID NOs: 135-139 and 186-196.

[0017] In some embodiments of the protein, the first Fc polypeptide comprises Ala at position 234, Ala at position 235, Trp at position 366, Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser at position 389, Ser at position 413, Glu at position 415, Glu at position 416, and Phe at position 421 according to EU numbering, and has at least 90% (e.g., 91%, 92%, and the second Fc polypeptide comprises a Ser at position 366, an Ala at position 368, and a Val at position 407 according to EU numbering and a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 133.

[0018] In other embodiments of the protein, the first Fc polypeptide comprises a Ser at position 366, an Ala at position 368, and a Val at position 407 according to EU numbering and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 133, and the second Fc polypeptide comprises an A at position 234 according to EU numbering. at position 235, Ala at position 236, 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 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:137.

[0019] In another aspect, the disclosure provides a protein comprising: (a) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:1; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:159; and (iii) a light chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25; or (b) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 164; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 173; and (iii) a light chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25; or (c) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 164; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 174; and (iii) a light chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25; or (d) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 166; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 173; and (iii) a light chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25; or (e) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 165; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 173; and (iii) a light chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25; or (f) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 165; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 174; and (iii) a light chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25; or (g) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 167; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 173; and (iii) a light chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25; or (h) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 164; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 175; and (iii) a light chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25; or (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 164; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 176; and (iii) a light chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25; or (j) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 166; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 175; and (iii) a light chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25; or (k) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 165; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 175; and (iii) a light chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25; or (l) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 165; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 176; and (iii) a light chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25; or (m) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 167; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 175; and (iii) a light chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25.

[0020] In some embodiments of the protein, the first heavy chain polypeptide comprises a sequence that contains a TfR binding site, a modification that promotes heterodimerization, a modification that enhances HER2-mediated effector function, and / or a modification that decreases TfR-mediated effector function present in the first heavy chain polypeptide sequence and has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the first heavy chain polypeptide sequence. In other embodiments of the protein, the second heavy chain polypeptide comprises a sequence that contains a TfR binding site, a modification that promotes heterodimerization, a modification that enhances HER2-mediated effector function, and / or a modification that decreases TfR-mediated effector function present in the second heavy chain polypeptide sequence and has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the second heavy chain polypeptide sequence.

[0021] In yet another aspect, the disclosure provides a protein comprising: (a) a first Fc polypeptide fused at its N-terminus to the Fd portion of a Fab; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; wherein the Fd portion in (a) and / or (b) is fused to the scFv at its N-terminus; wherein the Fab binds to subdomain II of human HER2 and the scFv binds to subdomain IV of human HER2, or wherein the Fab binds to subdomain IV of human HER2 and the scFv binds to subdomain II of human HER2, and The first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or an I332E substitution according to EU numbering.

[0022] In some embodiments of the protein, the first Fc polypeptide and / or the second Fc polypeptide independently comprising the S239D and / or the I332E substitution are capable of enhancing HER2-mediated effector function.

[0023] In some embodiments of the protein, the first Fc polypeptide or the second Fc polypeptide comprises an S239D and / or I332E substitution according to EU numbering, in other embodiments, the first Fc polypeptide comprises an S239D and / or I332E substitution and the second Fc polypeptide comprises an S239D and / or I332E substitution according to EU numbering.

[0024] In some embodiments of the protein, (a) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (b) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (c) the first Fc polypeptide comprises an S239D and I332E substitution, and the second Fc polypeptide comprises an S239D substitution, according to EU numbering; (d) the second Fc polypeptide comprises the following substitution, according to EU numbering: S239D; (e) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (f) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (g) the first Fc polypeptide comprises an S239D and I332E substitution, and the second Fc polypeptide comprises an I332E substitution, according to EU numbering; (h) the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (i) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; (j) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; (k) the first Fc polypeptide comprises an S239D and an I332E substitution, and the second Fc polypeptide comprises an S239D and an I332E substitution, according to EU numbering; (l) the second Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E; (m) the first Fc polypeptide comprises the following substitution, according to EU numbering: S239D; (n) the first Fc polypeptide comprises a I332E substitution, according to EU numbering; or (o) the first Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E.

[0025] In certain embodiments of the protein, (a) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (b) the first Fc polypeptide comprises an S239D and I332E substitution, and the second Fc polypeptide comprises an S239D substitution, according to EU numbering; (c) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (d) the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (e) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; or (f) the first Fc polypeptide comprises a I332E substitution, according to EU numbering.

[0026] In certain embodiments of the protein, (a) the first Fc polypeptide comprises a I332E substitution and a serine at position 239, and the second Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, according to EU numbering; (b) the first Fc polypeptide comprises an S239D and an I332E substitution, and the second Fc polypeptide comprises an S239D substitution and an isoleucine at position 332, according to EU numbering; (c) the first Fc polypeptide comprises an S239D substitution and an isoleucine at position 332, and the second Fc polypeptide comprises an I332E substitution and a serine at position 239, according to EU numbering; (d) the first Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, and the second Fc polypeptide comprises a I332E substitution and a serine at position 239, according to EU numbering; (e) the first Fc polypeptide comprises an S239D substitution and an isoleucine at position 332, and the second Fc polypeptide comprises an S239D and an I332E substitution, according to EU numbering; or (f) the first Fc polypeptide comprises a I332E substitution and a serine at position 239, and the second Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, according to EU numbering.

[0027] In some embodiments of the protein, the Fab binds to subdomain II of human HER2 and the scFv binds to subdomain IV of human HER2, in other embodiments, the Fab binds to subdomain IV of human HER2 and the scFv binds to subdomain II of human HER2.

[0028] In some embodiments of the protein, the Fd portion in (a) and / or (b) is fused N-terminally to the scFv.

[0029] In some embodiments of the protein, the Fd portion in (a) and / or (b) is fused to the scFv via a first linker. In certain embodiments, the first linker is 1-20 amino acids in length, e.g., GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 116, (GGSG) 5 ), GGGGS (SEQ ID NO: 117, G 4 S), GGGGSGGGGS (SEQ ID NO: 118, (G 4 S) 2 ), GGGGSGGGGSGGGGS (SEQ ID NO: 119, (G 4 S) 3 ), GGGGSGGGGSGGGG (SEQ ID NO: 120, (G 4 S) 2 -G 4 ), GGGGSGGGGSGG (SEQ ID NO: 121), GGGGSGGGGGS (SEQ ID NO: 122), and GGGGGSGGGGGSGGGGS (SEQ ID NO: 123).

[0030] In some embodiments of the protein, the scFv comprises a V L Area and V H In some embodiments, the second linker comprises a region that is 1 to 20 amino acids long, such as, for example, GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 116, (GGSG) 5 ), GGGGS (SEQ ID NO: 117, G 4 S), GGGGSGGGGS (SEQ ID NO: 118, (G 4 S) 2 ), GGGGSGGGGSGGGGS (SEQ ID NO: 119, (G 4 S) 3 ), GGGGSGGGGSGGGG (SEQ ID NO: 120, (G 4 S) 2 -G 4 ), GGGGSGGGGSGG (SEQ ID NO: 121), GGGGSGGGGGS (SEQ ID NO: 122), and GGGGGSGGGGGSGGGGS (SEQ ID NO: 123).

[0031] In some embodiments of the protein, the first Fc polypeptide and / or the second Fc polypeptide specifically binds to the transferrin receptor (TfR), e.g., comprises any of the sequence modifications described herein that create a TfR binding site. In some embodiments, the first Fc polypeptide and the second Fc polypeptide each comprise a modification that promotes heterodimerization. In certain embodiments, the first Fc polypeptide comprises a T366W substitution according to EU numbering, and the second Fc polypeptide comprises a T366S, L368A, and Y407V substitution. In other embodiments, the first Fc polypeptide comprises a T366S, L368A, and Y407V substitution according to EU numbering, and the second Fc polypeptide comprises a T366W substitution.

[0032] In some embodiments of the protein, the first Fc polypeptide and / or the second Fc polypeptide independently comprise a modification that reduces TfR-mediated effector function. In certain embodiments, the modification that reduces effector function is a substitution of L234A and L235A according to EU numbering. As an example, the first Fc polypeptide may specifically bind to TfR and comprise a substitution of L234A and L235A according to EU numbering, the first Fc polypeptide may further comprise a substitution of P329G or P329S, and the second Fc polypeptide may comprise Leu at positions 234 and 235 and Proline at position 329. As another example, the second Fc polypeptide may specifically bind to TfR and comprise substitutions L234A and L235A according to EU numbering, the second Fc polypeptide may further comprise substitutions P329G or P329S, and the first Fc polypeptide may comprise Leu at positions 234 and 235 and Proline at position 329.

[0033] In some embodiments of the protein, the hinge region or a portion thereof is linked to the N-terminus of the first Fc polypeptide and / or the second Fc polypeptide.

[0034] In some embodiments of the protein, the first Fc polypeptide and / or the second Fc polypeptide independently comprise a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence selected from the group consisting of SEQ ID NOs: 131-149 and 183-196. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence selected from SEQ ID NOs: 133 and 183-185. In certain other embodiments, the first Fc polypeptide or the second Fc polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence selected from SEQ ID NOs: 137 and 186-196.

[0035] In some embodiments of the protein, the first Fc polypeptide and / or the second Fc polypeptide independently comprise a Tyr at position 384, a Thr at position 386, a Glu at position 387, a Trp at position 388, a Ser at position 389, a Ser at position 413, a Glu at position 415, a Glu at position 416, and a Phe at position 421 according to EU numbering, and a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence selected from SEQ ID NOs: 135-139 and 186-196.

[0036] In some embodiments of the protein, the first Fc polypeptide comprises Ala at position 234, Ala at position 235, Trp at position 366, Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser at position 389, Ser at position 413, Glu at position 415, Glu at position 416, and Phe at position 421 according to EU numbering, and has at least 90% (e.g., 91%, 92%, and the second Fc polypeptide comprises a Ser at position 366, an Ala at position 368, and a Val at position 407 according to EU numbering and a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 133.

[0037] In other embodiments of the protein, the first Fc polypeptide comprises a Ser at position 366, an Ala at position 368, and a Val at position 407 according to EU numbering and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 133, and the second Fc polypeptide comprises an A at position 234 according to EU numbering. at position 235, Ala at position 236, 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 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:137.

[0038] In a further aspect, the disclosure provides a protein comprising: (a) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:6, (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:160, and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26, or (b) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:6; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:161; and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26; or (c) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:6; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:162; and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26; or (d) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 169; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 177; and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26; or (e) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 169; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 178; and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26; or (f) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 171; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 177; and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26; or (g) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 170; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 177; and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26; or (h) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 170; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 178; and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26; or (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 172, (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 177, and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26, or (j) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 169; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 179; and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26; or (k) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 169; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 180; and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26; or (l) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 171; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 179; and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26; or (m) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 170; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 179; and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26; or (n) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 170; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 180; and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26; or (o) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 172, (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 179, and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26, or (p) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 169; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 181; and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26; or (q) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 169, (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 182, and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26; or (r) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 171; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 181; and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26; or (s) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 170; (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 181; and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26; or (t) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 170, (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 182, and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26, or (u) (i) a first heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 172, (ii) a second heavy chain polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 181, and (iii) two light chain polypeptides each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26.

[0039] In some embodiments of the protein, the first heavy chain polypeptide comprises a sequence that contains a TfR binding site, a modification that promotes heterodimerization, a modification that enhances HER2-mediated effector function, and / or a modification that decreases TfR-mediated effector function present in the first heavy chain polypeptide sequence and has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the first heavy chain polypeptide sequence. In other embodiments of the protein, the second heavy chain polypeptide comprises a sequence that contains a TfR binding site, a modification that promotes heterodimerization, a modification that enhances HER2-mediated effector function, and / or a modification that decreases TfR-mediated effector function present in the second heavy chain polypeptide sequence and has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the second heavy chain polypeptide sequence.

[0040] In another aspect of the disclosure, the disclosure provides a pharmaceutical composition comprising any of the proteins described herein and a pharma- ceutically acceptable carrier.

[0041] In another aspect of the disclosure, the disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding a protein described herein.

[0042] In another aspect of the disclosure, the disclosure provides a vector comprising the polynucleotide of the preceding aspect.

[0043] In another aspect of the disclosure, the disclosure provides a host cell comprising the polynucleotide or the vector.

[0044] In another aspect of the disclosure, the disclosure provides a method for treating cancer or treating brain metastasis of cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein or pharmaceutical composition thereof described herein.

[0045] In some embodiments of the method, the protein is administered in combination with chemotherapy or radiation therapy.

[0046] In some embodiments of the method, the cancer is a metastatic cancer. In some embodiments, the cancer is a breast cancer. In some embodiments, the cancer is a HER2-positive cancer. [Brief description of the drawings]

