Transferrin receptor binding proteins for treating brain tumors

Abstract

Provided is a novel multispecific antibody comprising at least one of a first antigen-binding region and a second antigen-binding region, each capable of specifically binding to human epidermal growth factor receptor 2 (HER2), and a third antigen-binding region capable of specifically binding to transferrin receptor (TfR).

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 395,391, filed August 5, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] (Reference to electronically submitted sequence listing) This application contains an electronically submitted Sequence Listing, the contents of which (JBI6745WOPCT1_Sequence Listing.xml, size: 261,328 bytes; and creation date: July 18, 2023) are incorporated herein by reference in their entirety.

[0003] FIELD OF THE INVENTION The present disclosure relates to novel multispecific antibodies comprising a first means capable of binding to a first antigen and a second means capable of binding to a second antigen on a specific tissue, for example, on the blood-brain barrier. [Background technology]

[0004] Numerous approaches have been investigated to improve the delivery of therapeutic monoclonal antibodies (mAbs). For example, the blood-brain barrier (BBB), which prevents harmful substances from entering the brain and is essential for brain homeostasis, poses a formidable obstacle to the efficient delivery of drugs to the brain. Large molecules such as monoclonal antibodies and other biological therapeutics have great potential as therapeutic and diagnostic agents for treating and detecting pathologies in the central nervous system (CNS). However, their pathway into the brain is hindered by the BBB. Anti-transferrin receptor 1 (TfR1) monoclonal antibodies have been used to deliver drugs to the brain (Burkhart, et al., Progress in neurobiology, 181, 101665, 2019). However, safety issues and poor pharmacokinetics (PK) of anti-TfR1 monoclonal antibodies have hindered their clinical development as BBB carriers.

[0005] Therefore, there is a need for a platform that can be used to efficiently shuttle drugs to target tissues with improved safety and pharmacokinetics. Summary of the Invention

[0006] In one aspect, provided herein is a multispecific antibody, or antigen-binding fragment thereof, comprising at least one of a first antigen-binding region and a second antigen-binding region, each capable of specifically binding to human epidermal growth factor receptor 2 (HER2), and a third antigen-binding region capable of specifically binding to transferrin receptor (TfR).

[0007] In certain embodiments, the first antigen-binding region comprises a first heavy chain variable region (VH1) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively, and a first light chain variable region (VL1) comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; and the second antigen-binding region comprises a second heavy chain variable region (VL1) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively. and a second light chain variable region (VL2) comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and a third light chain variable region (VL3) comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and the third antigen-binding region comprises a first single chain variable fragment (scFv1) having a third heavy chain variable region (VH3) comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and a third light chain variable region (VL3) comprising a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 have any of the amino acid sequences in Table 2.

[0008] In certain embodiments, VH1 comprises an amino acid sequence identical to the VH of the HC sequence identified as SEQ ID NO: 1; VL1 comprises an amino acid sequence identical to the VL of the LC sequence identified as SEQ ID NO: 2; VH2 comprises an amino acid sequence identical to the VH of the HC sequence identified as SEQ ID NO: 11; VL2 comprises an amino acid sequence identical to the VL of the LC sequence identified as SEQ ID NO: 12; and VH3 and VL3 are selected from the following: (i) SEQ ID NO: 19; (ii) SEQ ID NO: 26; (iii) SEQ ID NO: 33; (iv) SEQ ID NO: 40, (v) SEQ ID NO: 47, (vi) SEQ ID NO: 54, (vii) SEQ ID NO: 59, (viii) SEQ ID NO: 66; (ix) SEQ ID NO: 72, (x) SEQ ID NO: 79, (xi) SEQ ID NO: 82, (xii) SEQ ID NO: 85, (xiii) SEQ ID NO: 87, (xiv) SEQ ID NO: 94, (xv) SEQ ID NO: 101, (xvi) SEQ ID NO: 104, (xvii) SEQ ID NO: 111, (xviii) SEQ ID NO: 118, (xix) SEQ ID NO: 125, (xx) SEQ ID NO: 132, (xxi) SEQ ID NO: 138, (xxii) SEQ ID NO: 144, (xxiii) SEQ ID NO: 150, (xxiv) SEQ ID NO: 155, (xxv) SEQ ID NO: 158, (xxvi) SEQ ID NO: 164, (xxvii) SEQ ID NO: 169, (xxviii) SEQ ID NO: 175, (xxix) SEQ ID NO: 183, (xxx) SEQ ID NO: 188, (xxxi) SEQ ID NO: 193, (xxxii) SEQ ID NO: 198, or (xxxiii) SEQ ID NO: 204, comprising amino acid sequences identical to the VH and VL of each of the scFvs identified as

[0009] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises (a) a first heavy chain (HC1) comprising VH1, a first heavy chain constant region comprising a first Fc region (Fc1), and an scFv1; and (b) a first light chain (LC1) comprising VL1 and a light chain constant region.

[0010] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises (a) a second heavy chain (HC2) comprising a first heavy chain constant region comprising a VH1 and a second Fc region (Fc2), and (b) a second light chain (LC2) comprising a VL1 and a light chain constant region.

[0011] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof further comprises a second Fc region (Fc2).

[0012] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising a first heavy chain constant region comprising a VH2 and a first Fc region (Fc1); (b) a first light chain (LC1) comprising a VL2 and a light chain constant region; and (c) a second heavy chain (HC2) comprising a second heavy chain constant region comprising an scFv1 and a second Fc region (Fc2).

[0013] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding region, a second antigen-binding region, and a third antigen-binding region.

[0014] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising a VH1, a first heavy chain constant region comprising a first Fc region (Fc1), and an scFv1; (b) a first light chain (LC1) comprising a VL1 and a light chain constant region; and (c) a second single chain variable fragment (scFv2) comprising a first heavy chain constant region comprising a second Fc region (Fc2), wherein the scFv2 comprises a VH2 and a VL2.

[0015] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising a VH2, a first heavy chain constant region comprising a first Fc region (Fc1), and an scFv1; (b) a first light chain (LC1) comprising a VL2 and a light chain constant region; and (c) a second single-chain variable fragment (scFv2) and a first heavy chain constant region comprising a second Fc region (Fc2), wherein the scFv2 comprises a second heavy chain (HC2) comprising a VH1 and a VL1.

[0016] In certain embodiments, scFv1 and / or scFv2 comprise at least one of (a) a first disulfide bond between a structurally conserved, surface-exposed VH cysteine (Cys) and a first L Cys, and b) a second disulfide bond between a structurally conserved, surface-exposed VL Cys and a second L Cys.

[0017] In certain embodiments, scFv1 and scFv2 each independently comprise a first disulfide bond and a second disulfide bond.

[0018] In certain embodiments, the scFv2 comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 3 or 4.

[0019] In certain embodiments, scFv1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19, 26, 33, 40, 47, 54, 59, 66, 72, 79, 82, 83, 84, 85, 86, 87, 94, 101, 104, 111, 118, 125, 132, 138, 144, 150, 155, 158, 164, 169, 175, 183, 188, 193, 198, and 204.

[0020] In certain embodiments, each of Fc1 and Fc2 comprises one or more heterodimer mutations or one or more knob and hole mutations.

[0021] In certain embodiments, the heterodimer mutations comprise amino acid modifications at positions T350, L351, F405, and Y407 in one of Fc1 and Fc2, and amino acid modifications at positions T350, T366, K392, and T394 in the other of Fc1 and Fc2, wherein the amino acid modification at position T350 is T350V, T350I, T350L, or T350M, the amino acid modification at position L351 is L351Y, and the amino acid modification at position F405 is F405A. , F405V, F405T, or F405S; the amino acid modification at position Y407 is Y407V, Y407A, or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V, or T366M; the amino acid modification at position K392 is K392F, K392L, or K392M; and the amino acid modification at position T394 is T394W, where the numbering of amino acid residues is according to the EU index as described in Kabat.

[0022] In certain embodiments, one of Fc1 and Fc2 comprises the mutations T350V, L351Y, F405A, and Y407V, and the other of Fc1 and Fc2 comprises the mutations T350V, T366L, K392L, and T394W.

[0023] In certain embodiments, Fc1 and Fc2 each comprise one or more knob and hole mutations.

[0024] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain with amino acid modifications that enhance binding of the multispecific antibody or antigen-binding fragment thereof to the neonatal Fc receptor (RcRn), preferably the amino acid modifications enhance binding at acidic pH, more preferably the Fc domain has M252Y / S254T / T256E (YTE) mutations, where the numbering of the amino acid residues is according to the EU index as set forth in Kabat.

[0025] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain with amino acid modifications that reduce or eliminate effector function, preferably the Fc domain has one or more amino acid modifications at positions L234, L235, D265, D270, N297, E318, K320, K322, P331, and P329, such as one, two, three, or four of L234A, L235A, D265S, and P331S, where the numbering of the amino acid residues is according to the EU index as set forth in Kabat.

[0026] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain with one or more of the following amino acid modifications: M252Y, S254T, T256E, L234A, L235A, and D265S, where the numbering of the amino acid residues is according to the EU index as set forth in Kabat.

[0027] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain that does not have amino acid modifications that reduce or eliminate effector function.

[0028] and a first heavy chain, a light chain, and a second heavy chain, each having an amino acid sequence at least 90% identical to (a) SEQ ID NO:211, SEQ ID NO:12, and SEQ ID NO:212, respectively, or (b) SEQ ID NO:213, SEQ ID NO:12, and SEQ ID NO:214, respectively; Also provided is a multispecific antibody, in which a first antigen-binding region is capable of specifically binding to a first epitope of HER2, a second antigen-binding region is capable of specifically binding to a second epitope of HER2, and a third antigen-binding region is capable of specifically binding to TfR.