[0047] [Figure 1A] FIG. 1 is a schematic diagram showing an exemplary bispecific protein with a "Fab-Fc polypeptide / scFv-Fc polypeptide" structure, in which the scFv is fused via a hinge or partial hinge region to the N-terminus of an Fc polypeptide carrying a TfR binding site (star) and a knob mutation, and the other Fc polypeptide carrying a hole mutation. [Figure 1B] FIG. 1 is a schematic diagram showing an exemplary bispecific protein with a "Fab-Fc polypeptide / scFv-Fc polypeptide" structure, in which the scFv is fused via a hinge or partial hinge region to the N-terminus of a hole mutation of an Fc polypeptide, and the other Fc polypeptide has a TfR binding site (star) and a knob mutation. [Figure 2A] FIG. 1 is a schematic diagram showing an exemplary bispecific protein with the "mAb / HC N- or C-terminal scFv" structure, in which the scFv is fused to the C-terminus of an Fc polypeptide carrying a hole mutation, and the other Fc polypeptide carrying a TfR binding site (star) and a knob mutation. [Figure 2B] Schematic diagram showing an exemplary bispecific protein with the "mAb / HC N- or C-terminal scFv" structure, in which the scFv is fused via a linker to the N-terminus of an Fd portion fused to an Fd portion of a heavy chain comprising an Fc polypeptide with a hole mutation and another Fc polypeptide with a TfR binding site (asterisk) and a knob mutation. [Figure 2C]Schematic diagram showing an exemplary bispecific protein with the "mAb / HC N- or C-terminal scFv" structure, in which two scFvs are each fused to the N-terminus of the Fd portion of the heavy chain via a linker. The TfR binding site of the Fc polypeptide is indicated by an asterisk. [Figure 2D] Schematic diagram showing an exemplary bispecific protein with the "mAb / HC N- or C-terminal scFv" structure, in which two scFvs are each fused via a linker to the C-terminus of the Fc polypeptide of the heavy chain. The TfR binding site of the Fc polypeptide is indicated by an asterisk. [Figure 3A] Schematic diagram showing an exemplary bispecific protein with the "mAb / LC N- or C-terminal scFv" structure, in which the scFv is fused to the C-terminus of the light chain via a linker. The TfR binding site of the Fc polypeptide is indicated by an asterisk. [Figure 3B] Schematic diagram showing an exemplary bispecific protein with the "mAb / LC N- or C-terminal scFv" structure, in which the scFv is fused to the N-terminus of the light chain via a linker. The TfR binding site of the Fc polypeptide is indicated by an asterisk. [Figure 3C] Schematic diagram showing an exemplary bispecific protein with the "mAb / LC N- or C-terminal scFv" structure, in which two scFvs are each fused to the N-terminus of the light chain via a linker. The TfR binding site of the Fc polypeptide is indicated by an asterisk. [Figure 3D] Schematic diagram showing an exemplary bispecific protein with the "mAb / LC N- or C-terminal scFv" structure, in which two scFvs are each fused to the C-terminus of the light chain via a linker. The TfR binding site of the Fc polypeptide is indicated by an asterisk. [Figure 4]Schematic diagram showing an exemplary bispecific protein with the structure "mAb / HC and LC N-terminal VH VL" in which two VH regions are each fused to the N-terminus of the Fd portion of a heavy chain and two VL regions are each fused to the N-terminus of a light chain. The VH and VL regions form an Fv fragment. The TfR binding site of the Fc polypeptide is indicated by an asterisk. [Diagram 5] Schematic diagram showing an exemplary bispecific protein with the "mAb / HC C-term VH VL" structure, in which the VH region is fused to the C-terminus of an Fc polypeptide carrying a hole mutation, and the VL region is fused to the C-terminus of an Fc polypeptide carrying a TfR binding site (star) and a knob mutation. The VH and VL regions form an Fv fragment. [Figure 6A] 1 shows the inhibition of cancer cell proliferation on day 6 in a BT474 cell proliferation inhibition assay by bispecific proteins having a "Fab-Fc polypeptide / scFv-Fc polypeptide" structure. [Figure 6B] 1 shows the inhibition of cancer cell proliferation on day 3 in an OE19 cell proliferation inhibition assay by bispecific proteins having a "Fab-Fc polypeptide / scFv-Fc polypeptide" structure. [Figure 7] 1 is a plot showing the plasma PK profiles of anti-HER2 bispecific proteins and anti-HER2 controls in C57 / BL6 mice. [Figure 8A] 13 is a plot showing quantification of reticulocytes in TfRmu / huKI mice administered anti-HER2 bispecific protein or anti-HER2 control intravenously. [Figure 8B] 1 is a plot showing the plasma PK profiles of anti-HER2 bispecific proteins and anti-HER2 controls in TfRmu / huKI mice. [Figure 8C] 13 is a plot showing the brain PK profile of anti-HER2 bispecific protein and anti-HER2 control in TfRmu / huKI mice. [Figure 8D]1 is a plot showing the percentage of brain versus plasma concentrations of anti-HER2 bispecific protein and anti-HER2 control in TfRmu / huKI mice. [Figure 9A] 1 shows a proliferation inhibition assay of BT474 cells with NRG1 by anti-HER2 bispecific protein and anti-HER2 control. [Figure 9B] 1 shows a proliferation inhibition assay of BT474 cells with NRG1 by anti-HER2 bispecific protein and anti-HER2 control. [Figure 9C] 1 shows a proliferation inhibition assay of BT474 cells with NRG1 by anti-HER2 bispecific protein and anti-HER2 control. [Figure 9D] 1 shows a proliferation inhibition assay of BT474 cells without NRG1 by anti-HER2 bispecific proteins and anti-HER2 controls. [Figure 9E] 1 shows a proliferation inhibition assay of BT474 cells without NRG1 by anti-HER2 bispecific proteins and anti-HER2 controls. [Figure 9F] 1 shows a proliferation inhibition assay of BT474 cells without NRG1 by anti-HER2 bispecific proteins and anti-HER2 controls. [Figure 9G] 1 shows a proliferation inhibition assay of OE19 cells by anti-HER2 bispecific proteins and anti-HER2 controls. [Figure 9H] 1 shows a proliferation inhibition assay of OE19 cells by anti-HER2 bispecific proteins and anti-HER2 controls. [Figure 9I] 1 shows a proliferation inhibition assay of OE19 cells by anti-HER2 bispecific proteins and anti-HER2 controls. [Figure 9J] 1 shows a proliferation inhibition assay of ZR75 cells by anti-HER2 bispecific proteins and anti-HER2 controls. [Figure 9K] 1 shows a proliferation inhibition assay of ZR75 cells by anti-HER2 bispecific proteins and anti-HER2 controls. [Figure 9L] 1 shows a proliferation inhibition assay of ZR75 cells by anti-HER2 bispecific proteins and anti-HER2 controls. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Detailed Description I. Introduction In one aspect, a bispecific protein is provided that can bind to both subdomain II of human HER2 and subdomain IV of human HER2. The bispecific protein can generally be produced without mispairing or steering of light chains. In some embodiments, the bispecific protein binds to each target subdomain of human HER2 in a monovalent manner. In some embodiments, the bispecific protein binds to one target subdomain of human HER2 in a monovalent manner and to the other target subdomain of human HER2 in a bivalent manner (e.g., monovalently to subdomain II and bivalently to subdomain IV, or monovalently to subdomain IV and bivalently to subdomain II). In some embodiments, the bispecific protein binds to each target subdomain of human HER2 in a bivalent manner. Various structures of bispecific proteins are described in further detail herein.

[0049] In some embodiments, the bispecific protein comprises an scFv that binds to subdomain II (or subdomain IV) of human HER2 and a Fab that binds to subdomain IV (or subdomain II) of human HER2 (see, e.g., the "Fab-Fc Polypeptide / scFv-Fc Polypeptide" structures in Section III). In some embodiments, the bispecific protein comprises one or more scFvs attached to the N-terminus or C-terminus of the heavy chain of the bispecific protein, where the scFv binds to subdomain II (or subdomain IV) of human HER2 and the Fab of the bispecific protein binds to subdomain IV (or subdomain II) of human HER2 (see, e.g., the "N- or C-terminal scFv of mAb / HC" structures in Section III). In some embodiments, the bispecific protein comprises one or more scFvs attached to the N-terminus or C-terminus of the light chain of the bispecific protein, where the scFv binds subdomain II (or subdomain IV) of human HER2 and the Fab of the bispecific protein binds subdomain IV (or subdomain II) of human HER2 (see, e.g., the "mAb / HC N- or C-terminal scFv mAb / LC N- or C-terminal scFv" structures in Section III). In some embodiments, the bispecific protein comprises one or more scFvs attached to the N-terminus of the heavy chain of the bispecific protein, where the scFv binds subdomain II (or subdomain IV) of human HER2 and the Fab of the bispecific protein binds subdomain IV (or subdomain II) of human HER2 (see, e.g., the "mAb / HC N- or C-terminal scFv mAb / LC N- or C-terminal scFv" structures in Section III). H Area (or V L region) and the V of the Fv fragment connected to the N-terminus of the light chain L Area (or V H and the Fv fragment binds to subdomain II (or subdomain IV) of human HER2, and the Fab of the bispecific protein binds to subdomain IV (or subdomain II) of human HER2 (see, for example, the "N-terminal V domain of mAb / HC and LC" in Section III). H V L In yet another embodiment, the bispecific protein comprises a VFv fragment connected to the C-terminus of one of the two heavy chains of the bispecific protein. H Area (or V L region) and the V of the Fv fragment connected to the C-terminus of the other of the two heavy chainsL Area (or V H and the Fv fragment binds to subdomain II (or subdomain IV) of human HER2, and the Fab of the bispecific protein binds to subdomain IV (or subdomain II) of human HER2 (see, for example, the "C-terminal V domain of mAb / HC" in Section III). H V L ” structure).

[0050] Previous treatments have failed to control brain metastasis of HER2-positive breast cancer, mainly due to the inability of the therapeutics to cross the blood-brain barrier (BBB) ​​and access the brain parenchyma. Thus, there is a need for new therapeutics that can cross the BBB and target HER2 in the brain parenchyma. We have previously described the use of transferrin receptor (TfR) binding as a method to enable BBB delivery across brain endothelium, as TfR expression is highly expressed in brain endothelial cells and can enable BBB delivery by receptor-mediated transcytosis. Interestingly, TfR is highly expressed in various cancers, including HER2-positive breast cancer. The mechanism by which cancer cells acquire increased TfR expression may be related to tumor cell proliferation and increased metabolic demands, such as iron uptake. Indeed, a published microarray dataset has demonstrated a correlation between TfR expression and breast cancer prognosis (Miller et al., Cancer Res. 71:6728, 2011). There are also several reports on the use of TfR as a pharmacological target for various types of cancer.

[0051] In some embodiments, the bispecific protein comprises one or more modified Fc polypeptides (i.e., TfR-binding Fc polypeptides) that specifically bind to a BBB receptor, e.g., TfR. In some embodiments, the bispecific protein is capable of transport across the BBB. In some embodiments, the anti-HER2 bispecific proteins that bind to both HER2 and TfR described herein may provide additional anti-tumor effects when binding to HER2-positive tumor cells that also express high levels of TfR, compared to other therapeutic agents that only bind to HER2. Specifically, these proteins can bind to both TfR and HER2 simultaneously, thereby increasing their potency and / or efficacy.

[0052] In some embodiments, the bispecific protein comprises a modified Fc polypeptide dimer that specifically binds to TfR and has reduced effector function (e.g., ADCC or CDC) upon binding to TfR, but maintains or enhances effector function (e.g., ADCC or CDC) upon binding to HER2.

[0053] II. Definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an antibody" optionally includes a combination of two or more such molecules, and the like.

[0054] As used herein, the terms "about" and "approximately," when used to modify a quantity specified in a numerical value or range, indicate that reasonable deviations from that numerical value and values ​​known to one of ordinary skill in the art, for example, ±20%, ±10%, or ±5%, are within the intended meaning of the stated value.

[0055] As used herein, the term "antibody" refers to a protein having an immunoglobulin fold and specifically binds to an antigen through its variable region. The term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single chain antibodies, multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, and human antibodies. As used herein, the term "antibody" also includes Fab, F(ab') and / or F(ab') antibodies. 2 The term also includes antibody fragments that retain antigen-binding specificity, including, but not limited to, Fv, scFv, and bivalent scFv. Antibodies may contain light chains, which are classified as either kappa or lambda. Antibodies may contain heavy chains, which are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively.

[0056] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The "variable light chain" (VLC) is L ) and "variable heavy chain" (V H ) refer to these light and heavy chains respectively.

[0057] The term "variable region" or "variable domain" refers to the domain in an antibody heavy or light chain that is derived from germline variable (V), diversity (D), or joining (J) genes (and not from constant (Cμ and Cδ) gene segments) and confers to the antibody its antigen-binding specificity. Typically, antibody variable regions contain four conserved "framework" regions interspersed with three hypervariable "complementarity determining regions".

[0058] The term "complementarity determining region" or "CDR" refers to the three hypervariable regions in each chain that interrupt the four framework regions established by the light and heavy chain variable regions. The CDRs are primarily responsible for antibody binding to an epitope of an antigen. The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are also usually identified by the chain in which a particular CDR is located. Thus, the V H CDR3 or CDR-H3 is located in the variable region of the antibody heavy chain in which it is found, whereas V L CDR1 or CDR-L1 is the CDR1 from the variable region of the antibody light chain in which it is found.

[0059] The "framework regions" or "FRs" of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, functions to position and align the CDRs in three-dimensional space. Framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBASE2" germline variable gene sequence database for human and mouse sequences.

[0060] The amino acid sequences of the CDRs and framework regions can be determined using various definitions known in the art, such as Kabat, Chothia, the International ImMunoGeneTics database (IMGT), AbM, and observed antigen contact ("Contact"). In some embodiments, the CDRs are determined according to the Contact definition. See MacCallum et al., J. Mol. Biol., 262:732-745, 1996. In some embodiments, the CDRs are determined by a combination of the Kabat, Chothia, and / or Contact CDR definitions.

[0061] The term "Fd portion" refers to the N-terminal portion of an immunoglobulin heavy chain. Typically, the Fd portion includes the heavy chain variable (VH) region and the heavy chain constant (CH1) region.

[0062] The term "Fab" refers to an antigen-binding fragment consisting of a light chain variable region, a light chain constant region, a heavy chain variable region, and a heavy chain CH1 constant region.

[0063] The term "single-chain variable fragment" or "scFv" refers to an antigen-binding fragment consisting of a heavy chain variable region and a light chain variable region linked via a peptide linker. scFvs lack the constant region.

[0064] The term "Fv fragment" refers to an antigen-binding fragment consisting of a heavy chain variable region and a light chain variable region which together form a binding site for an antigen.

[0065] The term "epitope" refers to the area or region of a molecule, for example, an antigen, to which the CDR of an antibody specifically binds, and may include a few amino acids, or a portion of a few amino acids, for example, 5 or 6, or more, for example, 20 or more amino acids, or a portion of those amino acids. In some cases, the epitope includes non-protein components, for example, from carbohydrates, nucleic acids, or lipids. In some cases, the epitope is a three-dimensional portion. Thus, for example, when the target is a protein, the epitope may consist of contiguous amino acids (for example, a linear epitope), or may consist of amino acids from different parts of the protein that are brought into close proximity by protein folding (for example, a discontinuous or conformational epitope).

[0066] As used herein, the phrase "recognizes an epitope," when used in reference to an antibody, means that the CDRs of the antibody interact with or specifically bind to the antigen at the epitope or a portion of the antigen that contains the epitope.

[0067] A "humanized antibody" is a chimeric immunoglobulin derived from a non-human source (e.g., mouse) that contains minimal sequence derived from the non-human immunoglobulin outside the CDRs. In general, a humanized antibody contains at least one (e.g., two) variable domains, the CDR regions of which correspond substantially to those of a non-human immunoglobulin and the framework regions of which correspond substantially to those of a human immunoglobulin sequence. In some cases, certain framework region residues of the human immunoglobulin can be replaced with corresponding residues from the non-human species, e.g., to improve specificity, affinity, and / or serum half-life. A humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), usually of a human immunoglobulin sequence. Methods for humanizing antibodies are known in the art.

[0068] A "human antibody" or "fully human antibody" is an antibody that has human heavy and light chain sequences, typically derived from human germline genes. In some embodiments, the antibody is produced by human cells, by non-human animals utilizing the human antibody repertoire (e.g., transgenic mice genetically engineered to express human antibody sequences), or by phage display platforms.

[0069] The term "specifically binds" refers to a molecule (e.g., a Fab, scFv, or modified Fc polypeptide (or target-binding portion thereof)) that binds to an epitope or target in a sample with greater affinity, greater avidity, and / or for a longer period of time than it binds to another epitope or non-target compound (e.g., a structurally distinct antigen). In some embodiments, a Fab, scFv, or modified Fc polypeptide (or target-binding portion thereof) that specifically binds to an epitope or target refers to a molecule that specifically binds to an epitope or target with greater affinity, greater avidity, and / or for a longer period of time than it binds to another epitope or non-target compound (e.g., a structurally distinct antigen). or a non-target compound, e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 100-fold, 1000-fold, 10,000-fold, or more. As used herein, the terms "specific binding," "specifically binds," or "is specific for" a particular epitope or target refer to, for example, the equilibrium dissociation constant K for the epitope or target to which it binds. D But, for example, 10 -4 M or less, e.g., 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M. One of skill in the art will recognize that a Fab or scFv that specifically binds to a target from one species may also specifically bind to an ortholog of that target.

[0070] The term "binding affinity" is used herein to refer to the strength of a non-covalent interaction between two molecules, e.g., between a Fab or scFv and an antigen, or between a modified Fc polypeptide (or target binding portion thereof) and a target. Thus, for example, the term can refer to a 1:1 interaction between a Fab or scFv and an antigen, or between a modified Fc polypeptide (or target binding portion thereof) and a target, unless otherwise specified or clear from the context. Binding affinity is measured by the equilibrium dissociation constant (K D ), which can be quantified by measuring the binding rate constant (k a ,time -1 M -1 ) divided by the dissociation rate constant (k d ,time -1 ) refers to D can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) techniques (e.g., a Biacore™ system), equilibrium exclusion binding techniques such as KinExA®, and BioLayer interferometry (e.g., using the ForteBio® Octet platform). As used herein, "binding affinity" includes not only formal binding affinities, e.g., those that reflect a 1:1 interaction between a Fab or scFv and an antigen, or between a modified Fc polypeptide (or target binding portion thereof) and a target, but also K, which may reflect tight binding. D Also included are apparent affinities, where:

[0071] As used herein, "transferrin receptor" or "TfR" refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is set forth in SEQ ID NO: 150. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee, accession number XP_003310238.1; rhesus monkey, NP_001244232.1; dog, NP_001003111.1; cattle, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP_990587.1). The term "transferrin receptor" also encompasses allelic variants of exemplary reference sequences, e.g., human sequences encoded by genes at the transferrin receptor protein 1 chromosomal locus. The full-length transferrin receptor protein includes a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain.

[0072] As used herein, the term "Fc polypeptide" refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide characterized by an Ig fold as a structural domain. An Fc polypeptide comprises a constant region sequence comprising at least a CH2 domain and / or a CH3 domain, and may include at least a portion of the hinge region, but does not include the variable region.

[0073] A "modified Fc polypeptide" refers to an Fc polypeptide that has at least one mutation, e.g., a substitution, deletion, or insertion, relative to a wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retains the overall Ig fold or structure of a native Fc polypeptide.

[0074] As used herein, "FcRn" refers to the fetal Fc receptor. Binding of an Fc polypeptide to FcRn reduces the clearance and extends the serum half-life of the Fc polypeptide. The human FcRn protein is a heterodimer consisting of a protein approximately 50 kDa in size similar to major histocompatibility (MHC) class I proteins and β2-microglobulin, approximately 15 kDa in size.

[0075] As used herein, "FcRn binding site" refers to a region of an Fc polypeptide that binds to FcRn. In human IgG, the FcRn binding site, as numbered by the EU index, includes L251, M252, I253, S254, R255, T256, M428, H433, N434, H435, and Y436. These positions correspond to positions 21-26, 198, and 203-206 of SEQ ID NO:130.

[0076] As used herein, a "native FcRn-binding site" refers to a region of a polypeptide that binds to FcRn and has the same amino acid sequence as a region of a naturally occurring Fc polypeptide that binds to FcRn.

[0077] As used herein, the terms "CH3 domain" and "CH2 domain" refer to polypeptides of immunoglobulin constant region domains. For the purposes of this application, a polypeptide of the CH3 domain refers to the segment of amino acids from about position 341 to about position 447 as numbered according to the EU numbering scheme, and a polypeptide of the CH2 domain refers to the segment of amino acids from about position 231 to about position 340 as numbered according to the EU numbering scheme, not including the sequence of the hinge region. Polypeptides of the CH2 and CH3 domains may also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, where the numbering of the CH2 domain is 1-110 and the numbering of the CH3 domain is 1-107 according to the numbering of the IMGT Scientific chart (IMGT website). The CH2 and CH3 domains are part of the Fc region of an immunoglobulin. Fc region, as used herein, refers to the segment of amino acids from about 231 to about 447, numbered according to the EU numbering scheme, but can include at least a portion of the hinge region of an antibody. An exemplary hinge region sequence is the human IgG1 hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO: 127).

[0078] The terms "wild-type," "native," and "naturally occurring" as used with respect to a CH3 or CH2 domain refer to a domain having a sequence that occurs in nature.

[0079] As used herein, the term "mutant" when used in reference to a mutant polypeptide or mutant polynucleotide is used interchangeably with "variant". Variants with respect to a given wild-type CH3 or CH2 domain reference sequence can include naturally occurring allelic variants. A "non-naturally" occurring CH3 or CH2 domain refers to a variant or mutant domain that does not naturally occur in a cell and is produced by genetic modification of a native CH3 domain or CH2 domain polynucleotide or polypeptide, for example, using genetic engineering or mutagenesis techniques. A "variant" includes any domain that contains at least one amino acid mutation with respect to the wild type. Mutations can include substitutions, insertions, and deletions.

[0080] The term "isolated" as used with respect to a nucleic acid or protein indicates that the nucleic acid or protein is essentially free of other cellular components with which it is naturally associated. It is preferably in a homogeneous state. Purity and homogeneity are usually measured using analytical chemistry techniques, such as electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high performance liquid chromatography). In some embodiments, an isolated nucleic acid or protein is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure.

[0081] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof. "Amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid."Amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0082] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. An amino acid polymer can contain entirely L-amino acids, entirely D-amino acids, or a mixture of L- and D-amino acids.

[0083] As used herein, the term "protein" refers to either a dimer (i.e., two) or multimer (i.e., three or more) of a polypeptide or a single-chain polypeptide. The single-chain polypeptides of a protein can be linked by covalent bonds, e.g., disulfide bonds, or by non-covalent interactions.

[0084] As used herein, the term "linker" refers to a moiety that links (e.g., covalently links) two peptides or polypeptides (e.g., between an Fc polypeptide and an scFv) to connect or fuse the peptides or polypeptides. In some embodiments, the linker comprises a chemical bond. In some embodiments, the linker comprises a peptide having a length of one or more amino acid residues. A linker suitable for connecting or fusing peptides or polypeptides can be selected based on the properties of the linker, such as the length, hydrophobicity, flexibility, rigidity, or cleavability of the linker.

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

[0086] The term "conservative substitution" or "conservative variation" refers to an alteration that results in the replacement of one amino acid with another amino acid that can be classified as having similar characteristics. Examples of classifications of conservative amino acid groups defined in this way include the "charged / polar group" including Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H), the "aromatic group" including Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H), and the "aliphatic group" including Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine ​​or C). Within each group, subgroups may also be identified. For example, the group of charged or polar amino acids can be divided into subgroups including the "positively charged subgroup" containing Lys, Arg, and His, the "negatively charged subgroup" containing Glu and Asp, and the "polar subgroup" containing Asn and Gln. In another example, the aromatic or cyclic group can be divided into subgroups including the "nitrogen ring subgroup" containing Pro, His, and Trp, and the "phenyl subgroup" containing Phe and Tyr. In another further example, the aliphatic group can be divided into subgroups, such as the "aliphatic non-polar subgroup" containing Val, Leu, Gly, and Ala, and the "aliphatic slightly polar subgroup" containing Met, Ser, Thr, and Cys. Examples of conservative mutations include substitutions of amino acids within the above subgroups, such as, but not limited to, Lys with Arg or vice versa so that a positive charge may be maintained, Glu with Asp or vice versa so that a negative charge may be maintained, Ser with Thr or vice versa so that a free -OH may be maintained, and a free -NH 2In some embodiments, the amino acid substitutions include an amino acid substitution of Gln with Asn, or vice versa, such that the amino acid sequence can be maintained. In some embodiments, a hydrophobic amino acid is substituted for a naturally occurring hydrophobic amino acid, e.g., in the active site, to preserve hydrophobicity.

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

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

[0089] The phrases "corresponding to," "determined with respect to," or "numbered with respect to," when used in the context of identifying a given amino acid residue in a polypeptide sequence, refer to the position of the residue in a particular reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. Thus, for example, an amino acid residue in a modified Fc polypeptide "corresponds to" an amino acid in SEQ ID NO: 130 if that residue matches an amino acid in SEQ ID NO: 130 when optimally aligned against SEQ ID NO: 130. A polypeptide that is aligned to a reference sequence need not be the same length as the reference sequence.

[0090] The terms "subject," "individual," and "patient," as used interchangeably herein, refer to mammals, including, but not limited to, humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In one embodiment, the patient is a human.

[0091] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. "Treating" or "treatment" may refer to any indication of success in treating or ameliorating a neurodegenerative disease (e.g., Alzheimer's disease or another neurodegenerative disease described herein), including any objective or subjective parameter, such as relief, mitigation, improvement of patient survival, increase in survival time or survival rate, reduction of symptoms or making the disease more tolerable to the patient, slowing down the rate of deterioration or decline, or improving the patient's mental and physical health. The treatment or amelioration of symptoms can be based on objective or subjective parameters. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient at different time points before or during treatment.

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

[0093] As used herein, a "therapeutic amount" or "therapeutically effective amount" of an agent (eg, any of the proteins described herein) is an amount of the agent that treats a disease in a subject.

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

[0095] III. Anti-HER2 Bispecific Proteins In one aspect, a bispecific protein is provided that has the ability to specifically bind to both subdomain II of human HER2 and subdomain IV of human HER2, in some embodiments, one or both of the Fc polypeptides of the bispecific protein is a modified Fc polypeptide (e.g., modified to promote TfR binding and / or improve heterodimerization of the Fc polypeptide).

[0096] Fab-Fc / scFv-Fc Polypeptides In some embodiments, the bispecific protein comprises an Fc polypeptide fused to a portion of a Fab and an scFv. A schematic diagram of such a bispecific protein is shown in Figures 1A and 1B. In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to the Fd portion of a Fab; (b) a second Fc polypeptide N-terminally fused to a single chain variable fragment (scFv), wherein the first and second Fc polypeptides form an Fc dimer; and (c) a light chain polypeptide which pairs with the Fd portion described in (a) to form a Fab; wherein the Fab binds to subdomain II of human HER2 and the scFv binds to subdomain IV of human HER2, or the Fab binds to subdomain IV of human HER2 and the scFv binds to subdomain II of human HER2. In some embodiments, the Fab of the protein binds to subdomain II of human HER2 and the scFv binds to subdomain IV of human HER2. In other embodiments, the Fab of the protein binds to subdomain IV of human HER2 and the scFv binds to subdomain II of human HER2.

[0097] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0098] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises substitutions of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a S239D and I332E substitution and the second Fc polypeptide comprises a S239D and I332E substitution. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a S239D and I332E substitution. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a S239D substitution. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a I332E substitution.In some embodiments, the first Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E.

[0099] The Fab is formed from the pairing of the Fd portion of the Fab fused to the N-terminus of the first Fc polypeptide with a light chain. In some embodiments, a Fab that specifically binds to subdomain II of human HER2 has a V sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 108. H In some embodiments, a Fab that specifically binds to subdomain IV of human HER2 comprises a V region having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 109. H Includes the area.

[0100] In some embodiments, the second Fc polypeptide of the bispecific protein is fused to an scFv fragment at its N-terminus. In some embodiments, the second Fc polypeptide is fused to the scFv fragment at its N-terminus via a first linker. In some embodiments, the first linker has a length of about 1 to about 50 amino acids, e.g., about 1 to about 40, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 2 to about 40, about 2 to about 30, about 2 to about 20, about 2 to about 10, about 5 to about 40, about 5 to about 30, about 5 to about 25, or about 5 to about 20 amino acids. In some embodiments, the first linker has a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 amino acids. Various linkers are described in more detail herein. In some embodiments, the first linker is GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 116, (GGSG) 5 ), GGGGS (SEQ ID NO: 117, G 4 S), GGGGSGGGGS (SEQ ID NO: 118, (G4 S) 2 ), GGGGSGGGGSGGGGS (SEQ ID NO: 119, (G 4 S) 3 ), GGGGSGGGGSGGGG (SEQ ID NO: 120, (G 4 S) 2 -G 4 ), GGGGSGGGGSGG (SEQ ID NO: 121), GGGGSGGGGGS (SEQ ID NO: 122), and GGGGGSGGGGGSGGGGGS (SEQ ID NO: 123).

[0101] In some embodiments, the scFvs of the bispecific protein are V H Area and V L In some embodiments, the V of the scFv L Area and V H The orientation of the domain is (N-terminus)-V L Area-V H In another embodiment, the V of the scFv is linked to the N-terminus of the Fc polypeptide via the first linker. L Area and V H The orientation of the domain is (N-terminus)-V H Area-V L Region-(C-terminus), where the C-terminus of the scFv is linked to the N-terminus of the Fc polypeptide via the first linker.

[0102] In some embodiments, the V of the scFv L Area and V HThe regions are connected via a second linker. In some embodiments, the second linker has a length of about 10 to about 25 amino acids, e.g., about 10 to about 20, about 12 to about 25, about 12 to about 20, about 14 to about 25, or about 14 to about 20 amino acids. In some embodiments, the second linker has a length of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some embodiments, the second linker comprises a flexible linker. Various linkers are described in more detail herein. In some embodiments, the second linker is GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 116, (GGSG) 5 ), GGGGS (SEQ ID NO: 117, G 4 S), GGGGSGGGGS (SEQ ID NO: 118, (G 4 S) 2 ), GGGGSGGGGSGGGGS (SEQ ID NO: 119, (G 4 S) 3 ), GGGGSGGGGSGGGG (SEQ ID NO: 120, (G 4 S) 2 -G 4 ), GGGGSGGGGSGG (SEQ ID NO: 121), GGGGSGGGGGS (SEQ ID NO: 122), and GGGGGSGGGGGSGGGGGS (SEQ ID NO: 123).

[0103] In some embodiments, the V of the scFv L Area and V H In some embodiments, both of the V L Area and corresponding V H The Cys substitutions in the regions may form disulfide bonds to help stabilize the structure of the scFv. In some embodiments, the scFv comprises the variable domain numbering system V according to Kabat. H 44 and V L In some embodiments, the scFv comprises a cysteine ​​at each of positions 100 and 110. H 44 and V L It contains a disulfide bond between the cysteines at position 100.

[0104] For example, anti-HER2DII V L The region may have a Gln to Cys substitution at position 100 of SEQ ID NO: 110. In certain embodiments, the V of anti-HER2 DII containing the Cys substitution L The region may have the sequence of SEQ ID NO: 114. In some embodiments, the V L The region may have a Gln to Cys substitution at position 100 of SEQ ID NO: 111. In certain embodiments, the V of anti-HER2 DIV containing the Cys substitution L The region may have the sequence of SEQ ID NO:115.

[0105] For example, anti-HER2DII V H The region may have a Gly to Cys substitution at position 44 of SEQ ID NO: 108. In certain embodiments, the V of anti-HER2 DII containing the Cys substitution H The region may have the sequence of SEQ ID NO: 112. In some embodiments, the V H The region may have a Gly to Cys substitution at position 44 of SEQ ID NO: 109. In certain embodiments, the V of anti-HER2 DIV containing the Cys substitution H The region may have the sequence of SEQ ID NO:113.

[0106] In some embodiments, in part (a) of a bispecific protein having the structure of "Fab-Fc polypeptide / scFv-Fc polypeptide", the first Fc polypeptide is fused to the Fd portion of the Fab at the N-terminus via a hinge region or a partial hinge region. In some embodiments, in part (b) of a bispecific protein having the structure of "Fab-Fc polypeptide / scFv-Fc polypeptide", the second Fc polypeptide is fused to the scFv at the N-terminus via a hinge region or a partial hinge region. An exemplary hinge region sequence is the human IgG1 hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO: 127). A partial hinge region refers to a partial hinge region having a portion of the sequence of SEQ ID NO: 127, for example, the sequence of DKTHTCPPCP (SEQ ID NO: 128).

[0107] In a further embodiment, in part (b) of a bispecific protein having the structure "Fab-Fc polypeptide / scFv-Fc polypeptide", a hinge region (e.g., SEQ ID NO: 127) or a partial hinge region (e.g., SEQ ID NO: 128) is fused at the N-terminus of the second Fc polypeptide. In certain embodiments, when the hinge region is fused at the N-terminus of the second Fc polypeptide, the hinge region may comprise a Cys to Ser mutation at position 5 relative to the sequence of SEQ ID NO: 127. For example, a hinge region with a Cys to Ser mutation may have the sequence of EPKSSDKTHTCPPCP (SEQ ID NO: 129).

[0108] In bispecific proteins having a "Fab-Fc polypeptide / scFv-Fc polypeptide" structure, the first Fc polypeptide and / or the second Fc polypeptide can specifically bind to the transferrin receptor (e.g., a TfR-binding Fc polypeptide). Different Fc polypeptides and modifications thereof are described in further detail herein. In some embodiments, the first Fc polypeptide and the second Fc polypeptide can each include a modification that promotes heterodimerization. For example, according to EU numbering, the first Fc polypeptide can include a substitution of T366W, and the second Fc polypeptide can include a substitution of T366S, L368A, and Y407V. In another example, according to EU numbering, the first Fc polypeptide can include a substitution of T366S, L368A, and Y407V, and the second Fc polypeptide can include a substitution of T366W. Additionally, the first Fc polypeptide and / or the second Fc polypeptide can independently comprise a modification that reduces TfR-mediated effector function, i.e., reduces effector function upon TfR binding, e.g., the modification that reduces TfR-mediated effector function is, according to EU numbering, (i) L234A and L235A substitutions or (ii) L234A and L235A and P329G or P329S substitutions.