[0029] In certain embodiments, the first heavy chain, light chain, and second heavy chain each comprise the amino acid sequence of (a) SEQ ID NO:211, SEQ ID NO:12, and SEQ ID NO:212, respectively, or (b) SEQ ID NO:213, SEQ ID NO:12, and SEQ ID NO:214, respectively.

[0030] Another general aspect of the present application relates to an isolated nucleic acid sequence encoding the multispecific antibody or antigen-binding fragment thereof of the present application. Also provided are a vector comprising the isolated nucleic acid of the present application, and a host cell comprising the isolated nucleic acid or vector of the present application.

[0031] Another general aspect of the present application relates to a method for producing a multispecific antibody or antigen-binding fragment thereof, the method comprising culturing a cell containing a nucleic acid of the present application under conditions to produce the multispecific antibody or antigen-binding fragment thereof, and recovering the multispecific antibody or antigen-binding fragment thereof.

[0032] Further provided is a pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof of the present application and a pharmaceutically acceptable carrier.

[0033] Yet another general aspect of the present invention relates to a method for treating or detecting a disorder, preferably cancer, in a subject in need thereof, comprising administering to the subject a multispecific antibody or antigen-binding fragment of the present application, or a pharmaceutical composition of the present application.

[0034] Other aspects, features, and advantages of the present invention will become apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments, and the appended claims. [Brief explanation of the drawings]

[0035] [Figure 1] FIG. 1 shows a diagram of a biparatopic multispecific antibody fused to a tissue targeting module. [Figure 2A-1] Targeted transport mechanisms—transcytosis and phagocytosis for target clearance. (A) Antibodies bind to receptors on the cell surface and, through selective modulation of intracellular trafficking, enable transcytosis across the BBB or deliver bound antigens to lysosomes for degradation. The feature of TEM (Transcytosis Enabling Module) is that it enhances antibody exposure in the brain while inducing non-FcγR phagocytosis to eliminate target cells in a robust and safe manner. (B) Diagram of the molecular structure of TEM mAb, in which a single TfR scFv is fused to the C-terminus of one heavy chain of a bivalent, monovalent, or biparatopic therapeutic mAb targeting HER2 by using a short, flexible linker. [Figure 2A-2] Targeted transport mechanisms—transcytosis and phagocytosis for target clearance. (A) Antibodies bind to receptors on the cell surface and, through selective modulation of intracellular trafficking, enable transcytosis across the BBB or deliver bound antigens to lysosomes for degradation. The feature of TEM (Transcytosis Enabling Module) is that it enhances antibody exposure in the brain while inducing non-FcγR phagocytosis to eliminate target cells in a robust and safe manner. (B) Diagram of the molecular structure of TEM mAb, in which a single TfR scFv is fused to the C-terminus of one heavy chain of a bivalent, monovalent, or biparatopic therapeutic mAb targeting HER2 by using a short, flexible linker. [Figure 2B]Targeted transport mechanisms—transcytosis and phagocytosis for target clearance. (A) Antibodies bind to receptors on the cell surface and, through selective modulation of intracellular trafficking, enable transcytosis across the BBB or deliver bound antigens to lysosomes for degradation. The feature of TEM (Transcytosis Enabling Module) is that it enhances antibody exposure in the brain while inducing non-FcγR phagocytosis to eliminate target cells in a robust and safe manner. (B) Diagram of the molecular structure of TEM mAb, in which a single TfR scFv is fused to the C-terminus of one heavy chain of a bivalent, monovalent, or biparatopic therapeutic mAb targeting HER2 by using a short, flexible linker. [Figure 3A] Figure 1 shows the binding of HER2-TfR-J-mut, HER2-TfR-K-mut, mono-HER2-TfR-J-mut, and mono-HER2-TfR-J-mut, as well as additional biparatopic antibodies, to (A) BT474, (B) MBA-MB-361, and (C) HCC1954 cells. Binding was compared to trastuzumab, silent trastuzumab, silent pertuzumab, and isotype IgG1 (CNTO3930). [Figure 3B] Figure 1 shows the binding of HER2-TfR-J-mut, HER2-TfR-K-mut, mono-HER2-TfR-J-mut, and mono-HER2-TfR-J-mut, as well as additional biparatopic antibodies, to (A) BT474, (B) MBA-MB-361, and (C) HCC1954 cells. Binding was compared to trastuzumab, silent trastuzumab, silent pertuzumab, and isotype IgG1 (CNTO3930). [Figure 3C] Figure 1 shows the binding of HER2-TfR-J-mut, HER2-TfR-K-mut, mono-HER2-TfR-J-mut, and mono-HER2-TfR-J-mut, as well as additional biparatopic antibodies, to (A) BT474, (B) MBA-MB-361, and (C) HCC1954 cells. Binding was compared to trastuzumab, silent trastuzumab, silent pertuzumab, and isotype IgG1 (CNTO3930). [Figure 4A] Figure 4 shows the kinetic curves of spheroid formation by (A) MDA-MB-361 NR, (B) BT474 NR, and (C) BT474 clone 5 cells co-cultured with iMG at an E:T ratio of 1:1 in the presence of anti-HER2 TEM mAbs, trastuzumab, silent trastuzumab, or isotype IgG1 (all at 10 μg / mL). Each symbol represents the mean ± SEM of n = 3 wells, normalized to the value at time 0 for each condition. Figure 4D–4F show values from day 14 for (D) MDA-MB-361, (E) BT474, (F) and BT474 clone 5, which are presented separately for statistical analysis. [Figure 4B] Figure 4 shows the kinetic curves of spheroid formation by (A) MDA-MB-361 NR, (B) BT474 NR, and (C) BT474 clone 5 cells co-cultured with iMG at an E:T ratio of 1:1 in the presence of anti-HER2 TEM mAbs, trastuzumab, silent trastuzumab, or isotype IgG1 (all at 10 μg / mL). Each symbol represents the mean ± SEM of n = 3 wells, normalized to the value at time 0 for each condition. Figure 4D–4F show values from day 14 for (D) MDA-MB-361, (E) BT474, (F) and BT474 clone 5, which are presented separately for statistical analysis. [Figure 4C] Figure 4 shows the kinetic curves of spheroid formation by (A) MDA-MB-361 NR, (B) BT474 NR, and (C) BT474 clone 5 cells co-cultured with iMG at an E:T ratio of 1:1 in the presence of anti-HER2 TEM mAbs, trastuzumab, silent trastuzumab, or isotype IgG1 (all at 10 μg / mL). Each symbol represents the mean ± SEM of n = 3 wells, normalized to the value at time 0 for each condition. Figure 4D–4F show values from day 14 for (D) MDA-MB-361, (E) BT474, (F) and BT474 clone 5, which are presented separately for statistical analysis. [Figure 4D]Figure 4 shows the kinetic curves of spheroid formation by (A) MDA-MB-361 NR, (B) BT474 NR, and (C) BT474 clone 5 cells co-cultured with iMG at an E:T ratio of 1:1 in the presence of anti-HER2 TEM mAbs, trastuzumab, silent trastuzumab, or isotype IgG1 (all at 10 μg / mL). Each symbol represents the mean ± SEM of n = 3 wells, normalized to the value at time 0 for each condition. Figure 4D–4F show values from day 14 for (D) MDA-MB-361, (E) BT474, (F) and BT474 clone 5, which are presented separately for statistical analysis. [Figure 4E] Figure 4 shows the kinetic curves of spheroid formation by (A) MDA-MB-361 NR, (B) BT474 NR, and (C) BT474 clone 5 cells co-cultured with iMG at an E:T ratio of 1:1 in the presence of anti-HER2 TEM mAbs, trastuzumab, silent trastuzumab, or isotype IgG1 (all at 10 μg / mL). Each symbol represents the mean ± SEM of n = 3 wells, normalized to the value at time 0 for each condition. Figure 4D–4F show values from day 14 for (D) MDA-MB-361, (E) BT474, (F) and BT474 clone 5, which are presented separately for statistical analysis. [Figure 4F] Figure 4 shows the kinetic curves of spheroid formation by (A) MDA-MB-361 NR, (B) BT474 NR, and (C) BT474 clone 5 cells co-cultured with iMG at an E:T ratio of 1:1 in the presence of anti-HER2 TEM mAbs, trastuzumab, silent trastuzumab, or isotype IgG1 (all at 10 μg / mL). Each symbol represents the mean ± SEM of n = 3 wells, normalized to the value at time 0 for each condition. Figure 4D–4F show values from day 14 for (D) MDA-MB-361, (E) BT474, (F) and BT474 clone 5, which are presented separately for statistical analysis. [Figure 5A]Figures 5C-5D show kinetic curves of red fluorescence for BT474 clone 5 labeled with 1 μg / mL pHrodo red and cocultured with human iMG at (A) an E:T ratio of 1:1 or (B) an E:T ratio of 1:4 in the presence of trastuzumab, anti-HER2 TEM mAb, or control (all at 20 nM). Figures 5C-5D show that values at 72 hours are also plotted, and statistical differences are indicated. Each symbol or column represents the mean + / - SEM of n=3 wells. Statistical differences were determined by one-way ANOVA; *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001; ns=not significant. [Figure 5B] Figures 5C-5D show kinetic curves of red fluorescence for BT474 clone 5 labeled with 1 μg / mL pHrodo red and cocultured with human iMG at (A) an E:T ratio of 1:1 or (B) an E:T ratio of 1:4 in the presence of trastuzumab, anti-HER2 TEM mAb, or control (all at 20 nM). Figures 5C-5D show that values at 72 hours are also plotted, and statistical differences are indicated. Each symbol or column represents the mean + / - SEM of n=3 wells. Statistical differences were determined by one-way ANOVA; *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001; ns=not significant. [Figure 5C] Figures 5C-5D show kinetic curves of red fluorescence for BT474 clone 5 labeled with 1 μg / mL pHrodo red and cocultured with human iMG at (A) an E:T ratio of 1:1 or (B) an E:T ratio of 1:4 in the presence of trastuzumab, anti-HER2 TEM mAb, or control (all at 20 nM). Figures 5C-5D show that values at 72 hours are also plotted, and statistical differences are indicated. Each symbol or column represents the mean + / - SEM of n=3 wells. Statistical differences were determined by one-way ANOVA; *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001; ns=not significant. [Figure 5D]Figures 5C-5D show kinetic curves of red fluorescence for BT474 clone 5 labeled with 1 μg / mL pHrodo red and cocultured with human iMG at (A) an E:T ratio of 1:1 or (B) an E:T ratio of 1:4 in the presence of trastuzumab, anti-HER2 TEM mAb, or control (all at 20 nM). Figures 5C-5D show that values at 72 hours are also plotted, and statistical differences are indicated. Each symbol or column represents the mean + / - SEM of n=3 wells. Statistical differences were determined by one-way ANOVA; *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001; ns=not significant. [Figure 6A] Figure 6C shows the kinetic curves of spheroid area formed by MDA-MB-361 cells co-cultured with iMG at an E:T ratio of 1:1 for 192 hours in the presence of (A) 80 nM and (B) 8 nM anti-HER2 TEM mAb, trastuzumab, or isotype IgG1. Figure 6C shows the values at 97 hours plotted for both the 80 nM and 8 nM concentrations. [Figure 6B] Figure 6C shows the kinetic curves of spheroid area formed by MDA-MB-361 cells co-cultured with iMG at an E:T ratio of 1:1 for 192 hours in the presence of (A) 80 nM and (B) 8 nM anti-HER2 TEM mAb, trastuzumab, or isotype IgG1. Figure 6C shows the values at 97 hours plotted for both the 80 nM and 8 nM concentrations. [Figure 6C] Figure 6C shows the kinetic curves of spheroid area formed by MDA-MB-361 cells co-cultured with iMG at an E:T ratio of 1:1 for 192 hours in the presence of (A) 80 nM and (B) 8 nM anti-HER2 TEM mAb, trastuzumab, or isotype IgG1. Figure 6C shows the values at 97 hours plotted for both the 80 nM and 8 nM concentrations. [Figure 7A]IL-1β, IL-10, and TNFα concentrations determined at 20 and 44 hours in conditioned medium from cocultures of (A) HCC1954 or (B) MDA-MB-361 and human iMG at an E:T ratio of 1:1 are shown in the presence of 20 nM trastuzumab (blue), HER2-TfRk-mut G1 (red), biparatopic HER2-TfRk-mut G1 (open red), or no antibody treatment (black). Each dot represents a value from a replicate, and the horizontal line in each column represents the mean. One-way ANOVA was performed to determine statistical differences. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001, ns=not significant. [Figure 7B] IL-1β, IL-10, and TNFα concentrations determined at 20 and 44 hours in conditioned medium from cocultures of (A) HCC1954 or (B) MDA-MB-361 and human iMG at an E:T ratio of 1:1 are shown in the presence of 20 nM trastuzumab (blue), HER2-TfRk-mut G1 (red), biparatopic HER2-TfRk-mut G1 (open red), or no antibody treatment (black). Each dot represents a value from a replicate, and the horizontal line in each column represents the mean. One-way ANOVA was performed to determine statistical differences. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001, ns=not significant. [Figure 8A]TEM promotes NCP in peripheral immune cells. (A) Killing kinetics mediated by trastuzumab and TEM in cocultures of human PBMCs and the trastuzumab-resistant BT474 clone 5 cell line. (B) Killing kinetics mediated by trastuzumab and TEM in cocultures of M2a macrophages and the BT474 cell line. (C) Representative photographs of M2a macrophages (blue) and the BT474 cell line (yellow) after 168 hours of coculture with trastuzumab and TEM. (D) pHrodo Red phagocytosis kinetics in cocultures of M2a macrophages (blue) and pHrodo-labeled BT474 clone 5 cells (yellow) with trastuzumab and TEM. (E) Sum of pHrodo Red signal in M2a macrophages reflecting the total number of phagocytic events over 14 hours of coculture. In all panels, mAb concentration = 80 nM. In panel A, the E:T ratio is 10:1, while in panels B, C, D, E, the E:T ratio is 3:1. [Figure 8B] TEM promotes NCP in peripheral immune cells. (A) Killing kinetics mediated by trastuzumab and TEM in cocultures of human PBMCs and the trastuzumab-resistant BT474 clone 5 cell line. (B) Killing kinetics mediated by trastuzumab and TEM in cocultures of M2a macrophages and the BT474 cell line. (C) Representative photographs of M2a macrophages (blue) and the BT474 cell line (yellow) after 168 hours of coculture with trastuzumab and TEM. (D) pHrodo Red phagocytosis kinetics in cocultures of M2a macrophages (blue) and pHrodo-labeled BT474 clone 5 cells (yellow) with trastuzumab and TEM. (E) Sum of pHrodo Red signal in M2a macrophages reflecting the total number of phagocytic events over 14 hours of coculture. In all panels, mAb concentration = 80 nM. In panel A, the E:T ratio is 10:1, while in panels B, C, D, E, the E:T ratio is 3:1. [Figure 8C]TEM promotes NCP in peripheral immune cells. (A) Killing kinetics mediated by trastuzumab and TEM in cocultures of human PBMCs and the trastuzumab-resistant BT474 clone 5 cell line. (B) Killing kinetics mediated by trastuzumab and TEM in cocultures of M2a macrophages and the BT474 cell line. (C) Representative photographs of M2a macrophages (blue) and the BT474 cell line (yellow) after 168 hours of coculture with trastuzumab and TEM. (D) pHrodo Red phagocytosis kinetics in cocultures of M2a macrophages (blue) and pHrodo-labeled BT474 clone 5 cells (yellow) with trastuzumab and TEM. (E) Sum of pHrodo Red signal in M2a macrophages reflecting the total number of phagocytic events over 14 hours of coculture. In all panels, mAb concentration = 80 nM. In panel A, the E:T ratio is 10:1, while in panels B, C, D, E, the E:T ratio is 3:1. [Figure 8D-1] TEM promotes NCP in peripheral immune cells. (A) Killing kinetics mediated by trastuzumab and TEM in cocultures of human PBMCs and the trastuzumab-resistant BT474 clone 5 cell line. (B) Killing kinetics mediated by trastuzumab and TEM in cocultures of M2a macrophages and the BT474 cell line. (C) Representative photographs of M2a macrophages (blue) and the BT474 cell line (yellow) after 168 hours of coculture with trastuzumab and TEM. (D) pHrodo Red phagocytosis kinetics in cocultures of M2a macrophages (blue) and pHrodo-labeled BT474 clone 5 cells (yellow) with trastuzumab and TEM. (E) Sum of pHrodo Red signal in M2a macrophages reflecting the total number of phagocytic events over 14 hours of coculture. In all panels, mAb concentration = 80 nM. In panel A, the E:T ratio is 10:1, while in panels B, C, D, E, the E:T ratio is 3:1. [Figure 8D-2]TEM promotes NCP in peripheral immune cells. (A) Killing kinetics mediated by trastuzumab and TEM in cocultures of human PBMCs and the trastuzumab-resistant BT474 clone 5 cell line. (B) Killing kinetics mediated by trastuzumab and TEM in cocultures of M2a macrophages and the BT474 cell line. (C) Representative photographs of M2a macrophages (blue) and the BT474 cell line (yellow) after 168 hours of coculture with trastuzumab and TEM. (D) pHrodo Red phagocytosis kinetics in cocultures of M2a macrophages (blue) and pHrodo-labeled BT474 clone 5 cells (yellow) with trastuzumab and TEM. (E) Sum of pHrodo Red signal in M2a macrophages reflecting the total number of phagocytic events over 14 hours of coculture. In all panels, mAb concentration = 80 nM. In panel A, the E:T ratio is 10:1, while in panels B, C, D, E, the E:T ratio is 3:1. [Figure 8E] TEM promotes NCP in peripheral immune cells. (A) Killing kinetics mediated by trastuzumab and TEM in cocultures of human PBMCs and the trastuzumab-resistant BT474 clone 5 cell line. (B) Killing kinetics mediated by trastuzumab and TEM in cocultures of M2a macrophages and the BT474 cell line. (C) Representative photographs of M2a macrophages (blue) and the BT474 cell line (yellow) after 168 hours of coculture with trastuzumab and TEM. (D) pHrodo Red phagocytosis kinetics in cocultures of M2a macrophages (blue) and pHrodo-labeled BT474 clone 5 cells (yellow) with trastuzumab and TEM. (E) Sum of pHrodo Red signal in M2a macrophages reflecting the total number of phagocytic events over 14 hours of coculture. In all panels, mAb concentration = 80 nM. In panel A, the E:T ratio is 10:1, while in panels B, C, D, E, the E:T ratio is 3:1. [Figure 9]PK of TEM mAb in non-human primates is shown. TEM and control IgG1 mAb were administered to cynomolgus monkeys at 10 mg / kg by slow bolus IV injection. Blood for PK was collected 1, 6, 24, 72, and 168 hours after administration and processed to serum via the test facility laboratory protocol. After the final blood collection, animals were euthanized at 72 and 168 hours (n=2 per time point). Approximately 200 mg of tissue was isolated from defined brain locations (frontal lobe, hippocampus, and temporal lobe). [Figure 10A] Figure 10 shows the in vivo biodistribution of Zr89-DFO*-HER2xTfR antibody (HER2xTfR) in C57BL6 (B6) and human TfR knock-in (TfR) mice. Figure 10A shows the SUV of Zr89-DFO*-HER2xTfR antibody in tissues on day 1. Figure 10B shows the SUV of Zr89-DFO*-HER2xCDTfR antibody in tissues on day 5. Figure 10C shows the SUV of Zr89-DFO*-HER2xCDTfR antibody in tissues on day 7. [Figure 10B] Figure 10 shows the in vivo biodistribution of Zr89-DFO*-HER2xTfR antibody (HER2xTfR) in C57BL6 (B6) and human TfR knock-in (TfR) mice. Figure 10A shows the SUV of Zr89-DFO*-HER2xTfR antibody in tissues on day 1. Figure 10B shows the SUV of Zr89-DFO*-HER2xCDTfR antibody in tissues on day 5. Figure 10C shows the SUV of Zr89-DFO*-HER2xCDTfR antibody in tissues on day 7. [Figure 10C] Figure 10 shows the in vivo biodistribution of Zr89-DFO*-HER2xTfR antibody (HER2xTfR) in C57BL6 (B6) and human TfR knock-in (TfR) mice. Figure 10A shows the SUV of Zr89-DFO*-HER2xTfR antibody in tissues on day 1. Figure 10B shows the SUV of Zr89-DFO*-HER2xCDTfR antibody in tissues on day 5. Figure 10C shows the SUV of Zr89-DFO*-HER2xCDTfR antibody in tissues on day 7. [Figure 11A]Figure 11A shows brain and heart uptake of HER2xTfR and Zr89-DFO*-HER2 (HER2) antibodies in C57BL6 (BL6) and human TfR knock-in (huTfR KI) mice. Figure 11A shows brain SUV on day 1. Figure 11B shows brain SUV on day 5. Figure 11C shows brain-to-heart ratio of SUV on day 1. Figure 11D shows brain-to-heart ratio of SUV on day 5. [Figure 11B] Figure 11A shows brain and heart uptake of HER2xTfR and Zr89-DFO*-HER2 (HER2) antibodies in C57BL6 (BL6) and human TfR knock-in (huTfR KI) mice. Figure 11A shows brain SUV on day 1. Figure 11B shows brain SUV on day 5. Figure 11C shows brain-to-heart ratio of SUV on day 1. Figure 11D shows brain-to-heart ratio of SUV on day 5. [Figure 11C] Figure 11A shows brain and heart uptake of HER2xTfR and Zr89-DFO*-HER2 (HER2) antibodies in C57BL6 (BL6) and human TfR knock-in (huTfR KI) mice. Figure 11A shows brain SUV on day 1. Figure 11B shows brain SUV on day 5. Figure 11C shows brain-to-heart ratio of SUV on day 1. Figure 11D shows brain-to-heart ratio of SUV on day 5. [Figure 11D] Figure 11A shows brain and heart uptake of HER2xTfR and Zr89-DFO*-HER2 (HER2) antibodies in C57BL6 (BL6) and human TfR knock-in (huTfR KI) mice. Figure 11A shows brain SUV on day 1. Figure 11B shows brain SUV on day 5. Figure 11C shows brain-to-heart ratio of SUV on day 1. Figure 11D shows brain-to-heart ratio of SUV on day 5. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention relates to a multispecific antibody comprising at least one of a first antigen-binding region and a second antigen-binding region, each capable of specifically binding to human epidermal growth factor receptor 2 (HER2), and a third antigen-binding region capable of specifically binding to transferrin receptor (TfR).