[0109] In certain embodiments of a bispecific protein having the structure "Fab-Fc polypeptide / scFv-Fc polypeptide", the first Fc polypeptide (or the second Fc polypeptide) is a TfR-binding Fc polypeptide comprising substitutions of T366W, L234A and L235A (optionally with substitutions of P329G or P329S), and optionally, S239D and / or I332E, according to EU numbering, and the second Fc polypeptide (or the first Fc polypeptide) comprises substitutions of T366S, L368A, and Y407V, and optionally, S239D and / or I332E, according to EU numbering. For example, the first Fc polypeptide (or the second Fc polypeptide) can comprise a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of the sequences of SEQ ID NOs: 137 and 186 to 196, and the second Fc polypeptide (or the first Fc polypeptide) can comprise a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of the sequences of SEQ ID NOs: 133 and 183 to 185.

[0110] In certain embodiments, the first Fc polypeptide is a TfR-binding Fc polypeptide and comprises a substitution of L234A and L235A (optionally comprising a substitution of P329G or P329S) according to EU numbering, and the second Fc polypeptide does not comprise a substitution of L234A or L325A (or a substitution of P329G or P329S, if present in the first Fc polypeptide). In certain embodiments, the first Fc polypeptide does not comprise a substitution of L234A or L325A (or a substitution of P329G or P329S, if present in the second Fc polypeptide), and the second Fc polypeptide is a TfR-binding Fc polypeptide and comprises a substitution of L234A and L235A (optionally comprising a substitution of P329G or P329S) according to EU numbering.

[0111] Exemplary Bispecific "Fab-Fc Polypeptide / scFv-Fc Polypeptide" Proteins In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain II of human HER2; (b) a second Fc polypeptide fused at its N-terminus to a single chain variable fragment (scFv) that binds subdomain IV of human HER2, wherein the first and second Fc polypeptides form an Fc dimer; and (c) a light chain polypeptide which pairs with the Fd portion described in (a) to form a Fab.

[0112] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0113] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0114] In one example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:1, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:159, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 164, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 173, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 164, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 174, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25.In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 166, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 173, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 165, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 173, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 165, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 174, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25.In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 167, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 173, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 164, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 175, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 164, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 176, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25.In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 166, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 175, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 165, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 175, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 165, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 176, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25.In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 167, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 175, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25.

[0115] In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:1, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:21 or 22, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:2, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:20, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:3, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:19, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25.In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:4 or 5, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:18, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25.

[0116] In yet another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:1, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:23, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25. In yet another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:5, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:24, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25. In both of these examples, the scFv portion comprises an anti-HER2 DIV VFv having a Gln to Cys substitution. L V of anti-HER2DIV with region (SEQ ID NO: 115) and Gly to Cys substitution HIn both constructs, the hinge region of (b) contains a Cys to Ser mutation at position 5 (EPKSSDKTHTCPPCP (SEQ ID NO: 129)).

[0117] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain IV of human HER2; (b) a second Fc polypeptide fused at its N-terminus to a single chain variable fragment (scFv) that binds subdomain II of human HER2, wherein the first and second Fc polypeptides form an Fc dimer; and (c) a light chain polypeptide which pairs with the Fd portion described in (a) to form a Fab.

[0118] In one embodiment, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0119] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0120] In one example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:6, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:15, 16, or 17, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:7, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:14, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:8, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:13, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26.In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:9 or 10, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:11 or 12, and (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26.

[0121] In some embodiments of the above bispecific proteins, the first Fc polypeptide comprises Leu at positions 234 and 235, according to EU numbering. In some embodiments, the second Fc polypeptide comprises Leu at positions 234 and 235, according to EU numbering. In certain embodiments, the protein comprises a cis-LALA configuration, as described below.

[0122] In some embodiments of the bispecific protein, one or both of the Fc polypeptides may have their C-terminal lysine removed (e.g., the Lys residue at position 447 of the Fc polypeptide according to EU numbering). In some embodiments, removal of the C-terminal lysine of the Fc polypeptide may improve the stability of the bispecific protein.

[0123] N- or C-terminal scFv of mAb / HC In some embodiments, the bispecific protein comprises an Fc polypeptide fused at each N-terminus to a Fab that specifically binds to subdomain II or IV of human HER2, and one or both Fc polypeptides are fused at their C-terminus to an scFv that specifically binds to subdomain II or IV of human HER2. In some embodiments, the bispecific protein comprises an Fc polypeptide fused at each N-terminus to a Fab that specifically binds to subdomain II or IV of human HER2, and one or both Fabs are fused at their N-terminus to an scFv that specifically binds to subdomain II or IV of human HER2. Schematic diagrams of such bispecific proteins are shown in Figures 2A-2D. In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to the Fd portion of a Fab; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; wherein the first Fc polypeptide and / or the second Fc polypeptide is C-terminally fused to an scFv; or the Fd portion in (a) and / or (b) is fused to an scFv at the N-terminus, or the first Fc polypeptide or the second Fc polypeptide is fused to an scFv at the C-terminus, and the Fd portion in (a) or (b) is fused to an scFv at the N-terminus; The Fab binds to subdomain II of human HER2 and the scFv binds to subdomain IV of human HER2, or the Fab binds to subdomain IV of human HER2 and the scFv binds to subdomain II of human HER2. In some embodiments, the Fab binds to subdomain II of human HER2 and the scFv binds to subdomain IV of human HER2. In other embodiments, the Fab binds to subdomain IV of human HER2 and the scFv binds to subdomain II of human HER2.

[0124] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0125] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises substitutions of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a S239D and I332E substitution and the second Fc polypeptide comprises a S239D and I332E substitution. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a S239D and I332E substitution. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a S239D substitution. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a I332E substitution.In some embodiments, the first Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E.

[0126] In some embodiments, a Fab that specifically binds to subdomain II of human HER2 has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 108. H In some embodiments, a Fab that specifically binds to subdomain IV of human HER2 comprises a V region having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 109. H Includes the area.

[0127] In certain embodiments, the first Fc polypeptide and / or the second Fc polypeptide are fused to the scFv at the C-terminus. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide are fused to the scFv at the C-terminus. In certain embodiments, each of the first Fc polypeptide and the second Fc polypeptide are fused to the scFv at the C-terminus. In certain embodiments, the Fd portion in (a) and / or (b) is fused to the scFv at the N-terminus. In certain embodiments, the Fd portion in (a) or (b) is fused to the scFv at the N-terminus. In certain embodiments, each of the Fd portions in (a) and (b) is fused to the scFv at the N-terminus. In further embodiments, the first Fc polypeptide or the second Fc polypeptide is fused to the scFv at the C-terminus and the Fd portion in (a) or (b) is fused to the scFv at the N-terminus.

[0128] In some embodiments, when the first Fc polypeptide and the second Fc polypeptide are each fused to the scFv at the C-terminus, the two scFvs may comprise the same sequence. In some embodiments, when the Fd portion in (a) and the Fd portion in (b) are each fused to the scFv at the N-terminus, the two scFvs may comprise the same sequence. In some embodiments, when the first Fc polypeptide or the second Fc polypeptide are fused to the scFv at the C-terminus, and the Fd portion in (a) or (b) is fused to the scFv at the N-terminus, the two scFvs may comprise the same sequence.

[0129] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide is fused to the scFv at the C-terminus via a first linker. In other embodiments, the Fd portion in (a) and / or the Fd portion in (b) is fused to the scFv at the N-terminus via a first linker. In some embodiments, the first linker has a length of about 1 to about 50 amino acids, for example, about 1 to about 40, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 2 to about 40, about 2 to about 30, about 2 to about 20, about 2 to about 10, about 5 to about 40, about 5 to about 30, about 5 to about 25, or about 5 to about 20 amino acids. In some embodiments, the first linker has a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 amino acids. Various linkers are described in more detail herein. In certain embodiments, the first linker is GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 116, (GGSG) 5 ), GGGGS (SEQ ID NO: 117, G 4 S), GGGGSGGGGS (SEQ ID NO: 118, (G 4 S) 2 ), GGGGSGGGGSGGGGS (SEQ ID NO: 119, (G 4 S) 3 ), GGGGSGGGGSGGGG (SEQ ID NO: 120, (G 4 S)2 -G 4 ), GGGGSGGGGSGG (SEQ ID NO: 121), GGGGSGGGGGS (SEQ ID NO: 122), and GGGGGSGGGGGSGGGGGS (SEQ ID NO: 123).

[0130] In some embodiments, the scFvs of the bispecific protein are V H Area and V L In some embodiments, the V of the scFv L Area and V H The orientation of the domain is (N-terminus)-V L Area-V H In another embodiment, the V of the scFv is a region-(C-terminus), and the C-terminus of the scFv is linked to the N-terminus of the Fd portion in (a) or (b) via the first linker. L Area and V H The orientation of the domain is (N-terminus)-V H Area-V L Region-(C-terminus), wherein the C-terminus of the scFv is linked to the N-terminus of the Fd moiety in (a) or (b) via the first linker. In some embodiments, the V L Area and V H The orientation of the domain is (N-terminus)-V L Area-V H In another embodiment, the V of the scFv is a C-terminus region (C-terminus), and the N-terminus of the scFv is linked to the C-terminus of the first Fc polypeptide or the second Fc polypeptide via the first linker. L Area and V H The orientation of the domain is (N-terminus)-V H Area-V L Region-(C-terminus), wherein the N-terminus of the scFv is linked to the C-terminus of the first Fc polypeptide or the second Fc polypeptide via the first linker.

[0131] In some embodiments, the V of the scFv L Area and V HThe second linker connecting the regions has a length of about 10 to about 25 amino acids, e.g., about 10 to about 20, about 12 to about 25, about 12 to about 20, about 14 to about 25, or about 14 to about 20 amino acids. In some embodiments, the second linker has a length of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some embodiments, the second linker comprises a flexible linker. Various linkers are described in further detail herein. In some embodiments, the second linker is GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 116, (GGSG) 5 ), GGGGS (SEQ ID NO: 117, G 4 S), GGGGSGGGGS (SEQ ID NO: 118, (G 4 S) 2 ), GGGGSGGGGSGGGGS (SEQ ID NO: 119, (G 4 S) 3 ), GGGGSGGGGSGGGG (SEQ ID NO: 120, (G 4 S) 2 -G 4 ), GGGGSGGGGSGG (SEQ ID NO: 121), GGGGSGGGGGS (SEQ ID NO: 122), and GGGGGSGGGGGSGGGGGS (SEQ ID NO: 123).

[0132] In some embodiments, the V of the scFv L Area and V H In some embodiments, both of the V L Area and corresponding V H The Cys substitutions in the regions may form disulfide bonds to help stabilize the structure of the scFv. In some embodiments, the scFv comprises the variable domain numbering system V according to Kabat. H 44 and V L In some embodiments, the scFv comprises a cysteine ​​at each of positions 100 and 110. H 44 and V L It contains a disulfide bond between the cysteines at position 100.

[0133] For example, anti-HER2DII V L The region may have a Gln to Cys substitution at position 100 of SEQ ID NO: 110. In certain embodiments, the V of anti-HER2 DII containing the Cys substitution L The region may have the sequence of SEQ ID NO: 114. In some embodiments, the V L The region may have a Gln to Cys substitution at position 100 of SEQ ID NO: 111. In certain embodiments, the V of anti-HER2 DIV containing the Cys substitution L The region may have the sequence of SEQ ID NO:115.

[0134] For example, anti-HER2DII V H The region may have a Gly to Cys substitution at position 44 of SEQ ID NO: 108. In certain embodiments, the V of anti-HER2 DII containing the Cys substitution H The region may have the sequence of SEQ ID NO: 112. In some embodiments, the V H The region may have a Gly to Cys substitution at position 44 of SEQ ID NO: 109. In certain embodiments, the V of anti-HER2 DIV containing the Cys substitution H The region may have the sequence of SEQ ID NO:113.

[0135] In some embodiments, in part (a) of a bispecific protein having the structure "N- or C-terminal scFv of mAb / HC", the first Fc polypeptide is fused to the Fd portion of the Fab at the N-terminus via a hinge region or partial hinge region. In some embodiments, in part (b) of a bispecific protein having the structure "N- or C-terminal scFv of mAb / HC", the second Fc polypeptide is fused to the Fd portion of the Fab at the N-terminus via a hinge region or partial hinge region. An exemplary hinge region sequence is the human IgG1 hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO: 127). A partial hinge region refers to a partial hinge region having a portion of the sequence of SEQ ID NO: 127, for example, the sequence of DKTHTCPPCP (SEQ ID NO: 128).

[0136] In bispecific proteins with the structure of "mAb / HC N- or C-terminal scFv", the first Fc polypeptide and / or the second Fc polypeptide can specifically bind to the transferrin receptor (e.g., TfR-binding Fc polypeptide). Different Fc polypeptides and modifications thereof are described in further detail herein. In some embodiments, the first Fc polypeptide and the second Fc polypeptide can each include a modification that promotes heterodimerization. For example, according to EU numbering, the first Fc polypeptide can include a substitution of T366W, and the second Fc polypeptide can include a substitution of T366S, L368A, and Y407V. In another example, according to EU numbering, the first Fc polypeptide can include a substitution of T366S, L368A, and Y407V, and the second Fc polypeptide can include a substitution of T366W. Additionally, the first Fc polypeptide and / or the second Fc polypeptide can independently comprise a modification that reduces TfR-mediated effector function, i.e., reduces effector function upon TfR binding, e.g., the modification that reduces TfR-mediated effector function is, according to EU numbering, (i) L234A and L235A substitutions or (ii) L234A and L235A and P329G or P329S substitutions.

[0137] In certain embodiments of a bispecific protein having the structure "mAb / HC N- or C-terminal scFv", the first Fc polypeptide (or the second Fc polypeptide) is a TfR-binding Fc polypeptide comprising substitutions of T366W, L234A and L235A (optionally with substitutions of P329G or P329S), and optionally, S239D and / or I332E, according to EU numbering, and the second Fc polypeptide (or the first Fc polypeptide) comprises substitutions of T366S, L368A, and Y407V, and optionally, S239D and / or I332E, according to EU numbering. For example, the first Fc polypeptide (or the second Fc polypeptide) can comprise a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of the sequences of SEQ ID NOs: 137 and 186 to 196, and the second Fc polypeptide (or the first Fc polypeptide) can comprise a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of the sequences of SEQ ID NOs: 133 and 183 to 185.

[0138] In certain embodiments, the first Fc polypeptide is a TfR-binding Fc polypeptide and comprises a substitution of L234A and L235A (optionally comprising a substitution of P329G or P329S) according to EU numbering, and the second Fc polypeptide does not comprise a substitution of L234A or L325A (or a substitution of P329G or P329S, if present in the first Fc polypeptide). In certain embodiments, the first Fc polypeptide does not comprise a substitution of L234A or L325A (or a substitution of P329G or P329S, if present in the second Fc polypeptide), and the second Fc polypeptide is a TfR-binding Fc polypeptide and comprises a substitution of L234A and L235A (optionally comprising a substitution of P329G or P329S) according to EU numbering.

[0139] Exemplary bispecific "N- or C-terminal scFv of mAb / HC" proteins In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain II of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; wherein the first Fc polypeptide or the second Fc polypeptide is fused at its C-terminus to an scFv that binds to subdomain IV of human HER2.

[0140] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0141] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0142] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 27 or 28, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 4 or 5, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:29, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:3, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:30, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:2, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25.In yet another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:31 or 32, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:1, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25.

[0143] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain IV of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; wherein the first Fc polypeptide or the second Fc polypeptide is fused at its C-terminus to an scFv that binds to subdomain II of human HER2.