[0037] definition The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and / or commonly employed by those skilled in the art using conventional techniques, e.g., Sambrook et al: A Laboratory Manual (3rd ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009), Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010), and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2nd ed. 2010). Unless otherwise defined herein, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. For purposes of interpreting this specification, the following explanations of terms shall apply, and where appropriate, terms used in the singular shall also include the plural and vice versa. In the event that any explanation of a term provided herein conflicts with any document incorporated by reference, the explanation of the term provided below shall prevail.

[0038] The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and are used in the broadest sense, specifically encompassing, for example, monoclonal antibodies (including agonist, antagonist, neutralizing, and full-length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain antibodies, and fragments thereof, as described below. Antibodies may be human, humanized, chimeric, and / or affinity matured, and may also be antibodies from other species, e.g., mouse, rabbit, llama, etc. The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides that are capable of binding to a specific molecular antigen and are composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids, and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2nd ed. 1995); and Kuby, Immunology (3rd ed. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, antibodies derived from Camelidae species (e.g., llamas and alpacas) or humanized variants thereof, intracellular antibodies, anti-idiotype (anti-Id) antibodies, and functional fragments of any of the above (e.g., antigen-binding fragments), and refer to portions of antibody heavy or light chain polypeptides that retain some or all of the binding activity of the antibody from which the fragment is derived.Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen-binding domains or molecules containing an antigen-binding site that binds to an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989), Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules. The antibodies can be agonist or antagonist antibodies. The antibody may be neither an agonist nor an antagonist.

[0039] An "antigen" is a structure to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with a cell, e.g., present on or within a cell.

[0040] An "intact" antibody is one that contains an antigen-binding site as well as a CL and at least the heavy chain constant regions CH1, CH2, and CH3. The constant region may include a human constant region or a variant thereof. In certain embodiments, an intact antibody has one or more effector functions.

[0041] The terms "binding" or "binding" refer to interactions between molecules, including, for example, forming a complex. The interaction can be a non-covalent interaction, including, for example, hydrogen bonding, ionic bonding, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the association of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site of an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The dissociation rate (k off ) and association rate (k on ) and the ratio (k off / k on ) is the dissociation constant K D and has an inverse relationship with affinity. D The lower the value, the higher the affinity of the antibody. D The value of k varies for different complexes of antibody and antigen. on and k off The dissociation constant K of the antibodies provided herein depends on both D Affinity can be determined using any of the methods provided herein or any other method known to those skilled in the art. The affinity at one binding site does not necessarily reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeating antigenic determinants, such as a multivalent antigen, comes into contact with an antibody containing multiple binding sites, the interaction of the antibody with the antigen at one site will increase the probability of reaction at a second site. The strength of multiple interactions between such a multivalent antibody and an antigen is called avidity.

[0042] In the context of the binding molecules described herein, terms such as "binds to," "specifically binds to," and similar terms are also used interchangeably herein to refer to binding molecules of an antigen-binding domain that specifically binds to an antigen, such as a polypeptide. Binding molecules or antigen-binding domains that bind to or specifically bind to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other techniques known to those of skill in the art. In some embodiments, a binding molecule or antigen-binding domain binds to or specifically binds to an antigen if it binds to the antigen with higher affinity than any cross-reactive antigens as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response is at least two times the background signal or noise, and may exceed ten times the background signal or noise. For a discussion of binding specificity, see, e.g., Fundamental Immunology 332-36 (Paul, ed., 2d ed. 1989). In certain embodiments, the extent of binding of a binding molecule or antigen-binding domain to a "non-target" protein is less than about 10% of the binding of the binding molecule or antigen-binding domain to its particular target antigen, as determined, for example, by fluorescence activated cell sorting (FACS) analysis or RIA. Binding molecules or antigen-binding domains that bind to an antigen include those that can bind the antigen with sufficient affinity so that the binding molecule is useful, for example, as an antigen-targeted therapeutic and / or diagnostic agent. In certain embodiments, a binding molecule or antigen-binding domain that binds to an antigen has a dissociation constant (K) of 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM or less. DIn certain embodiments, the binding molecule or antigen-binding domain binds to an epitope of an antigen that is conserved among antigens of different species.

[0043] In certain embodiments, binding molecules or antigen-binding domains may comprise "chimeric" sequences in which a portion of the heavy and / or light chain is identical to or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567 and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55). Chimeric sequences may include humanized sequences.

[0044] In certain embodiments, a binding molecule or antigen-binding domain may comprise a portion of a "humanized" form of a non-human (e.g., camelid, murine, non-human primate) antibody comprising sequences from a human immunoglobulin (e.g., recipient antibody) in which native CDR residues are replaced by residues from a corresponding CDR of a non-human species (e.g., donor antibody) such as camel, mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some cases, one or more FR region residues of the human immunoglobulin sequence are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. The heavy or light chain of a humanized antibody can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, a humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-29 (1988); Presta, Curr. Op. Struct. Biol., 2:593-96 (1992); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); U.S. Patent Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

[0045] In certain embodiments, a binding molecule or antigen-binding domain can comprise a portion of a "fully human antibody" or "human antibody," which terms are used interchangeably herein to refer to an antibody comprising a human variable region and, for example, a human constant region. A binding molecule can comprise antibody sequences. In specific embodiments, these terms refer to antibodies comprising variable and constant regions of human origin. A "fully human" antibody can also encompass, in certain embodiments, antibodies that bind to a polypeptide and that are encoded by nucleic acid sequences that are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequences. The term "fully human antibody" includes antibodies having variable and constant regions that correspond to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or is produced using any of the techniques for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)) and yeast display libraries (Chao, et al., Nature Protocols, 1:755-68 (2006)).Furthermore, human monoclonal antibodies can be prepared using the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985), Boerner et al., J. Immunol. 147(1):86-95 (1991), and van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., mice, that have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous gene loci have been disabled (see, e.g., Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995); Bruggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997); and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology. See also, e.g., Li, et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006) (regarding human antibodies generated by human B-cell hybridoma technology).

[0046] In certain embodiments, the binding molecule or antigen-binding domain, antigen-binding protein may comprise a portion of a "recombinant human antibody," which term includes human antibodies prepared, expressed, created, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector introduced into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from animals (e.g., mice or cows) that are transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, LD et al., Nucl. Acids Res. 20:6287-6295 (1992)), or antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

[0047] In certain embodiments, a binding molecule or antigen-binding domain can comprise a portion of a "monoclonal antibody," a term used herein to refer to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for minor naturally occurring mutations and well-known post-translational modifications, such as amino acid isomerization and deamidation, oxidation of methionine, and deamidation of asparagine and glutamine, and each monoclonal antibody typically recognizes a single epitope on an antigen. In specific embodiments, a "monoclonal antibody," as used herein, is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to a particular method for making the antibody. For example, monoclonal antibodies useful in the present disclosure can be prepared by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991). Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art. See, for example, Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).

[0048] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). In the case of IgG, the four-chain unit generally has a size of approximately 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus followed by three constant domains (CH) for the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain and heavy-chain variable domains. The pairing of VH and VL together forms a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology 71 (Stites, et al. eds., 8th ed. 1994) and Immunobiology (Janeway, et al. eds., 5th ed. 2001).

[0049] The term "Fab" or "Fab region" refers to the region of an antibody that binds to an antigen. Conventional IgGs typically contain two Fab regions, each located in one of the two arms of the Y-shaped IgG structure. Each Fab region typically consists of one variable region and one constant region from each of the heavy and light chains. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL regions in the Fab region can be arranged in various ways to confer antigen-binding capability according to the present disclosure. For example, the VH and CH1 regions can be on one polypeptide, while the VL and CL regions can be on separate polypeptides, as in the Fab region of a conventional IgG. Alternatively, the VH, CH1, VL, and CL regions can all be present on the same polypeptide and oriented in a different order, as described in more detail below.

[0050] The terms "variable region," "variable domain," "V region," or "V domain" refer to a portion of an antibody light or heavy chain located generally at the amino terminus of the light or heavy chain, approximately 120-130 amino acids in length for heavy chains and approximately 100-110 amino acids in length for light chains, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable region of a heavy chain may be referred to as "VH." The variable region of a light chain may be referred to as "VL." The term "variable" refers to the fact that certain segments of the variable region vary significantly in sequence among antibodies. The V region mediates antigen binding and determines the specificity of a particular antibody for a particular antigen. However, variability is not uniform across the 110-amino acid span of the variable region. Instead, V regions consist of less variable (e.g., relatively invariant) stretches of approximately 15-30 amino acids called framework regions (FRs) separated by shorter regions of greater variability (e.g., extreme variability) called "hypervariable regions," each approximately 9-12 amino acids in length. The heavy and light chain variable regions each contain four FRs, which adopt a largely β-sheet structure connected by three hypervariable regions, which form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions within each chain are held together in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant region is not directly involved in binding the antibody to an antigen, but exhibits various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable region varies significantly in sequence among different antibodies. In a specific embodiment, the variable region is a human variable region.

[0051] The terms "variable region residue numbering according to Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering system used for the heavy or light chain variable regions of the antibody compilation of Kabat et al. (supra). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by aligning the antibody's sequence with the "standard" Kabat numbering sequence at the regions of homology. The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 in the light chain, approximately residues 1-113 in the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0052] The term "heavy chain" when used with respect to antibodies refers to a polypeptide chain of approximately 50 to 70 kDa, the amino-terminal portion of which contains a variable region of approximately 120 to 130 amino acids and the carboxy-terminal portion of which contains a constant region. The constant region can be one of five distinct types (e.g., isotypes), designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains vary in size, with α, δ, and γ containing approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with light chains, these distinct types of heavy chains give rise to five well-known classes (e.g., isotypes) of antibodies: IgA, IgD, IgE, IgG, and IgM (including the four subclasses of IgG, i.e., IgG1, IgG2, IgG3, and IgG4).

[0053] The term "light chain" when used in reference to an antibody refers to a polypeptide chain of approximately 25 kDa, which contains a variable region of about 100 to about 110 or more amino acids at its amino terminal end and a constant region at its carboxy terminal end. The approximate length of a light chain is 211 to 217 amino acids. There are two different types, called kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain.

[0054] As used herein, the terms "hypervariable region," "hypervariable region (HVR)," "complementarity determining region," and "Complementarity Determining Region (CDR)" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of an immunoglobulin (Ig or antibody) VH β-sheet framework, or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of an antibody VL β-sheet framework. CDR1, CDR2, and CDR3 in a VH domain are also referred to as HCDR1, HCDR2, and HCDR3, respectively. CDR1, CDR2, and CDR3 in a VL domain are also referred to as LCDR1, LCDR2, and LCDR3, respectively. Thus, CDRs are variable region sequences interspersed within framework region sequences.

[0055] CDR regions are well known to those skilled in the art and are defined by well-known numbering systems. For example, Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (see, for example, Kabat et al. (supra); see Nick Deschacht et al., J Immunol 2010,184:5696-5704). "Chothia" instead refers to the position of structural loops (see, for example, Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). The ends of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, vary from H32 to H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Dubel, eds., 2nd ed. 2010)). The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc, et al., Dev. Comp. Immunol. 27(1):55-77(2003)). IMGT is an integrated information system dedicated to immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. As used herein, CDRs are referred to in terms of both amino acid sequence and location within the light or heavy chain.Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved among species and resides in structures called loops, CDR and framework residues are readily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and replace CDR residues from one species of immunoglobulin into an acceptor framework, typically from a human antibody. An additional numbering system (AHon) was developed by Honegger and Pluckthun, J. Mol. Biol. 309:657-70 (2001). For example, the correspondence between numbering systems, including Kabat numbering and the IMGT specific numbering system, is well known to those skilled in the art (see, e.g., Kabat (supra), Chothia and Lesk (supra), Martin (supra), Lefranc et al., supra). Residues from each of these hypervariable regions or CDRs are illustrated in Table 1 below.

[0056] [Table 1]

[0057] The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms "CDR" and "complementarity-determining region" of a given antibody or region thereof, such as a variable region, and individual CDRs of an antibody or region thereof (e.g., CDR-H1, CDR-H2) should be understood to encompass the complementarity-determining regions defined by any of the known schemes described hereinabove. In some cases, a scheme for identifying a particular CDR or CDRs is specified, such as CDRs defined by the IMGT, Kabat, Chothia, or Contact methods. In other cases, a specific amino acid sequence of the CDR is included. Note that CDR regions can also be defined by a combination of various numbering systems, for example, a combination of the Kabat numbering system and the Chothia numbering system, or a combination of the Kabat numbering system and the IMGT numbering system. Thus, a term such as "CDR1 shown in a particular VH" includes any CDR1 defined by a system, including, but not limited to, the exemplary CDR numbering systems described above. Given a variable region (eg, VH or VL), one of skill in the art will understand that the CDRs within the region may be defined by different numbering systems or combinations thereof.

[0058] The hypervariable regions may include "extended hypervariable regions" such as: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH.

[0059] The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains that are not directly involved in binding the antibody to an antigen, but which exhibit various effector functions, such as interaction with Fc receptors. This term refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of the immunoglobulin, the variable region, which contains the antigen-binding site. The constant region can include the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0060] The term "framework" or "FR" refers to variable domain residues that flank the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain, diabodies, linear, and bispecific antibodies. FR residues are variable domain residues other than hypervariable region or CDR residues.

[0061] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native-sequence Fc regions, recombinant Fc regions, and variant Fc regions. While the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is often defined to stretch from the amino acid residue at Cys226, or from Pro230, to the carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, an intact antibody composition can include an antibody population in which all K447 residues have been removed, an antibody population in which the K447 residue has not been removed, and an antibody population having a mixture of antibodies with and without the K447 residue. A "functional Fc region" possesses the "effector functions" of a native-sequence Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require that the Fc region be combined with a binding region or domain (e.g., an antibody variable region or domain) and can be assessed using a variety of assays known to those of skill in the art. A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, the variant Fc region comprises at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions, or from about 1 to about 5 amino acid substitutions in the native-sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region herein may have at least about 80% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide, or at least about 90% homology thereto, for example, at least about 95% homology thereto.

[0062] As used herein, "epitope" is a term of art that refers to a localized region of an antigen to which a binding molecule (e.g., an antibody) can specifically bind. An epitope can be a linear or conformational epitope, a non-linear epitope, or a discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be consecutive amino acids of a polypeptide (a "linear" epitope), or an epitope can include amino acids from two or more non-contiguous regions of a polypeptide (a "conformational," "non-linear," or "discontinuous" epitope). In general, it will be understood by those skilled in the art that a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids regardless of whether the amino acids are folded into the native three-dimensional protein structure. In other embodiments, the binding molecule requires that the amino acid residues that make up the epitope exhibit a particular conformation (eg, a bend, twist, turn, or fold) in order to recognize and bind to the epitope.

[0063] The term "biparatopic" antigen-binding molecule or "biparatopic" polypeptide, as used herein, is intended to mean a polypeptide comprising a first immunoglobulin single variable domain as defined herein and a second immunoglobulin single variable domain, wherein these two variable domains are capable of binding to two different epitopes of an antigen.

[0064] "Percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a particular peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0065] The term "specificity" refers to the selective recognition of an antigen-binding protein for a specific epitope of an antigen. Natural antibodies, for example, are monospecific. As used herein, the term "multispecific" refers to an antigen-binding protein having two or more antigen-binding sites, at least two of which bind different antigens. As used herein, "bispecific" refers to an antigen-binding protein having two different antigen-binding specificities. As used herein, the term "monospecific" antibody refers to an antigen-binding protein having one or more binding sites, each of which binds to the same antigen.

[0066] The term "valent" as used herein refers to the presence of a specific number of binding sites in an antigen-binding protein. For example, a natural antibody or a full-length antibody has two binding sites and is bivalent. Thus, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" refer to the presence of two, three, four, five, and six binding sites in an antigen-binding protein, respectively.

[0067] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The term also refers to amino acid polymers that are naturally modified or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. The definition also includes polypeptides containing one or more analogs of an amino acid, including, but not limited to, unnatural amino acids, as well as other modifications known in the art. Because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in certain embodiments, a "polypeptide" can occur as a single chain or as two or more related chains.

[0068] "Polynucleotide" or "nucleic acid," as used interchangeably herein, refer to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to a short, generally single-stranded, synthetic polynucleotide, generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is fully applicable to oligonucleotides as well. Cells that produce the binding molecules of the present disclosure can include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acid encoding the antibody has been introduced. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end. The left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of the nascent RNA transcript is referred to as the transcription direction. The region of the DNA strand with the same sequence as the RNA transcript at the 5' to 5' end of the RNA transcript is referred to as the "upstream sequence," and the region of the DNA strand with the same sequence as the RNA transcript at the 3' to 3' end of the RNA transcript is referred to as the "downstream sequence."