[0144] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0145] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0146] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 33 or 34, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 9 or 10, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:35, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:8, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:36, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:7, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26.In yet another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:37, 38, or 39; (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:6; and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26.

[0147] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain II of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; wherein the first Fc polypeptide and the second Fc polypeptide are each fused at their C-terminus to an scFv that binds to subdomain IV of human HER2.

[0148] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0149] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0150] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 27 or 28, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 31 or 32, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:29, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:30, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25.

[0151] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain IV of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, the first Fc polypeptide and the second Fc polypeptide are each fused at their C-terminus to an scFv that binds to subdomain II of human HER2.

[0152] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0153] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0154] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 33 or 34, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 37, 38, or 39, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:35, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:36, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26.

[0155] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain II of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, the Fd portion in (a) or (b) is fused at the N-terminus to an scFv that binds to subdomain IV of human HER2.

[0156] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0157] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0158] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 40, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 4 or 5, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:41, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:3, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:42, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:2, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25.In yet another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:43 or 44, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:1, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25.

[0159] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain IV of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, the Fd portion in (a) or (b) is fused at the N-terminus to an scFv that binds to subdomain II of human HER2.

[0160] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0161] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0162] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:6, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:160, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:6, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:161, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:6, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:162, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26.In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 169, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 177, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 169, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 178, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 171, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 177, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26.In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 170, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 177, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 170, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 178, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 172, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 177, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26.In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 169, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 179, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 169, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 180, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 171, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 179, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26.In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 170, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 179, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 170, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 180, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 172, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 179, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26.In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 169, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 181, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 169, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 182, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:171, and (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:181. and (c) each of the two light chain polypeptides comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 170, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 181, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 170, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 182, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 172, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 181, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26.

[0163] In another example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 45 or 46, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 9 or 10, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:47, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:8, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:48, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:7, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26.In yet another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:49 or 50, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:6, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26.

[0164] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain II of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, each of the Fd moieties in (a) and (b) is fused at its N-terminus to an scFv that binds to subdomain IV of human HER2.

[0165] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0166] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0167] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 40, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 43 or 44, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:41, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:42, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25.

[0168] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain IV of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, each of the Fd moieties in (a) and (b) is fused at its N-terminus to an scFv that binds to subdomain II of human HER2.

[0169] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0170] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0171] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 45, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 49 or 50, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:47, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:48, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26.

[0172] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain II of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, the first Fc polypeptide or the second Fc polypeptide is fused at the C-terminus to an scFv that binds to subdomain IV of human HER2, and the Fd portion in (a) or (b) is fused at the N-terminus to the scFv.

[0173] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0174] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0175] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 40, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 31 or 32, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:41, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:30, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:42, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:29, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25.In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 43 or 44, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 27 or 28, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25.

[0176] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain IV of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, the first Fc polypeptide or the second Fc polypeptide is fused at the C-terminus to an scFv that binds to subdomain II of human HER2, and the Fd portion in (a) or (b) is fused at the N-terminus to the scFv.

[0177] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0178] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0179] In one example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:45, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:37, 38, or 39, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:47, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:36, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:48, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:35, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26.In yet another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 49 or 50, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 33 or 34, and each of the two light chain polypeptides of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26.

[0180] In some embodiments of the above bispecific proteins, the first Fc polypeptide comprises Leu at positions 234 and 235, according to EU numbering. In some embodiments, the second Fc polypeptide comprises Leu at positions 234 and 235, according to EU numbering. In certain embodiments, the protein comprises a cis-LALA configuration, as described below.

[0181] In some embodiments of the bispecific protein, one or both of the Fc polypeptides may have their C-terminal lysine removed (e.g., the Lys residue at position 447 of the Fc polypeptide according to EU numbering). In some embodiments, removal of the C-terminal lysine of the Fc polypeptide may improve the stability of the bispecific protein.

[0182] N- or C-terminal scFv of mAb / LC In some embodiments, the bispecific protein comprises a light chain fused at its N-terminus and / or C-terminus to an scFv that specifically binds to subdomain II or IV of human HER2. Schematic diagrams of such bispecific proteins are shown in Figures 3A-3D. In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to the Fd portion of a Fab; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; wherein one or both of the light chain polypeptides are N-terminally fused to an scFv; or one or both of the light chain polypeptides are C-terminally fused to an scFv, or a first light chain polypeptide is N-terminally fused to the scFv and a second light chain polypeptide is C-terminally fused to the scFv; The Fab binds to subdomain II of human HER2 and the scFv binds to subdomain IV of human HER2, or the Fab binds to subdomain IV of human HER2 and the scFv binds to subdomain II of human HER2.

[0183] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0184] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises substitutions of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a S239D and I332E substitution and the second Fc polypeptide comprises a S239D and I332E substitution. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a S239D and I332E substitution. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a S239D substitution. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a I332E substitution.In some embodiments, the first Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E.

[0185] In some embodiments, a Fab that specifically binds to subdomain II of human HER2 has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 108. H In some embodiments, a Fab that specifically binds to subdomain IV of human HER2 comprises a V region having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 109. H Includes the area.

[0186] In certain embodiments, one or both of the light chain polypeptides are fused to the scFv at the N-terminus. In certain embodiments, one of the light chain polypeptides is fused to the scFv at the N-terminus. In certain embodiments, each of the two light chain polypeptides is fused to the scFv at the N-terminus. In certain embodiments, one or both of the light chain polypeptides are fused to the scFv at the C-terminus. In certain embodiments, one of the light chain polypeptides is fused to the scFv at the C-terminus. In certain embodiments, each of the two light chain polypeptides is fused to the scFv at the C-terminus. In further embodiments, the first light chain polypeptide is fused to the scFv at the N-terminus and the second light chain polypeptide is fused to the scFv at the C-terminus.

[0187] In some embodiments, when both light chain polypeptides are each fused C-terminally to the scFv, the two scFvs may comprise the same sequence. In some embodiments, when both light chain polypeptides are each fused N-terminally to the scFv, the two scFvs may comprise the same sequence.

[0188] In some embodiments, one or both of the light chain polypeptides are fused to the scFv via a first linker. In some embodiments, the first linker has a length of about 1 to about 50 amino acids, for example, about 1 to about 40, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 2 to about 40, about 2 to about 30, about 2 to about 20, about 2 to about 10, about 5 to about 40, about 5 to about 30, about 5 to about 25, or about 5 to about 20 amino acids. In some embodiments, the first linker has a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 amino acids. Various linkers are described in further detail herein. In certain embodiments, the first linker is GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 116, (GGSG) 5 ), GGGGS (SEQ ID NO: 117, G 4 S), GGGGSGGGGS (SEQ ID NO: 118, (G 4 S) 2 ), GGGGSGGGGSGGGGS (SEQ ID NO: 119, (G 4 S) 3 ), GGGGSGGGGSGGGG (SEQ ID NO: 120, (G 4 S) 2 -G 4 ), GGGGSGGGGSGG (SEQ ID NO: 121), GGGGSGGGGGS (SEQ ID NO: 122), and GGGGGSGGGGGSGGGGGS (SEQ ID NO: 123).

[0189] In some embodiments, the scFvs of the bispecific protein are V H Area and V L In some embodiments, the V of the scFv L Area and V H The orientation of the domain is (N-terminus)-V L Area-V H In another embodiment, the V of the scFv is linked to the N-terminus of the light chain polypeptide via the first linker.L Area and V H The orientation of the domain is (N-terminus)-V H Area-V L In some embodiments, the V of the scFv is a C-terminus, and the C-terminus of the scFv is linked to the N-terminus of the light chain polypeptide via the first linker. L Area and V H The orientation of the domain is (N-terminus)-V L Area-V H In another embodiment, the V of the scFv is a C-terminus region (C-terminus), and the N-terminus of the scFv is linked to the C-terminus of the light chain polypeptide via the first linker. L Area and V H The orientation of the domain is (N-terminus)-V H Area-V L Region-(C-terminus), where the N-terminus of the scFv is linked to the C-terminus of the light chain polypeptide via the first linker.

[0190] In some embodiments, the V of the scFv L Area and V H The second linker connecting the regions has a length of about 10 to about 25 amino acids, e.g., about 10 to about 20, about 12 to about 25, about 12 to about 20, about 14 to about 25, or about 14 to about 20 amino acids. In some embodiments, the second linker has a length of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some embodiments, the second linker comprises a flexible linker. Various linkers are described in further detail herein. In some embodiments, the second linker is GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 116, (GGSG) 5 ), GGGGS (SEQ ID NO: 117, G 4 S), GGGGSGGGGS (SEQ ID NO: 118, (G 4 S) 2 ), GGGGSGGGGSGGGGS (SEQ ID NO: 119, (G 4 S) 3 ), GGGGSGGGGSGGGG (SEQ ID NO: 120, (G 4 S) 2-G 4 ), GGGGSGGGGSGG (SEQ ID NO: 121), GGGGSGGGGGS (SEQ ID NO: 122), and GGGGGSGGGGGSGGGGGS (SEQ ID NO: 123).

[0191] In some embodiments, the V of the scFv L Area and V H In some embodiments, both of the V L Area and corresponding V H The Cys substitutions in the regions may form disulfide bonds to help stabilize the structure of the scFv. In some embodiments, the scFv comprises the variable domain numbering system V according to Kabat. H 44 and V L In some embodiments, the scFv comprises a cysteine ​​at each of positions 100 and 110. H 44 and V L It contains a disulfide bond between the cysteines at position 100.

[0192] For example, anti-HER2DII V L The region may have a Gln to Cys substitution at position 100 of SEQ ID NO: 110. In certain embodiments, the V of anti-HER2 DII containing the Cys substitution L The region may have the sequence of SEQ ID NO: 114. In some embodiments, the V L The region may have a Gln to Cys substitution at position 100 of SEQ ID NO: 111. In certain embodiments, the V of anti-HER2 DIV containing the Cys substitution L The region may have the sequence of SEQ ID NO:115.

[0193] For example, anti-HER2DII V H The region may have a Gly to Cys substitution at position 44 of SEQ ID NO: 108. In certain embodiments, the V of anti-HER2 DII containing the Cys substitution H The region may have the sequence of SEQ ID NO: 112. In some embodiments, the V HThe region may have a Gly to Cys substitution at position 44 of SEQ ID NO: 109. In certain embodiments, the V of anti-HER2 DIV containing the Cys substitution H The region may have the sequence of SEQ ID NO:113.

[0194] In some embodiments, in part (a) of a bispecific protein having the structure "N- or C-terminal scFv of mAb / LC", the first Fc polypeptide is fused to the Fd portion of the Fab at the N-terminus via a hinge region or partial hinge region. In some embodiments, in part (b) of a bispecific protein having the structure "N- or C-terminal scFv of mAb / LC", the second Fc polypeptide is fused to the Fd portion of the Fab at the N-terminus via a hinge region or partial hinge region. An exemplary hinge region sequence is the human IgG1 hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO: 127). A partial hinge region refers to a partial hinge region having a portion of the sequence of SEQ ID NO: 127, for example, the sequence of DKTHTCPPCP (SEQ ID NO: 128).

[0195] In bispecific proteins with the structure of "mAb / LC N- or C-terminal scFv", the first Fc polypeptide and / or the second Fc polypeptide can specifically bind to the transferrin receptor (e.g., TfR-binding Fc polypeptide). Different Fc polypeptides and modifications thereof are described in further detail herein. In some embodiments, the first Fc polypeptide and the second Fc polypeptide can each include a modification that promotes heterodimerization. For example, according to EU numbering, the first Fc polypeptide can include a substitution of T366W, and the second Fc polypeptide can include a substitution of T366S, L368A, and Y407V. In another example, according to EU numbering, the first Fc polypeptide can include a substitution of T366S, L368A, and Y407V, and the second Fc polypeptide can include a substitution of T366W. Additionally, the first Fc polypeptide and / or the second Fc polypeptide can independently comprise a modification that reduces TfR-mediated effector function, i.e., reduces effector function upon TfR binding, e.g., the modification that reduces TfR-mediated effector function is, according to EU numbering, (i) L234A and L235A substitutions or (ii) L234A and L235A and P329G or P329S substitutions.

[0196] In certain embodiments of a bispecific protein having the structure "mAb / HC N- or C-terminal scFv", the first Fc polypeptide (or the second Fc polypeptide) is a TfR-binding Fc polypeptide comprising substitutions of T366W, L234A and L235A (optionally with substitutions of P329G or P329S), and optionally, S239D and / or I332E, according to EU numbering, and the second Fc polypeptide (or the first Fc polypeptide) comprises substitutions of T366S, L368A, and Y407V, and optionally, S239D and / or I332E, according to EU numbering. For example, the first Fc polypeptide (or the second Fc polypeptide) can comprise a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of the sequences of SEQ ID NOs: 137 and 186 to 196, and the second Fc polypeptide (or the first Fc polypeptide) can comprise a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of the sequences of SEQ ID NOs: 133 and 183 to 185.

[0197] In certain embodiments, the first Fc polypeptide is a TfR-binding Fc polypeptide and comprises a substitution of L234A and L235A (optionally comprising a substitution of P329G or P329S) according to EU numbering, and the second Fc polypeptide does not comprise a substitution of L234A or L325A (or a substitution of P329G or P329S, if present in the first Fc polypeptide). In certain embodiments, the first Fc polypeptide does not comprise a substitution of L234A or L325A (or a substitution of P329G or P329S, if present in the second Fc polypeptide), and the second Fc polypeptide is a TfR-binding Fc polypeptide and comprises a substitution of L234A and L235A (optionally comprising a substitution of P329G or P329S) according to EU numbering.

[0198] Exemplary bispecific "N- or C-terminal scFv of mAb / LC" proteins In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain II of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, one of the light chain polypeptides is fused at its C-terminus to an scFv that binds to subdomain IV of human HER2.

[0199] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0200] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0201] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:1, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:4 or 5, and the first light chain polypeptide comprises a first light chain polypeptide having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:4 or 5, and The first light chain polypeptide is fused at the C-terminus to the scFv and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:52, and the second light chain polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:2, and (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:3, and the first light chain polypeptide comprises and the second light chain polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:52, and the second light chain polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25.

[0202] In some embodiments the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain IV of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, one of the light chain polypeptides is fused at its C-terminus to an scFv that binds to subdomain II of human HER2.

[0203] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0204] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0205] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 6, and (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 9 or 10, and the first light chain polypeptide. The first light chain polypeptide is fused at its C-terminus to the scFv and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:53 or 54, and the second light chain polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26. In yet another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:7, and (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:8, and the first light chain polypeptide comprises the s and the second light chain polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 53 or 54, and the second light chain polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26.

[0206] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain II of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, each of the two light chain polypeptides is fused at its C-terminus to an scFv that binds to subdomain IV of human HER2.

[0207] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0208] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0209] In one example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:1, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:4 or 5, and each of the two light chain polypeptides is fused at its C-terminus to the scFv and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:52. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:2, and (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:3, and each of the two light chain polypeptides is fused at its C-terminus to the scFv and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:52.

[0210] In some embodiments, the bispecific protein comprises: In some embodiments the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain IV of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, each of the two light chain polypeptides is fused at its C-terminus to an scFv that binds to subdomain II of human HER2.

[0211] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0212] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0213] In one example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 6, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 9 or 10, and each of the two light chain polypeptides is fused at its C-terminus to the scFv and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 53 or 54. In yet another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:7, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:8, and each of the two light chain polypeptides is fused at the C-terminus to the scFv and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:53 or 54.

[0214] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain II of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, one of the light chain polypeptides is fused at its N-terminus to an scFv that binds to subdomain IV of human HER2.

[0215] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0216] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0217] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:1, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:4 or 5, and the first light chain polypeptide comprises a first light chain polypeptide having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:4 or 5, and The first light chain polypeptide is fused at the N-terminus to the scFv and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:55, and the second light chain polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:2, and (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:3, and the first light chain polypeptide comprises and the second light chain polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:55, and the second light chain polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25.