[0069] An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or mixed nucleic acid, that is substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, that are naturally associated with the native sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. In certain embodiments, one or more nucleic acid molecules encoding an antibody described herein are isolated or purified. This term encompasses nucleic acid sequences that have been removed from their naturally occurring environment, including recombinant or cloned DNA isolates and chemically synthesized analogs, or analogs biologically synthesized in heterologous systems. A substantially pure molecule can include isolated forms of the molecule. Specifically, an "isolated" nucleic acid molecule encoding an antibody described herein is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced.

[0070] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes nucleotide sequences that are degenerate versions of each other and all nucleotide sequences that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns to the extent that, in some cases, the nucleotide sequence encoding the protein may contain introns.

[0071] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0072] As used herein, the term "operably linked," and similar phrases (e.g., genetically fused), when used with reference to nucleic acids or amino acids, refers to an operable linkage in which nucleic acid or amino acid sequences, respectively, are placed in a functional relationship with each other. For example, operably linked promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the correct production of a nucleic acid molecule (e.g., RNA). In some embodiments, operably linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operably linked peptide refers to one in which functional regions are positioned at an appropriate distance from each other to confer their intended function.

[0073] The term "vector" refers to a substance used to carry or contain a nucleic acid sequence, including, for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) described herein, to introduce the nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain operable selection sequences or markers capable of stable integration into a host cell chromosome. In addition, a vector may contain one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that may be included provide, for example, resistance to antibiotics or toxins, complement deficiencies in auxiliary auxotrophies, or supply critical nutrients absent from the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, as are well known in the art. When two or more nucleic acid molecules are coexpressed (e.g., both the heavy and light chains of an antibody, or antibody VH and VL), both nucleic acid molecules may be inserted, for example, into a single expression vector or into separate expression vectors. For expression in a single vector, the encoding nucleic acids can be operably linked to a common expression control sequence or to different expression control sequences, such as one inducible promoter and one constitutive promoter. Introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blot or amplification of mRNA by polymerase chain reaction (PCR), immunoblotting for expression of gene products, or other suitable analytical methods for testing expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that nucleic acid molecules will be expressed in amounts sufficient to produce the desired product, and will further understand that expression levels can be optimized to obtain sufficient expression using methods well known to those skilled in the art.

[0074] As used herein, the term "host" refers to an animal, such as a mammal (e.g., a human).

[0075] As used herein, the term "host cell" refers to a particular subject cell into which a nucleic acid molecule can be transfected, and to the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur during the subsequent generation or integration of the nucleic acid molecule into the host cell genome.

[0076] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0077] As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals, and more particularly for use in humans, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias.

[0078] The term "excipient" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, encapsulating material, etc. Excipients include, for example, encapsulating materials or additives such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavorings, wetting agents, lubricants, fragrances, preservatives, propellants, release agents, bactericides, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle.

[0079] In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; proteins such as low molecular weight (e.g., less than about 10 amino acid residues) polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990).

[0080] In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other components of the pharmaceutical formulation and suitable for use in contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed., Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed., Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed., Gibson Ed., CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutically acceptable excipient is nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffered solution.

[0081] In some embodiments, the excipient may be a sterile liquid, such as water or oil, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, or sesame oil. Water is an exemplary excipient when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid excipients, particularly for injectable solutions. Excipients may also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene, glycol, water, ethanol, or the like. If desired, the composition may further contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition may take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, or the like. Oral composition containing formulations can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0082] A composition comprising a pharmaceutical compound can include, for example, a binding molecule (eg, an antibody) in isolated or purified form, together with a suitable amount of an excipient.

[0083] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an antibody or therapeutic molecule, including drugs and antibodies or pharmaceutical compositions provided herein, that is sufficient to bring about a desired result.

[0084] The terms "subject" and "patient" can be used interchangeably herein. As used herein, in certain embodiments, a subject is a mammal, either non-primate or primate (e.g., human). In specific embodiments, a subject is a human. In one embodiment, a subject is a mammal, e.g., a human, diagnosed with a disease or disorder. In another embodiment, a subject is a mammal, e.g., a human, at risk of developing a disease or disorder.

[0085] "Administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance present outside the body to a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0086] As used herein, the terms "treatment" and "treatment / treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or condition resulting from the administration of one or more therapies. Treatment may be determined by assessing whether there has been a reduction, alleviation, and / or alleviation of one or more symptoms associated with the underlying disease, such that an improvement is observed in the patient, even though the patient may still be suffering from the underlying disease. The term "treating" includes both disease management and remission. The terms "manage," "managing," and "management" refer to the beneficial effects a subject derives from treatment, which does not necessarily result in a cure of the disease.

[0087] The terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of the occurrence (or recurrence) of a disease, disorder, condition, or associated symptom (e.g., diabetes or cancer).

[0088] As used herein, "delaying" the onset of cancer means postponing, preventing, slowing, retarding, stabilizing, and / or postponing the onset of the disease. This delay can be of varying duration, depending on the disease history and / or the individual receiving treatment. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. A method for "delaying" the onset of cancer is one that reduces the probability of disease development in a given time frame and / or reduces the extent of disease in a given time frame compared to the absence of the method. Such comparisons are typically based on clinical studies using a statistically significant number of individuals. Cancer development can be detected using standard methods, including, but not limited to, computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Development can also refer to the progression of cancer, which may be undetectable initially, and includes occurrence, recurrence, and onset.

[0089] The term "HER2" (also known as HER2 / neu and ErbB-2) refers to "human epidermal growth factor receptor 2." As used herein, it is intended to include variants, isoforms, and species homologs of HER2.

[0090] As used herein, the term "HER2-associated disease or disorder" refers to a disease or disorder involving cells or tissues in which HER2 is expressed or overexpressed. In some embodiments, a HER2-associated disease or disorder involves cells in which HER2 is aberrantly expressed. In other embodiments, a HER2-associated disease or disorder involves cells in which at least one of the activities of HER2 is deficient.

[0091] The "blood-brain barrier" or "BBB" refers to the physiological barrier between the peripheral circulation and the brain and spinal cord, formed by tight junctions in the brain capillary endothelial cell membranes, forming a tight barrier that restricts the transport of molecules into the brain. The BBB can restrict the transport of even very small molecules, such as urea (60 daltons), into the brain. Examples of BBBs include the BBB in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retinal barrier in the retina, all of which are continuous capillary barriers in the CNS. The BBB also encompasses the blood-CSF barrier (choroid plexus), which is composed of ependymal cells rather than capillary endothelial cells.

[0092] "Blood-brain barrier receptors" (abbreviated herein as "R / BBB") are extracellular membrane-bound receptor proteins that are expressed on brain endothelial cells and can transport molecules across the BBB or are used to transport exogenously administered molecules. Examples of R / BBB receptors include, but are not limited to, the large neutral amino acid transporter (LAT) complex, which includes the CD98 component, the transferrin receptor (TfR), the insulin receptor, the insulin-like growth factor receptor (IGF-R), the low-density lipoprotein receptors, including, but not limited to, low-density lipoprotein receptor-related protein 1 (LRP1) and low-density lipoprotein receptor-related protein 8 (LRP8), and heparin-binding epidermal growth factor-like growth factor (HB-EGF). An exemplary R / BBB receptor herein is the transferrin receptor (TfR).

[0093] As used herein, the term "transferrin receptor" or "TfR" refers to a cell surface receptor required for cellular iron uptake via the process of receptor-mediated endocytosis of transferrin carrier proteins. TfR is involved in iron uptake in vertebrates and is regulated in response to intracellular iron concentrations. TfR uptakes iron by internalizing transferrin-iron complexes via receptor-mediated endocytosis. Two transferrin receptors, transferrin receptor 1 and transferrin receptor 2, have been characterized in humans. Both receptors are transmembrane glycoproteins. TfR1 is a high-affinity, ubiquitously expressed receptor. TfR2 binds to transferrin with 25- to 30-fold lower affinity than TfR1. TfR2 expression is restricted to certain cell types and is not affected by intracellular iron concentrations. In one embodiment, the TfR is a human TfR comprising the amino acid sequence described, for example, in Schneider et al., Nature 311:675-678 (1984). The TfR may have a molecular weight of about 180,000 daltons and have two subunits, each with an apparent molecular weight of about 90,000 daltons. Preferably, the TfR is human TfR1.

[0094] The terms "about" and "approximately" refer to within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0095] As used in this disclosure and the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0096] Whenever an embodiment is described herein with the term "comprising," it is understood that other similar embodiments otherwise described in terms of "consisting of" and / or "consisting essentially of" are also provided. Whenever an embodiment is described herein with the phrase "consisting essentially of," it is also understood that similar embodiments otherwise described in terms of "consisting of" are also provided.

[0097] The term "between" when used in phrases such as "between A and B" or "between A and B" refers to a range that includes both A and B.

[0098] The term "and / or" used herein in phrases such as "A and / or B" is intended to include both A and B, A or B, A alone, and B alone. Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A alone, B alone, and C alone.

[0099] Multispecific antibodies with tissue-targeting moieties In one general aspect, the present application relates to an optimized platform for specific tissue delivery. In one aspect, the platform utilizes binding molecules, particularly antibodies or antigen-binding fragments thereof, that bind to targets expressed on specific tissues.

[0100] In one aspect, provided herein is a multispecific antibody comprising at least one of a first antigen-binding region and a second antigen-binding region, each capable of specifically binding to human epidermal growth factor receptor 2 (HER2), and a third antigen-binding region capable of specifically binding to transferrin receptor (TfR).

[0101] HER2 (also known as ErbB2 or Neu, UniProtKB / Swiss-Prot No. P04626) consists of 1,233 amino acids and is structurally similar to EGFR, possessing an extracellular domain consisting of four subdomains I-IV, a transmembrane domain, a juxtamembrane domain, an intracellular cytoplasmic tyrosine kinase, and a regulatory C-terminal domain (Yamamoto et al. (1986) Nature 319:230-234). HER2 is activated through the formation of heteromeric complexes with other ErbB family members, thereby indirectly regulated by EGFR and HER3 ligands (reviewed in Yarden et al. (2001) Nat Rev Mol Cell Biol. 2:127-137). HER2 is the preferred heterodimerization partner for three other ErbB receptors (Graus-Porta et al. (1997) EMBO J 16:1647-1655, Tzahar et al. (1996) Mol Cell Biol. 16:5276-5287), and by slowing the rate of ligand-receptor complex dissociation, HER2 enhances the affinity of other ErbB receptors for their ligands, thereby enhancing and prolonging signal transduction (Pedersen et al. (2009) Mol Cancer Res. 7:275-284). Heterodimerization of HER2 with other ligand-binding receptors of the ErbB family induces cross-phosphorylation, resulting in phosphorylation of C-terminal amino acids. These then function as scaffolds for signal transduction molecules (King et al. (1988) EMBO J 7:1647-1651). The most active HER2 heterodimer is the HER2-HER3 complex (Pinkas-Kramarski et al. (1996) EMBO J 15:2452-2467), in which HER2 complements kinase-deficient HER3 by providing an active kinase (Guy et al. (1994) Proc Natl Acad Sci USA 91:8132-8136).In contrast to EGFR, HER2 is internalization resistant (Hommelgaard et al. (2004) Mol Biol Cell 15:1557-1567), avoiding lysosomal degradation and thereby remaining at the plasma membrane.

[0102] In certain embodiments, the antigen-binding regions provided herein comprise one or more CDR sequences. The sequences of the CDRs can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact.

[0103] In some embodiments, a first antigen-binding region provided herein comprises a first heavy chain variable region (VH1) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 set forth in HC comprising the amino acid sequence of SEQ ID NO: 1, and a first light chain variable region (VL1) comprising LCDR1, LCDR2, and LCDR3 set forth in LC comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the first heavy chain variable region (VH1) comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively, and the first light chain variable region (VL1) comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively.

[0104] In certain embodiments, VH1 comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH of the HC sequence identified as SEQ ID NO: 1, and VL1 comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VL of the LC sequence identified as SEQ ID NO: 2. In certain embodiments, VH1 comprises an amino acid sequence identical to the VH of the HC sequence identified as SEQ ID NO: 1, and VL1 comprises an amino acid sequence identical to the VL of the LC sequence identified as SEQ ID NO: 2.

[0105] In some embodiments, the second antigen-binding region provided herein comprises a first heavy chain variable region (VH2) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 set forth in HC comprising the amino acid sequence of SEQ ID NO: 11, and a second light chain variable region (VL2) comprising LCDR1, LCDR2, and LCDR3 set forth in LC comprising the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the second heavy chain variable region (VH1) comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively, and the first light chain variable region (VL1) comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively.

[0106] In certain embodiments, VH1 comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH of the HC sequence identified as SEQ ID NO: 1, and VL1 comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VL of the LC sequence identified as SEQ ID NO: 2. In certain embodiments, VH1 comprises an amino acid sequence identical to the VH of the HC sequence identified as SEQ ID NO: 1, and VL1 comprises an amino acid sequence identical to the VL of the LC sequence identified as SEQ ID NO: 2.

[0107] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, for example, to score=100 and word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set, for example, to score 50 and word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When using BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998).Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. Typically, only exact matches are counted when calculating percent identity.

[0108] In some embodiments, the antibodies provided herein contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but an anti-HER2 antibody comprising that sequence retains the ability to bind to HER2. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the reference amino acid sequence. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). Optionally, the anti-HER2 antibodies or antigen-binding regions provided herein include post-translational modifications of the reference sequence.

[0109] In some embodiments, functional epitopes can be mapped, for example, by combinatorial alanine scanning, to identify amino acids in the HER2 protein required for interaction with the anti-HER2 antibodies provided herein. In some embodiments, the three-dimensional structure and crystal structure of an anti-HER2 antibody bound to HER2 may be used to identify the epitope. In some embodiments, the present disclosure provides antibodies that specifically bind to the same epitope as any of the anti-HER2 antibodies provided herein.

[0110] In certain embodiments, the multispecific antibodies of the present invention comprise a third antigen-binding moiety that binds to a primate TfR, such as a human TfR or a monkey TfR, and the antibody or antigen-binding fragment thereof is optimized for delivering a drug to the brain of a subject in need thereof. The relationship between the binding affinity of an anti-TfR antibody to TfR and transcytosis efficiency has previously been described, with improved transcytosis resulting from decreased affinity for TfR (Yu, Zhang et al. 2011, Sci Transl Med 3(84):84ra44). Surprisingly, a more nuanced relationship between affinity and transcytosis efficiency than previously described was discovered, with both on-rate and off-rate effects affecting brain concentration. In particular, a balanced off-rate that is neither too fast nor too slow is required for an anti-TfR antibody or its antigen-binding fragment to efficiently achieve optimal brain PK and PD of an active substance (e.g., a mAb). Anti-TfR antibodies and antigen-binding fragments thereof are described in WO202105358, which is incorporated herein by reference in its entirety.

[0111] In certain embodiments, the third antigen-binding region comprises a first single-chain variable fragment (scFv1) having a third heavy chain variable region (VH3) comprising HCDR1, HCDR2, and HCDR3, and a third light chain variable region (VL3) comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are identical to the respective CDRs of the scFvs identified in Table 2.

[0112] In certain embodiments, the third antigen-binding region comprises a first single-chain variable fragment (scFv1) having a third heavy chain variable region (VH3) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and a third light chain variable region (VL3) comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each have any of the amino acid sequences identified in Table 2.

[0113] [Table 2-1]

[0114] [Table 2-2]

[0115] [Table 2-3]

[0116] In certain embodiments, the VH3 and VL3 comprise amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the respective VH and VL of the scFv identified as follows: SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:33, SEQ ID NO:40, SEQ ID NO:47, SEQ ID NO:54, SEQ ID NO:59, SEQ ID NO:66, SEQ ID NO:72, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:94, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:111, SEQ ID NO:118, SEQ ID NO:125, SEQ ID NO:132, SEQ ID NO:138, SEQ ID NO:144, SEQ ID NO:150, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:164, SEQ ID NO:169, SEQ ID NO:175, SEQ ID NO:183, SEQ ID NO:188, SEQ ID NO:193, SEQ ID NO:198, or SEQ ID NO:204.

[0117] In certain embodiments, VH3 and VL3 comprise amino acid sequences identical to the respective VH and VL of the scFv identified as: SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:33, SEQ ID NO:40, SEQ ID NO:47, SEQ ID NO:54, SEQ ID NO:59, SEQ ID NO:66, SEQ ID NO:72, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:94, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:111, SEQ ID NO:118, SEQ ID NO:125, SEQ ID NO:132, SEQ ID NO:138, SEQ ID NO:144, SEQ ID NO:150, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:164, SEQ ID NO:169, SEQ ID NO:175, SEQ ID NO:183, SEQ ID NO:188, SEQ ID NO:193, SEQ ID NO:198, or SEQ ID NO:204.

[0118] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises (a) a first heavy chain (HC1) comprising a VH1, a first heavy chain constant region comprising a first Fc region (Fc1), and an scFv1; and (b) a first light chain (LC1) comprising a VL1 and a light chain constant region.

[0119] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises (a) a second heavy chain (HC2) comprising a first heavy chain constant region comprising a VH1 and a second Fc region (Fc2), and (b) a second light chain (LC2) comprising a VL1 and a light chain constant region.

[0120] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof further comprises a second Fc region (Fc2).

[0121] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising a first heavy chain constant region comprising a VH2 and a first Fc region (Fc1); (b) a first light chain (LC1) comprising a VL2 and a light chain constant region; and (c) a second heavy chain (HC2) comprising a second heavy chain constant region comprising an scFv1 and a second Fc region (Fc2).

[0122] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding region, a second antigen-binding region, and a third antigen-binding region.

[0123] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising a VH1, a first heavy chain constant region comprising a first Fc region (Fc1), and an scFv1; (b) a first light chain (LC1) comprising a VL1 and a light chain constant region; and (c) a second heavy chain (HC2) comprising a second single-chain variable fragment (scFv2) and a first heavy chain constant region comprising a second Fc region (Fc2), wherein the scFv2 comprises a VH2 and a VL2.

[0124] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising a VH2, a first heavy chain constant region comprising a first Fc region (Fc1), and an scFv1; (b) a first light chain (LC1) comprising a VL2 and a light chain constant region; and (c) a second single-chain variable fragment (scFv2) and a first heavy chain constant region comprising a second Fc region (Fc2), wherein the scFv2 comprises a second heavy chain (HC2) comprising a VH1 and a VL1.

[0125] In certain embodiments, the scFvs of the invention comprise a light chain variable region (L V ) covalently linked to the heavy chain variable region (H V ) scFv can retain the specificity of the original immunoglobulin even if the constant region is removed and a linker is introduced. In scFv, the domain order is H V -Linker L V , or L V -Linker H VThe linker may be either de novo designed or derived from a known protein structure to provide a length and conformation compatible with cross-linking the variable domains of the scFv without significant steric hindrance. The linker may be 10 to approximately 25 amino acids in length. Preferably, the linker is a peptide linker that extends approximately 3.5 nm (35 Å) between the carboxy terminus of one variable domain and the amino terminus of the other domain without affecting the ability of the domains to fold and form an intact antigen-binding site (Huston et al., Methods in Enzymology, vol. 203, pp. 46-88, 1991, incorporated herein by reference in its entirety). The linker preferably contains a hydrophilic sequence to prevent peptide insertion within or between variable domains throughout protein folding (Argos, Journal of Molecular Biology, vol. 211, no. 4, pp. 943-958, 1990). For example, the linker can include Gly and Ser residues, and / or with charged residues such as Glu, Thr, and Lys interspersed to enhance solubility. In one embodiment, the linker has the amino acid sequence of SEQ ID NO: 208 (GTEGKSSGSGSESKST). In another embodiment, the linker has the amino acid sequence of SEQ ID NO: 209 (GGSEGKSSGSGSESKSTGGS). Any other suitable linker may also be used, taking into account the present disclosure.