[0218] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain IV of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, one of the light chain polypeptides is fused at its N-terminus to an scFv that binds to subdomain II of human HER2.

[0219] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0220] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0221] In one example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 6, and (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 9 or 10, and the first light chain polypeptide comprises and the second light chain polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 56 or 57, and the second light chain polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:7, and (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:8, and the first light chain polypeptide is v and the second light chain polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:56 or 57, and the second light chain polypeptide comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26.

[0222] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain II of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, each of the two light chain polypeptides is fused at its N-terminus to an scFv that binds to subdomain IV of human HER2.

[0223] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0224] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0225] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:1, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:4 or 5, and each of the two light chain polypeptides is fused at its C-terminus to the scFv and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:55. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:2, and (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:3, and each of the two light chain polypeptides is fused at its C-terminus to the scFv and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:55.

[0226] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain IV of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, each of the two light chain polypeptides is fused at its N-terminus to an scFv that binds to subdomain II of human HER2.

[0227] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0228] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0229] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 6, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 9 or 10, and each of the two light chain polypeptides is fused at its C-terminus to the scFv and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 56 or 57. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:7, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:8, and each of the two light chain polypeptides is fused at its C-terminus to the scFv and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:56 or 57.

[0230] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain II of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, a first light chain polypeptide is fused at its N-terminus to an scFv that binds to subdomain IV of human HER2, and a second light chain polypeptide is fused at its C-terminus to said scFv.

[0231] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0232] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0233] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:1, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:4 or 5, and the first light chain polypeptide comprises a first light chain polypeptide fused to the scFv at its N-terminus and comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:55, and a second light chain polypeptide fused to the scFv at its C-terminus and comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:52. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:2, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:3, and the first light chain polypeptide is N-terminally linked to the scFv. and a second light chain polypeptide is fused at the C-terminus to the scFv and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:55, and a second light chain polypeptide is fused at the C-terminus to the scFv and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:52.

[0234] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain IV of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; Here, a first light chain polypeptide is fused at its N-terminus to an scFv that binds to subdomain II of human HER2, and a second light chain polypeptide is fused at its C-terminus to the scFv.

[0235] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0236] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0237] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 6, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 9 or 10, and a first light chain polypeptide is fused N-terminally to the scFv and has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:53 or 54, and a second light chain polypeptide is fused at the C-terminus to the scFv and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26.In yet another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:7, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:8, and a first light chain polypeptide is fused N-terminally to the scFv and has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:53 or 54, and a second light chain polypeptide is fused at the C-terminus to the scFv and comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26.

[0238] In some embodiments of the above bispecific proteins, the first Fc polypeptide comprises Leu at positions 234 and 235, according to EU numbering. In some embodiments, the second Fc polypeptide comprises Leu at positions 234 and 235, according to EU numbering. In certain embodiments, the protein comprises a cis-LALA configuration, as described below.

[0239] In some embodiments of the bispecific protein, one or both of the Fc polypeptides may have their C-terminal lysine removed (e.g., the Lys residue at position 447 of the Fc polypeptide according to EU numbering). In some embodiments, removal of the C-terminal lysine of the Fc polypeptide may improve the stability of the bispecific protein.

[0240] N-Terminal V of mAb / HC and LC H V L In some embodiments, the bispecific protein comprises a V fused at the N-terminus of each of the two heavy chains. H Area (or V L region) and V fused at the N-terminus of each of the two light chains L Area (or V H A schematic of such a bispecific protein is shown in Figure 4. In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to the Fd portion of a Fab; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; wherein each of the Fd moieties in (a) and (b) is an Fv fragment V at its N-terminus. H Area or V L It is integrated with the realm, Each of the two light chain polypeptides comprises at its N-terminus the V domain of the Fv fragment. H Area or V L It is fused with the other side of the realm, Applicable V H Area and corresponding V L the regions together form the Fv fragment, The Fab binds to subdomain II of human HER2 and the Fv fragment binds to subdomain IV of human HER2, or the Fab binds to subdomain IV of human HER2 and the Fv fragment binds to subdomain II of human HER2.

[0241] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0242] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises substitutions of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a S239D and I332E substitution and the second Fc polypeptide comprises a S239D and I332E substitution. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a S239D and I332E substitution. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a S239D substitution. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a I332E substitution.In some embodiments, the first Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E.

[0243] In some embodiments of the bispecific protein, each of the Fd moieties in (a) and (b) comprises at its N-terminus the V of an Fv fragment. H and each of the two light chain polypeptides is fused at its N-terminus to the V domain of the Fv fragment. L In another embodiment, each of the Fd moieties in (a) and (b) is fused at its N-terminus to the V domain of the Fv fragment. L and each of the two light chain polypeptides is fused at its N-terminus to the V domain of the Fv fragment. H It merges with the realm.

[0244] In some embodiments, a Fab that specifically binds to subdomain II of human HER2 has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 108. H In some embodiments, a Fab that specifically binds to subdomain IV of human HER2 comprises a V region having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 109. H Includes the area.

[0245] In some embodiments, the first linker is V H Area or V L In some embodiments, the second linker connects the V H Area or V LThe region is connected to the N-terminus of each of the two light chain polypeptides. In some embodiments, the first linker or the second linker has a length of about 1 to about 50 amino acids, for example, about 1 to about 40, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 2 to about 40, about 2 to about 30, about 2 to about 20, about 2 to about 10, about 5 to about 40, about 5 to about 30, about 5 to about 25, or about 5 to about 20 amino acids. In some embodiments, the first linker has a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 amino acids. Various linkers are described in more detail herein.In certain embodiments, the first linker comprises the sequence of ASTKGPSVF (SEQ ID NO: 125).In certain embodiments, the second linker comprises the sequence of RTVAAPSVFI (SEQ ID NO: 126).

[0246] In some embodiments, the N-terminal V H V L In part (a) of a bispecific protein having the structure "A", the first Fc polypeptide is fused at its N-terminus to the Fd portion of the Fab via a hinge region or partial hinge region. In some embodiments, the N-terminal V of the mAb / HC and LC is H V L In part (b) of a bispecific protein having the structure "(a)", the second Fc polypeptide is fused at its N-terminus to the Fd portion of the Fab via a hinge region or a partial hinge region. An exemplary hinge region sequence is the human IgG1 hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO: 127). A partial hinge region refers to a portion of the sequence of SEQ ID NO: 127, for example a partial hinge region having the sequence DKTHTCPPCP (SEQ ID NO: 128).

[0247] "mAb / HC and LC N-terminal V H V LIn a bispecific protein having the structure "T366W" and / or "Y407V", the first Fc polypeptide and / or the second Fc polypeptide can specifically bind to the transferrin receptor (e.g., a TfR-binding Fc polypeptide). Different Fc polypeptides and modifications thereof are described in further detail herein. In some embodiments, the first Fc polypeptide and the second Fc polypeptide can each comprise a modification that promotes heterodimerization. For example, according to EU numbering, the first Fc polypeptide can comprise a substitution of T366W and the second Fc polypeptide can comprise a substitution of T366S, L368A, and Y407V. In another example, according to EU numbering, the first Fc polypeptide can comprise a substitution of T366S, L368A, and Y407V and the second Fc polypeptide can comprise a substitution of T366W. Additionally, the first Fc polypeptide and / or the second Fc polypeptide can independently comprise a modification that reduces TfR-mediated effector function, i.e., reduces effector function upon TfR binding, e.g., the modification that reduces TfR-mediated effector function is, according to EU numbering, (i) L234A and L235A substitutions or (ii) L234A and L235A and P329G or P329S substitutions.

[0248] "mAb / HC and LC N-terminal V H V L", wherein the first Fc polypeptide (or the second Fc polypeptide) is a TfR-binding Fc polypeptide comprising substitutions of T366W, L234A and L235A (optionally with substitutions of P329G or P329S), and optionally, S239D and / or I332E, according to EU numbering, and the second Fc polypeptide (or the first Fc polypeptide) comprises substitutions of T366S, L368A, and Y407V, and optionally, S239D and / or I332E, according to EU numbering. For example, the first Fc polypeptide (or the second Fc polypeptide) can comprise a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of the sequences of SEQ ID NOs: 137 and 186 to 196, and the second Fc polypeptide (or the first Fc polypeptide) can comprise a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of the sequences of SEQ ID NOs: 133 and 183 to 185.

[0249] In certain embodiments, the first Fc polypeptide is a TfR-binding Fc polypeptide and comprises a substitution of L234A and L235A (optionally comprising a substitution of P329G or P329S) according to EU numbering, and the second Fc polypeptide does not comprise a substitution of L234A or L325A (or a substitution of P329G or P329S, if present in the first Fc polypeptide). In certain embodiments, the first Fc polypeptide does not comprise a substitution of L234A or L325A (or a substitution of P329G or P329S, if present in the second Fc polypeptide), and the second Fc polypeptide is a TfR-binding Fc polypeptide and comprises a substitution of L234A and L235A (optionally comprising a substitution of P329G or P329S) according to EU numbering.

[0250] Exemplary bispecific "mAb / HC and LC N-terminal V H V L "protein In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain II of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; wherein each of the Fd portions in (a) and (b) is a V of an Fv fragment that binds to subdomain IV of human HER2. H It is fused to the N-terminus of the Each of the two light chain polypeptides comprises at its N-terminus the V domain of the Fv fragment. L It is fused with the other side of the realm, Applicable V H Area and corresponding V L The regions combine to form the Fv fragment.

[0251] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0252] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0253] In one example, the bispecific protein comprises at the N-terminus the V of the Fv fragment. H a first polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:58, fused to a region (a) of the Fv fragment; H a second polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 61 or 62, and a V L and a third and fourth polypeptide each comprising a light chain polypeptide fused to a region and each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 78. In another example, the bispecific protein comprises a light chain polypeptide fused to a region of the Fv fragment at the N-terminus thereof. Ha first polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:59, fused to a region (a) of the Fv fragment; H a second polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 60, and a V L and a third and fourth polypeptide each comprising a light chain polypeptide fused to the region and each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:78.

[0254] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain IV of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; wherein each of the Fd portions in (a) and (b) is a V of an Fv fragment that binds to subdomain II of human HER2. H It is fused to the N-terminus of the Each of the two light chain polypeptides comprises at its N-terminus the V domain of the Fv fragment. L It is fused with the other side of the realm, Applicable V H Area and corresponding V L The regions combine to form the Fv fragment.

[0255] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0256] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0257] In one example, the bispecific protein comprises the V of the Fv fragment at the N-terminus. Ha first polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 63, fused to a region (a) of the Fv fragment; H a second polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 66 or 67, and a V L and a third and fourth polypeptide each comprising a light chain polypeptide fused to a region and each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 79. In another example, the first polypeptide comprises at its N-terminus the V of the Fv fragment. H and a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 64, wherein the second polypeptide comprises at its N-terminus the V of the Fv fragment. H and a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 65, and the third and fourth polypeptides each have at their N-terminus the V of the Fv fragment. L and a light chain polypeptide fused to a region, each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:79.

[0258] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain II of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; wherein each of the Fd portions in (a) and (b) is a V of an Fv fragment that binds to subdomain IV of human HER2. L It is fused to the N-terminus of the Each of the two light chain polypeptides comprises at its N-terminus the V domain of the Fv fragment. H It is fused with the other side of the realm, Applicable V H Area and corresponding V L The regions combine to form the Fv fragment.

[0259] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0260] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0261] In one example, the bispecific protein comprises the V of the Fv fragment at the N-terminus. L a first polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:68, fused to a region (a) of the Fv fragment; L a second polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 71 or 72, and a V H and a third and fourth polypeptide each comprising a light chain polypeptide fused to a region and each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 80. In another example, the bispecific protein comprises a light chain polypeptide fused to a region of the Fv fragment at the N-terminus thereof. La first polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:69, fused to a region (a) of the Fv fragment; L a second polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 70, and a V H and a third and fourth polypeptide each comprising a light chain polypeptide fused to the region and each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:80.

[0262] In some embodiments, the bispecific protein comprises: (a) a first Fc polypeptide fused at its N-terminus to an Fd portion of a Fab that binds to subdomain IV of human HER2; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; wherein each of the Fd portions in (a) and (b) is a V of an Fv fragment that binds to subdomain II of human HER2. L It is fused to the N-terminus of the Each of the two light chain polypeptides comprises at its N-terminus the V domain of the Fv fragment. H It is fused with the other side of the realm, Applicable V H Area and corresponding V L The regions combine to form the Fv fragment.

[0263] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0264] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0265] In one example, the bispecific protein comprises the V of the Fv fragment at the N-terminus. La first polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 73, fused to a region (a) of the Fv fragment; L a second polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 76 or 77, and a V H and a third and fourth polypeptide each comprising a light chain polypeptide fused to a region and each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 81. In another example, the bispecific protein comprises a light chain polypeptide fused to a region of the Fv fragment at the N-terminus thereof. L a first polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 74, fused to a region (a) of the Fv fragment; L a second polypeptide comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 75, and a V H and a third and fourth polypeptide each comprising a light chain polypeptide fused to the region and each comprising a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:81.

[0266] In some embodiments of the above bispecific proteins, the first Fc polypeptide comprises Leu at positions 234 and 235, according to EU numbering. In some embodiments, the second Fc polypeptide comprises Leu at positions 234 and 235, according to EU numbering. In certain embodiments, the protein comprises a cis-LALA configuration, as described below.

[0267] In some embodiments of the bispecific protein, one or both of the Fc polypeptides may have their C-terminal lysine removed (e.g., the Lys residue at position 447 of the Fc polypeptide according to EU numbering). In some embodiments, removal of the C-terminal lysine of the Fc polypeptide may improve the stability of the bispecific protein.

[0268] C-Terminal V of mAb / HC H V L In some embodiments, the bispecific protein comprises a VFc polypeptide fused at the C-terminus of one of the two Fc polypeptides. H (or V L region) and a V fused to the C-terminus of the other of the two Fc polypeptides L Area (or V H A schematic of such a bispecific protein is shown in Figure 5. In some embodiments, the bispecific protein comprises: (a) The Fd portion of the Fab is fused at the N-terminus to the V of the Fv fragment. H Area or V L a first Fc polypeptide fused at its C-terminus to a region; (b) a V fusion protein having an N-terminus with the Fd portion of Fab, H Area or V L a second Fc polypeptide fused at its C-terminus to the other of the regions, Applicable V H Area and corresponding V L the second Fc polypeptide, wherein the regions together form the Fv fragment, and the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides each pairing with a respective Fd portion described in (a) and (b) to form a Fab; wherein the Fab binds to subdomain II of human HER2 and the Fv fragment binds to subdomain IV of human HER2, or wherein the Fab binds to subdomain IV of human HER2 and the Fv fragment binds to subdomain II of human HER2.

[0269] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0270] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises substitutions of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a S239D and I332E substitution and the second Fc polypeptide comprises a S239D and I332E substitution. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a S239D and I332E substitution. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a S239D substitution. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a I332E substitution.In some embodiments, the first Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E.

[0271] In some embodiments of the bispecific protein, the first Fc polypeptide comprises the V of the Fv fragment. H and the second Fc polypeptide is fused to the V domain of the Fv fragment. L In some embodiments, the first Fc polypeptide is fused to the V region of the Fv fragment. L and the second Fc polypeptide is fused to the V domain of the Fv fragment. H It merges with the realm.

[0272] In some embodiments, a Fab that specifically binds to subdomain II of human HER2 has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 108. H In some embodiments, a Fab that specifically binds to subdomain IV of human HER2 comprises a V region having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 109. H Includes the area.

[0273] In some embodiments, the first linker is H Area or V LA region is connected to the C-terminus of the Fc polypeptide. In some embodiments, the first linker or the second linker has a length of about 1 to about 50 amino acids, for example, about 1 to about 40, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 2 to about 40, about 2 to about 30, about 2 to about 20, about 2 to about 10, about 5 to about 40, about 5 to about 30, about 5 to about 25, or about 5 to about 20 amino acids. In some embodiments, the first linker has a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 amino acids. Various linkers are described in further detail herein. In some embodiments, the first linker is GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 116, (GGSG) 5 ), GGGGS (SEQ ID NO: 117, G 4 S), GGGGSGGGGS (SEQ ID NO: 118, (G 4 S) 2 ), GGGGSGGGGSGGGGS (SEQ ID NO: 119, (G 4 S) 3 ), GGGGSGGGGSGGGG (SEQ ID NO: 120, (G 4 S) 2 -G 4 ), GGGGSGGGGSGG (SEQ ID NO: 121), GGGGSGGGGGS (SEQ ID NO: 122), and GGGGGSGGGGGSGGGGGS (SEQ ID NO: 123).

[0274] In some embodiments, the C-terminal V H V L In part (a) of a bispecific protein having the structure "A", the first Fc polypeptide is fused at its N-terminus to the Fd portion of the Fab via a hinge region or partial hinge region. In some embodiments, the C-terminal V of the mAb / HC is H V LIn part (b) of a bispecific protein having the structure "(a)", the second Fc polypeptide is fused at its N-terminus to the Fd portion of the Fab via a hinge region or a partial hinge region. An exemplary hinge region sequence is the human IgG1 hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO: 127). A partial hinge region refers to a portion of the sequence of SEQ ID NO: 127, for example a partial hinge region having the sequence DKTHTCPPCP (SEQ ID NO: 128).

[0275] "C-terminal V of mAb / HC H V L In a bispecific protein having the structure "T366W" and / or "Y407V", the first Fc polypeptide and / or the second Fc polypeptide can specifically bind to the transferrin receptor (e.g., a TfR-binding Fc polypeptide). Different Fc polypeptides and modifications thereof are described in further detail herein. In some embodiments, the first Fc polypeptide and the second Fc polypeptide can each comprise a modification that promotes heterodimerization. For example, according to EU numbering, the first Fc polypeptide can comprise a substitution of T366W and the second Fc polypeptide can comprise a substitution of T366S, L368A, and Y407V. In another example, according to EU numbering, the first Fc polypeptide can comprise a substitution of T366S, L368A, and Y407V and the second Fc polypeptide can comprise a substitution of T366W. Additionally, the first Fc polypeptide and / or the second Fc polypeptide can independently comprise a modification that reduces TfR-mediated effector function, i.e., reduces effector function upon TfR binding, e.g., the modification that reduces TfR-mediated effector function is, according to EU numbering, (i) L234A and L235A substitutions or (ii) L234A and L235A and P329G or P329S substitutions.

[0276] "mAb / HC N-terminal V H V L", wherein the first Fc polypeptide (or the second Fc polypeptide) is a TfR-binding Fc polypeptide comprising substitutions of T366W, L234A and L235A (optionally with substitutions of P329G or P329S), and optionally, S239D and / or I332E, according to EU numbering, and the second Fc polypeptide (or the first Fc polypeptide) comprises substitutions of T366S, L368A, and Y407V, and optionally, S239D and / or I332E, according to EU numbering. For example, the first Fc polypeptide (or the second Fc polypeptide) can comprise a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of the sequences of SEQ ID NOs: 137 and 186 to 196, and the second Fc polypeptide (or the first Fc polypeptide) can comprise a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of the sequences of SEQ ID NOs: 133 and 183 to 185.

[0277] In certain embodiments, the first Fc polypeptide is a TfR-binding Fc polypeptide and comprises a substitution of L234A and L235A (optionally comprising a substitution of P329G or P329S) according to EU numbering, and the second Fc polypeptide does not comprise a substitution of L234A or L325A (or a substitution of P329G or P329S, if present in the first Fc polypeptide). In certain embodiments, the first Fc polypeptide does not comprise a substitution of L234A or L325A (or a substitution of P329G or P329S, if present in the second Fc polypeptide), and the second Fc polypeptide is a TfR-binding Fc polypeptide and comprises a substitution of L234A and L235A (optionally comprising a substitution of P329G or P329S) according to EU numbering.

[0278] Exemplary bispecific "C-terminal V of mAb / HC H V L "protein In some embodiments, the bispecific protein comprises: (a) V of an Fv fragment that binds to subdomain IV of human HER2, fused at the N-terminus to the Fd portion of an Fab that binds to subdomain II of human HER2 H a first Fc polypeptide fused at its C-terminus to a region; (b) a Fab fragment fused at its N-terminus to the Fd portion of the Fab, and the V L A second Fc polypeptide fused at its C-terminus to the region.

[0279] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0280] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0281] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 82 or 83, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 99, 100, or 101, and each of the two light chains of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:84, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:98, and each of the two light chains of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:85, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:97, and each of the two light chains of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:25.In yet another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 86, 87, or 88, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 95 or 96, and each of the two light chains of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 25.

[0282] In some embodiments, the bispecific protein comprises: (a) V of an Fv fragment that binds to subdomain II of human HER2 and is N-terminally fused to the Fd portion of an Fab that binds to subdomain IV of human HER2 H a first Fc polypeptide fused at its C-terminus to a region; (b) a Fab fragment fused at its N-terminus to the Fd portion of the Fab, and the V L A second Fc polypeptide fused at its C-terminus to the region.

[0283] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain other embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution according to EU numbering. In certain embodiments, the first Fc polypeptide independently comprise an S239D and / or I332E substitution and the second Fc polypeptide independently comprise an S239D and / or I332E substitution are capable of enhancing HER2-mediated effector function, i.e., enhancing effector function by binding HER2.

[0284] In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and I332E and the second Fc polypeptide comprises a substitution of S239D. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the second Fc polypeptide comprises a substitution of I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of S239D and the second Fc polypeptide comprises a substitution of S239D and I332E. In some embodiments, according to EU numbering, the first Fc polypeptide comprises a substitution of I332E.

[0285] In one example of the bispecific protein, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 89 or 90, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 105, 106, or 107, and each of the two light chains of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:91, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:104, and each of the two light chains of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26. In another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:92, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:103, and each of the two light chains of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:26.In yet another example, (a) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 93 or 94, (b) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 102, and each of the two light chains of (c) comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 26.

[0286] In some embodiments of the above bispecific proteins, the first Fc polypeptide comprises Leu at positions 234 and 235, according to EU numbering. In some embodiments, the second Fc polypeptide comprises Leu at positions 234 and 235, according to EU numbering. In certain embodiments, the protein comprises a cis-LALA configuration, as described below.

[0287] In some embodiments of the bispecific protein, one or both of the Fc polypeptides may have their C-terminal lysine removed (e.g., the Lys residue at position 447 of the Fc polypeptide according to EU numbering). In some embodiments, removal of the C-terminal lysine of the Fc polypeptide may improve the stability of the bispecific protein.

[0288] IV. Fc Polypeptides and Modifications Thereof In some aspects, any of the anti-HER2 bispecific proteins described herein comprises an Fc polypeptide dimer, and one or both Fc polypeptides of the dimer comprise an amino acid modification compared to a wild-type Fc polypeptide. In some embodiments, the amino acid modification in the Fc polypeptide (e.g., modified Fc polypeptide) can provide binding of the Fc polypeptide dimer to a BBB receptor (e.g., TfR), promote heterodimerization of the two Fc polypeptides of the dimer, modulate effector function, extend serum half-life, affect glycosylation, and / or reduce immunogenicity in humans. In some embodiments, the Fc polypeptides present in the bispecific protein independently have at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to the corresponding wild-type Fc polypeptide (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide). Examples and descriptions of modified Fc polypeptides (e.g., TfR-binding Fc polypeptides) can be found, for example, in International Patent Publication No. WO2018 / 152326, which is incorporated by reference in its entirety.

[0289] Fc Polypeptide Modifications for BBB Receptor Binding Provided herein is an anti-HER2 bispecific protein that can be transported across the BBB. Such protein comprises a modified Fc polypeptide that binds to a BBB receptor. BBB receptors are expressed in the BBB endothelium, as well as other cell and tissue types. In some embodiments, the BBB receptor is TfR. The modified Fc polypeptide that binds to TfR is also referred to as having a TfR binding site.

[0290] The amino acid residues designated in the various Fc modifications, including those introduced into modified Fc polypeptides that bind to BBB receptors, e.g., TfR, are numbered herein using EU index numbering. Any Fc polypeptide, e.g., IgG1, IgG2, IgG3, or IgG4 Fc polypeptide, may have modifications, e.g., amino acid substitutions, at one or more positions described herein. In some embodiments, the domain modified for BBB (e.g., TfR) receptor binding activity is a human Ig CH3 domain, e.g., IgG1 CH3 domain. The CH3 domain may be from any IgG subtype, i.e., IgG1, IgG2, IgG3, or IgG4. In the context of IgG1 antibodies, the CH3 domain refers to the segment of amino acids from about position 341 to about position 447, numbered according to the EU numbering scheme.

[0291] In some embodiments, a modified Fc polypeptide that specifically binds to TfR can bind to the apical domain of TfR and bind to TfR without blocking or inhibiting the binding of transferrin to TfR. In some embodiments, the binding of transferrin to TfR is not substantially inhibited. In some embodiments, the binding of transferrin to TfR is inhibited by less than about 50% (e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%).

[0292] In some embodiments, a BBB (e.g., TfR) receptor binding Fc polypeptide present in a bispecific protein described herein comprises one or more, at least one, two, or three substitutions, and in some embodiments at least four, five, six, seven, eight, nine, or ten substitutions at amino acid positions including 266, 267, 268, 269, 270, 271, 295, 297, 298, and 299 according to the EU numbering scheme. In some embodiments, a BBB (e.g., TfR) receptor binding Fc polypeptide present in a bispecific protein described herein comprises at least one, two, or three substitutions, and in some embodiments at least four, five, six, seven, eight, or nine substitutions at amino acid positions including 274, 276, 283, 285, 286, 287, 288, 289, and 290 according to the EU numbering scheme. In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide present in the bispecific proteins described herein comprises at least one, two, or three substitutions, and in some embodiments at least 4, 5, 6, 7, 8, 9, or 10 substitutions at amino acid positions including 268, 269, 270, 271, 272, 292, 293, 294, 296, and 300 according to the EU numbering scheme. In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide present in the bispecific proteins described herein comprises at least one, two, or three substitutions, and in some embodiments at least 4, 5, 6, 7, 8, or 9 substitutions at amino acid positions including 272, 274, 276, 322, 324, 326, 329, 330, and 331 according to the EU numbering scheme. In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide present in the bispecific proteins described herein comprises at least one, two, or three substitutions, and in some embodiments at least four, five, six, or seven substitutions, including at amino acid positions 345, 346, 347, 349, 437, 438, 439, and 440, according to the EU numbering scheme.

[0293] In some embodiments, a BBB (e.g., TfR) receptor binding Fc polypeptide present in a bispecific protein described herein comprises at least one, two, or three substitutions, and in some embodiments at least four, five, six, seven, eight, or nine substitutions at amino acid positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 according to the EU numbering scheme.

[0294] In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide comprises at least one position having a substitution compared to SEQ ID NO: 130, 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, e.g., Trp or Gly (e.g., Trp), at position 388, Val, Ser, or Ala at position 389, an acidic amino acid, Ala, Ser, Leu, Thr, or Pro at position 413, a Thr or an acidic amino acid at position 416, or Trp, Tyr, His, or Phe at position 421. In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide may comprise conservative substitutions of particular amino acids at one or more positions of the set, e.g., amino acids of the same charge class, hydrophobic class, side chain ring structure class (e.g., aromatic amino acids), or size class, and / or polar or non-polar class. Thus, for example, Ile can be present at positions 384, 386, and / or 413. In some embodiments, the acidic amino acid at one, two, or each of positions 387, 413, and 416 is Glu. In other embodiments, the acidic amino acid at one, two, or each of positions 387, 413, and 416 is Asp. In some embodiments, two, three, four, five, six, seven, or all eight of positions 384, 386, 387, 388, 389, 413, 416, and 421 have an amino acid substitution as defined in this paragraph.

[0295] In some embodiments, an Fc polypeptide having modifications at amino acid positions 384, 386, 387, 388, 389, 390, 413, 416, and / or 421 includes a native Asn at position 390. In some embodiments, the Fc polypeptide includes a Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, or Asp at position 390. In some embodiments, the Fc polypeptide further includes one, two, three, or four substitutions at positions including 380, 391, 392, and 415. In some embodiments, a Trp, Tyr, Leu, or Gln may be present at position 380. In some embodiments, a Ser, Thr, Gln, or Phe may be present at position 391. In some embodiments, a Gln, Phe, or His may be present at position 392. In some embodiments, a Glu may be present at position 415.

[0296] In certain embodiments, the Fc polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions selected from the following: Trp, Leu or Glu at position 380, Tyr or Phe at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser, Ala, Val or Asn at position 389, Ser or Asn at position 390, Thr or Ser at position 413, Glu or Ser at position 415, Glu at position 416, and / or Phe at position 421. In some embodiments, the Fc polypeptide comprises all eleven of the following positions: Trp, Leu or Glu at position 380, Tyr or Phe at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser, Ala, Val or Asn at position 389, Ser or Asn at position 390, Thr or Ser at position 413, Glu or Ser at position 415, Glu at position 416, and / or Phe at position 421.

[0297] In certain embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide comprises Leu or Met at position 384, Leu, His, or Pro at position 386, Val at position 387, Trp at position 388, Val or Ala at position 389, Pro at position 413, Thr at position 416, and / or Trp at position 421. In some embodiments, the Fc polypeptide further comprises Ser, Thr, Gln, or Phe at position 391. In some embodiments, the Fc polypeptide further comprises Trp, Tyr, Leu, or Gln at position 380 and / or Gln, Phe, or His at position 392. In some embodiments, Trp is present at position 380 and / or Gln is present at position 392. In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide does not have a Trp at position 380.

[0298] In other embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide comprises a Tyr at position 384, a Thr at position 386, a Glu or Val at position 387, a Trp at position 388, a Ser at position 389, a Ser or Thr at position 413, a Glu at position 416, and / or a Phe at position 421. In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide comprises a natural Asn at position 390. In certain embodiments, the Fc polypeptide further comprises a Trp, Tyr, Leu, or Gln at position 380 and / or a Gln at position 415. In some embodiments, the Fc polypeptide further comprises a Trp at position 380 and / or a Glu at position 415.

[0299] In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide comprises one or more of the following substitutions: Trp at position 380, Thr at position 386, Trp at position 388, Val at position 389, Ser or Thr at position 413, Glu at position 415, and / or Phe at position 421.

[0300] In further embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide further comprises one, two, or three positions selected from the following: position 414 is Lys, Arg, Gly, or Pro, position 424 is Ser, Thr, Glu, or Lys, and position 426 is Ser, Trp, or Gly.

[0301] In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide has the sequence of SEQ ID NO: 135. In some embodiments of the bispecific proteins described herein, one of the two Fc polypeptides of the Fc polypeptide dimer can be a BBB (e.g., TfR) receptor binding Fc polypeptide having the sequence of SEQ ID NO: 135, while the other Fc polypeptide of the Fc polypeptide dimer can have the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO: 130). In other embodiments of the bispecific proteins described herein, both Fc polypeptides of the Fc polypeptide dimer can be BBB (e.g., TfR) receptor binding Fc polypeptides having the sequence of SEQ ID NO: 135.

[0302] In some embodiments of the bispecific proteins described herein, the first Fc polypeptide and / or the second Fc polypeptide independently comprise a Tyr at position 384, a Thr at position 386, a Glu at position 387, a Trp at position 388, a Ser at position 389, a Ser at position 413, a Glu at position 415, a Glu at position 416, and a Phe at position 421, according to EU numbering, and a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence selected from SEQ ID NOs: 135-139 and 186-196.

[0303] In some embodiments of the bispecific proteins described herein, one of the two Fc polypeptides of the Fc polypeptide dimer may be a BBB (e.g., TfR) receptor binding Fc polypeptide comprising a sequence having at least 90% identity to the sequence of SEQ ID NO: 135, comprising Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser at position 389, Ser at position 413, Glu at position 415, Glu at position 416, and Phe at position 421, according to EU numbering, while the other Fc polypeptide of the Fc polypeptide dimer may have the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO: 130).

[0304] In some embodiments of the bispecific proteins described herein, the first Fc polypeptide and / or the second Fc polypeptide independently comprise a Tyr at position 384, a Thr at position 386, a Glu at position 387, a Trp at position 388, an Ala at position 389, a Thr at position 413, a Glu at position 415, a Glu at position 416, and a Phe at position 421, according to EU numbering, and a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence selected from SEQ ID NOs: 140-144.

[0305] In some embodiments of the bispecific proteins described herein, one of the two Fc polypeptides of the Fc polypeptide dimer may be a BBB (e.g., TfR) receptor binding Fc polypeptide comprising a sequence having at least 90% identity to the sequence of SEQ ID NO: 140, comprising Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ala at position 389, Thr at position 413, Glu at position 415, Glu at position 416, and Phe at position 421, according to EU numbering, while the other Fc polypeptide of the Fc polypeptide dimer may have the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO: 130).

[0306] In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide has the sequence of SEQ ID NO: 140. In some embodiments of the bispecific proteins described herein, one of the two Fc polypeptides of the Fc polypeptide dimer can be a BBB (e.g., TfR) receptor binding Fc polypeptide having the sequence of SEQ ID NO: 140, while the other Fc polypeptide of the Fc polypeptide dimer can have the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO: 130). In other embodiments of the bispecific proteins described herein, both Fc polypeptides of the Fc polypeptide dimer can be BBB (e.g., TfR) receptor binding Fc polypeptides having the sequence of SEQ ID NO: 140.

[0307] In some embodiments, the BBB (e.g., TfR) receptor binding Fc polypeptide has the sequence of SEQ ID NO: 145. In some embodiments of the bispecific proteins described herein, one of the two Fc polypeptides of the Fc polypeptide dimer can be a BBB (e.g., TfR) receptor binding Fc polypeptide having the sequence of SEQ ID NO: 145, while the other Fc polypeptide of the Fc polypeptide dimer can have the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO: 130). In other embodiments of the bispecific proteins described herein, both Fc polypeptides of the Fc polypeptide dimer can be BBB (e.g., TfR) receptor binding Fc polypeptides having the seq...

Claims

1. below: (a) a first Fc polypeptide fused at its N-terminus to the Fd portion of a Fab; (b) a second Fc polypeptide N-terminally fused to a single-chain variable fragment (scFv), wherein the first and second Fc polypeptides form an Fc dimer; and (c) a light chain polypeptide that pairs with the Fd portion described in (a) above to form a Fab. A protein comprising: The Fab binds to subdomain II of human HER2 and the scFv binds to subdomain IV of human HER2, or the Fab binds to subdomain IV of human HER2 and the scFv binds to subdomain II of human HER2, and the first Fc polypeptide and / or the second Fc polypeptide independently comprise a S239D and / or I332E substitution according to EU numbering; The protein.

2. (a) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (b) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (c) the first Fc polypeptide comprises a S239D and I332E substitution, and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (d) the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (e) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (f) the first Fc polypeptide comprises a I332E substitution, and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (g) the first Fc polypeptide comprises a S239D and I332E substitution, and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (h) the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (i) the first Fc polypeptide comprises a S239D substitution, and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; (j) the first Fc polypeptide comprises a I332E substitution, and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; (k) the first Fc polypeptide comprises a S239D and I332E substitution, and the second Fc polypeptide comprises a S239D and I332E substitution, according to EU numbering; (l) the second Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E; (m) the first Fc polypeptide comprises a S239D substitution, according to EU numbering; (n) the first Fc polypeptide comprises a I332E substitution, according to EU numbering; or (o) the first Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E; The protein of claim 1.

3. (a) the first Fc polypeptide comprises a I332E substitution and a serine at position 239, and the second Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, according to EU numbering; (b) the first Fc polypeptide comprises an S239D and I332E substitution, and the second Fc polypeptide comprises an S239D substitution and an isoleucine at position 332, according to EU numbering; (c) the first Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, and the second Fc polypeptide comprises a I332E substitution and a serine at position 239, according to EU numbering; (d) the first Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, and the second Fc polypeptide comprises a I332E substitution and a serine at position 239, according to EU numbering; (e) the first Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, and the second Fc polypeptide comprises a S239D and I332E substitution, according to EU numbering; or (f) said first Fc polypeptide comprises a I332E substitution and a serine at position 239, and said second Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, according to EU numbering; The protein of claim 2.

4. The protein of claim 1, wherein the second Fc polypeptide is fused to the scFv via a first linker. (a) the first Fc polypeptide and / or the second Fc polypeptide specifically binds to the transferrin receptor (TfR); and / or (b) the first Fc polypeptide and the second Fc polypeptide each comprise a modification that promotes heterodimerization; and / or (c) the first Fc polypeptide and / or the second Fc polypeptide independently comprise a modification that reduces a TfR-mediated effector function; The protein of claim 1.

6. The protein of claim 1 , wherein a hinge region or a portion thereof is linked to the N-terminus of the first Fc polypeptide and / or the second Fc polypeptide. (a) the first Fc polypeptide and / or the second Fc polypeptide independently comprise a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 131-149 and 183-196; or (b) the first Fc polypeptide and / or the second Fc polypeptide, independently, comprise a Tyr at position 384, a Thr at position 386, a Glu at position 387, a Trp at position 388, a Ser at position 389, a Ser at position 413, a Glu at position 415, a Glu at position 416, and a Phe at position 421 according to EU numbering, and comprise a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 135-139 and 186-196; The protein of claim 1. (a) the first Fc polypeptide comprises Ala at position 234, Ala at position 235, Trp at position 366, Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser at position 389, Ser at position 413, Glu at position 415, Glu at position 416, and Phe at position 421 according to EU numbering, and comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 137; and the second Fc polypeptide comprises Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering, and comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 133; or (b) the first Fc polypeptide comprises Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering, and comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 133; and the second Fc polypeptide comprises Ala at position 234, Ala at position 235, Trp at position 366, Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser at position 389, Ser at position 413, Glu at position 415, Glu at position 416, and Phe at position 421 according to EU numbering, and comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 137; The protein of claim 1.

9. below: (a) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO:1 or a sequence having at least 90% identity to the sequence of SEQ ID NO:1, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO:159 or a sequence having at least 90% identity to the sequence of SEQ ID NO:159, and (iii) a light chain polypeptide comprising the sequence of SEQ ID NO:25 or a sequence having at least 90% identity to the sequence of SEQ ID NO:25; (b) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 164 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 164, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 173 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 173, and (iii) a light chain polypeptide comprising the sequence of SEQ ID NO: 25 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 25; (c) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 164 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 164, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 174 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 174, and (iii) a light chain polypeptide comprising the sequence of SEQ ID NO: 25 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 25; (d) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 166 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 166; (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 173 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 173; and (iii) a light chain polypeptide comprising the sequence of SEQ ID NO: 25 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 25; (e) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 165 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 165; (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 173 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 173; and (iii) a light chain polypeptide comprising the sequence of SEQ ID NO: 25 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 25; (f) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 165 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 165; (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 174 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 174; and (iii) a light chain polypeptide comprising the sequence of SEQ ID NO: 25 or a sequence having at least 90% identity to the sequence of SEQ ID NO:

25. (g) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 167 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 167; (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 173 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 173; and (iii) a light chain polypeptide comprising the sequence of SEQ ID NO: 25 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 25; (h) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 164 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 164; (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 175 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 175; and (iii) a light chain polypeptide comprising the sequence of SEQ ID NO: 25 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 25; (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 164 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 164, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 176 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 176, and (iii) a light chain polypeptide comprising the sequence of SEQ ID NO: 25 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 25; (j) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 166 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 166; (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 175 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 175; and (iii) a light chain polypeptide comprising the sequence of SEQ ID NO: 25 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 25; (k) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 165 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 165; (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 175 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 175; and (iii) a light chain polypeptide comprising the sequence of SEQ ID NO: 25 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 25; (l) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 165 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 165, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 176 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 176, and (iii) a light chain polypeptide comprising the sequence of SEQ ID NO: 25 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 25; or (m) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 167 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 167, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 175 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 175, and (iii) a light chain polypeptide comprising the sequence of SEQ ID NO: 25 or a sequence having at least 90% identity to the sequence of SEQ ID NO:

25. Proteins, including:

10. below: (a) a first Fc polypeptide fused at its N-terminus to the Fd portion of a Fab; (b) a second Fc polypeptide N-terminally fused to the Fd portion of the Fab, wherein the first and second Fc polypeptides form an Fc dimer; and (c) two light chain polypeptides, each pairing with a respective Fd moiety described in (a) and (b) to form a Fab; A protein comprising: the Fd portion in (a) and / or (b) is fused to the scFv at the N-terminus; The Fab binds to subdomain II of human HER2 and the scFv binds to subdomain IV of human HER2, or the Fab binds to subdomain IV of human HER2 and the scFv binds to subdomain II of human HER2, and the first Fc polypeptide and / or the second Fc polypeptide independently comprise a S239D and / or I332E substitution according to EU numbering; The protein.

11. (a) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (b) the first Fc polypeptide comprises a I332E substitution and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (c) the first Fc polypeptide comprises a S239D and I332E substitution, and the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (d) the second Fc polypeptide comprises a S239D substitution, according to EU numbering; (e) the first Fc polypeptide comprises a S239D substitution and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (f) the first Fc polypeptide comprises a I332E substitution, and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (g) the first Fc polypeptide comprises a S239D and I332E substitution, and the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (h) the second Fc polypeptide comprises a I332E substitution, according to EU numbering; (i) the first Fc polypeptide comprises a S239D substitution, and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; (j) the first Fc polypeptide comprises a I332E substitution, and the second Fc polypeptide comprises S239D and I332E substitutions, according to EU numbering; (k) the first Fc polypeptide comprises a S239D and I332E substitution, and the second Fc polypeptide comprises a S239D and I332E substitution, according to EU numbering; (l) the second Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E; (m) the first Fc polypeptide comprises a S239D substitution, according to EU numbering; (n) the first Fc polypeptide comprises a I332E substitution, according to EU numbering; or (o) the first Fc polypeptide comprises the following substitutions, according to EU numbering: S239D and I332E; The protein of claim 10.

12. (a) the first Fc polypeptide comprises a I332E substitution and a serine at position 239, and the second Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, according to EU numbering; (b) the first Fc polypeptide comprises an S239D and I332E substitution, and the second Fc polypeptide comprises an S239D substitution and an isoleucine at position 332, according to EU numbering; (c) the first Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, and the second Fc polypeptide comprises a I332E substitution and a serine at position 239, according to EU numbering; (d) the first Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, and the second Fc polypeptide comprises a I332E substitution and a serine at position 239, according to EU numbering; (e) the first Fc polypeptide comprises a S239D substitution and an isoleucine at position 332, and the second Fc polypeptide comprises a S239D and I332E substitution, according to EU numbering; or (f) said first Fc polypeptide comprises a I332E substitution and a serine at position 239, and said second Fc polypeptide comprises a serine at position 239 and an isoleucine at position 332, according to EU numbering; The protein of claim 11.

13. The Fd moiety in either (a) or (b), but not both, is fused N-terminally to the scFv; and / or The Fd portion in (a) and / or (b) is fused to the scFv via a first linker. The protein of claim 10. (a) the first Fc polypeptide and / or the second Fc polypeptide specifically binds to the transferrin receptor (TfR); and / or (b) the first Fc polypeptide and the second Fc polypeptide each comprise a modification that promotes heterodimerization; and / or (c) the first Fc polypeptide and / or the second Fc polypeptide independently comprise a modification that reduces a TfR-mediated effector function; The protein of claim 10.

15. The protein of claim 10, wherein a hinge region or a portion thereof is linked to the N-terminus of the first Fc polypeptide and / or the second Fc polypeptide. (a) the first Fc polypeptide and / or the second Fc polypeptide independently comprise a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 131-149 and 183-196; or (b) the first Fc polypeptide and / or the second Fc polypeptide, independently, comprise a Tyr at position 384, a Thr at position 386, a Glu at position 387, a Trp at position 388, a Ser at position 389, a Ser at position 413, a Glu at position 415, a Glu at position 416, and a Phe at position 421 according to EU numbering, and comprise a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 135-139 and 186-196; The protein of claim 10. (a) the first Fc polypeptide comprises Ala at position 234, Ala at position 235, Trp at position 366, Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser at position 389, Ser at position 413, Glu at position 415, Glu at position 416, and Phe at position 421 according to EU numbering, and comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 137; and the second Fc polypeptide comprises Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering, and comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 133; or (b) the first Fc polypeptide comprises Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering, and comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 133; and the second Fc polypeptide comprises Ala at position 234, Ala at position 235, Trp at position 366, Tyr at position 384, Thr at position 386, Glu at position 387, Trp at position 388, Ser at position 389, Ser at position 413, Glu at position 415, Glu at position 416, and Phe at position 421 according to EU numbering, and comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 137; The protein of claim 10.

18. below: (a) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO:6 or a sequence having at least 90% identity to the sequence of SEQ ID NO:6, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO:160 or a sequence having at least 90% identity to the sequence of SEQ ID NO:160, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO:26 or a sequence having at least 90% identity to the sequence of SEQ ID NO:26; (b) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO:6 or a sequence having at least 90% identity to the sequence of SEQ ID NO:6, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO:161 or a sequence having at least 90% identity to the sequence of SEQ ID NO:161, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO:26 or a sequence having at least 90% identity to the sequence of SEQ ID NO:26; (c) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO:6 or a sequence having at least 90% identity to the sequence of SEQ ID NO:6, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO:162 or a sequence having at least 90% identity to the sequence of SEQ ID NO:162, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO:26 or a sequence having at least 90% identity to the sequence of SEQ ID NO:26; (d) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 169 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 169, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 177 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 177, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (e) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 169 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 169, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 178 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 178, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (f) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 171 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 171, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 177 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 177, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (g) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 170 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 170, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 177 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 177, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (h) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 170 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 170, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 178 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 178, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 172 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 172, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 177 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 177, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (j) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 169 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 169, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 179 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 179, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (k) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 169 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 169, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 180 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 180, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (l) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 171 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 171, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 179 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 179, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (m) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 170 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 170, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 179 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 179, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (n) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 170 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 170, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 180 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 180, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (o) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 172 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 172, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 179 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 179, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (p) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 169 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 169, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 181 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 181, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (q) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 169 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 169, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 182 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 182, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (r) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 171 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 171, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 181 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 181, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (s) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 170 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 170, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 181 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 181, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; (t) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 170 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 170, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 182 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 182, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 26; or (u) (i) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 172 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 172, (ii) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 181 or a sequence having at least 90% identity to the sequence of SEQ ID NO: 181, and (iii) two light chain polypeptides each comprising the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity to the sequence of SEQ ID NO:

26. Proteins, including:

19. A pharmaceutical composition comprising the protein of any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

20. An isolated polynucleotide or vector comprising a nucleotide sequence encoding a protein according to any one of claims 1 to 18.

21. A host cell comprising the polynucleotide or vector of claim 20.

22. 20. A method for treating cancer or treating brain metastasis of cancer in a subject, comprising administering to the subject a therapeutically effective amount of a protein according to any one of claims 1 to 18.

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