[0126] In certain embodiments, the scFv of the present invention may be a stabilized scFv, referred to herein as a stapled Fv (spFv). "Staple" refers to an scFv linker containing one or two Cys residues capable of forming a disulfide bond with the anchor point Cys. As used herein, "VH cysteine" or "VH Cys" refers to a Cys residue present in the VH framework. "VL cysteine" or "VL Cys" refers to a Cys residue present in the VL framework. "Stabilized" refers to an scFv that retains equivalent binding to hK2 when compared to an unheated scFv sample, which indicates thermal stability. Stapled Fvs are described in WO 2021 / 030657, the entire contents of which are incorporated herein by reference.

[0127] In certain embodiments, the isolated single-chain variable fragment (scFv) comprises a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), wherein the scFv comprises a first disulfide bond between a structurally conserved, surface-exposed VH cysteine (Cys) and a first L Cys, a second disulfide bond between a structurally conserved, surface-exposed VL Cys and a second L Cys, or a first disulfide bond between a structurally conserved, surface-exposed VH Cys and a first L Cys and a second disulfide bond between a structurally conserved, surface-exposed VL Cys and a second L Cys.

[0128] In certain embodiments, scFv1 and scFv2 each independently comprise a first disulfide bond and a second disulfide bond.

[0129] In certain embodiments, the linker has the amino acid sequence of SEQ ID NO: 210 (GGGSGGSGGCPPCGGSGG).

[0130] In certain embodiments, the scFv further comprises a histidine at the N-terminus.

[0131] In certain embodiments, scFv1 has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or both an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 26, 33, 40, 47, 54, 59, 66, 72, 79, 82, 83, 84, 85, 86, 87, 94, 101, 104, 111, 118, 125, 132, 138, 144, 150, 155, 158, 164, 169, 175, 183, 188, 193, 198, and 204. , comprising an amino acid sequence having at least 96%, at least 97%, at least 98% or at least 99% sequence identity, preferably scFv1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 26, 33, 40, 47, 54, 59, 66, 72, 79, 82, 83, 84, 85, 86, 87, 94, 101, 104, 111, 118, 125, 132, 138, 144, 150, 155, 158, 164, 169, 175, 183, 188, 193, 198 and 204.

[0132] In certain embodiments, scFv2 comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, preferably, scFv2 comprises the amino acid sequence of SEQ ID NO: 3 or 4.

[0133] Antibody variants In some embodiments, amino acid sequence modifications of the HER2-binding antibodies described herein are contemplated. For example, it may be desirable to optimize the binding affinity and / or other biological properties of the antibody (including, but not limited to, specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility). Thus, in addition to the HER2-binding antibodies described herein, it is contemplated that variants of the HER2-binding antibodies described herein can be prepared. For example, antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesizing the desired antibody or polypeptide. Those skilled in the art, aware of the amino acid changes, can alter the post-translational processing of antibodies.

[0134] chemical modification In some embodiments, the antibodies provided herein are chemically modified, for example, by covalently attaching any type of molecule to the antibody. Antibody derivatives can include antibodies chemically modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, or conjugation to one or more immunoglobulin domains (e.g., Fc or portions of Fc). Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. Additionally, the antibody can contain one or more non-classical amino acids.

[0135] In some embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0136] When the antibodies provided herein are fused to an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are generally attached by an N-linkage to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the binding molecules provided herein may be made to generate variants with certain improved properties.

[0137] In other embodiments, when an antibody provided herein is fused to an Fc region, the antibody variant provided herein may have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (EU numbering of Fc region residues) in the Fc region. However, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108 and 2004 / 0093621. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Application Publication Nos. 2003 / 0157108, WO 2000 / 61739, WO 2001 / 29246, U.S. Patent Application Publication Nos. 2003 / 0115614, 2002 / 0164328, 2004 / 0093621, and 2004 / 0132140. , U.S. Patent Application Publication No. 2004 / 0110704, U.S. Patent Application Publication No. 2004 / 0110282, U.S. Patent Application Publication No. 2004 / 0109865, WO 2003 / 085119, WO 2003 / 084570, WO 2005 / 035586, WO 2005 / 035778, WO 2005 / 053742, WO 2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108; and WO 2004 / 056312), as well as knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0138] The binding molecules, including antibodies, provided herein further provide bisected oligosaccharides, for example, where a biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function. Examples of such variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Pat. No. 6,602,684 (Umana et al.), and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such variants may have improved CDC function. Such materials are described in WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[0139] In the antibodies and molecules comprising an Fc region of the present invention, one or more amino acid modifications may be introduced into the Fc region, thereby generating an Fc region variant. The Fc region variant may comprise the sequence of a human Fc region (e.g., the Fc region of a human IgG1, IgG2, IgG3, or IgG4) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0140] In some embodiments, the present application contemplates variants that retain some, but not all, effector functions, making them desirable candidates for applications where in vivo half-life of the binding molecule is important, but where certain effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the binding molecule lacks FcγR binding (and thus likely lacks ADCC activity) while retaining FcRn binding ability. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity.See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Additionally, FcRn binding and in vivo clearance / half-life measurements can be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0141] Binding molecules with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0142] Certain variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0143] In some embodiments, the variants include an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues). In some embodiments, changes can be made in the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0144] Binding molecules with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which account for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in U.S. Patent Application Publication No. 2005 / 0014934(A1) (Hinton et al.). These molecules comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution of one or more of residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 of the Fc region, e.g., a substitution of residue 434 of the Fc region (U.S. Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0145] In some embodiments, it may be desirable to generate cysteine engineered antibodies in which one or more residues of an antibody are replaced with cysteine residues. In some embodiments, the replaced residues are located at accessible sites of the antibody. As further described herein, by replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates.

[0146] Substitutions, deletions, or insertions The variations may be substitutions, deletions, or insertions of one or more codons encoding the antibody or polypeptide, resulting in a change in the amino acid sequence compared to the native sequence antibody or polypeptide. Target sites for substitutional mutagenesis include the CDRs and FRs.

[0147] Amino acid substitutions may result from substituting one amino acid for another amino acid with similar structural and / or chemical properties, such as substituting a serine for a leucine, e.g., a conservative amino acid substitution. Standard techniques known to those of skill in the art can be used to introduce mutations into the nucleotide sequences encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. Insertions or deletions can optionally range from about 1 to 5 amino acids. In certain embodiments, substitutions, deletions, or insertions comprise fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions compared to the original molecule. In certain embodiments, substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Acceptable variations can be determined by systematically making insertions, deletions, or substitutions of amino acids in a sequence and testing the resulting variants for activity exhibited by the parent antibody.

[0148] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing multiple residues, as well as intrasequence insertions of single or multiple amino acid residues. An exemplary terminal insertion is an antibody with an N-terminal methionyl residue.

[0149] Antibodies generated by conservative amino acid substitutions are included in the present disclosure. In conservative amino acid substitutions, an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. As discussed above, the art has defined families of amino acid residues with side chains that have similar charges. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and the activity of the protein determined. Conservative substitutions (e.g., within a group of amino acids with similar properties and / or side chains) can be made to maintain or not significantly alter properties. Exemplary substitutions are shown in Table 3 below.

[0150] [Table 3]

[0151] Amino acids can be grouped according to the similarity of their side chain properties (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H)). Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. For example, any cysteine residue not involved in maintaining the proper conformation of the antibody can be substituted with another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Non-conservative substitutions would involve exchanging a member of one of these classes for a member of another class.

[0152] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have a modification (e.g., an improvement) in a particular biological property (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated (e.g., using phage-display-based affinity maturation techniques such as those described herein). Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0153] Changes (e.g., substitutions) can be made in the CDRs, for example, to improve antibody affinity. Such changes can be made in CDR "hotspots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in the SDRs (a-CDRs), and the resulting mutant antibodies or fragments thereof are tested for binding affinity. Affinity maturation by constructing and reselecting from a secondary library is described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-directed approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. A more detailed description of affinity maturation is provided below.

[0154] In some embodiments, substitutions, insertions, or deletions can occur within one or more CDRs, so long as such changes do not significantly reduce the antibody's ability to bind antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not significantly reduce binding affinity can be made in a CDR. In some embodiments of the variant antibody sequences provided herein, each CDR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0155] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham and Wells, Science, 244:1081-1085 (1989). In this method, a target residue or group (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Mutants can be screened to determine whether they contain desired properties.

[0156] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0157] Modifications can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, PCR mutagenesis, etc. Site-directed mutagenesis (see, e.g., Carter, Biochem J. 237:1-7 (1986); and Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315-23 (1985)), or other known techniques can be performed on cloned DNA to produce antibody variant DNA.

[0158] Fc mutations Heterodimerization mutations introduced into the Fc domains of two heavy chains to promote heterodimer formation between two heavy chains, e.g., a heavy chain with and without a fusion of an anti-HER2 antibody or antigen-binding fragment thereof, or a heavy chain with an Fc domain of an anti-HER2 arm and a heavy chain with an Fc domain of a tissue-targeting arm. Examples of such Fc mutations include, but are not limited to, Zymework mutations (see, e.g., U.S. Pat. No. 10,457,742) and "knob-in-hole" mutations (see, e.g., Ridgway et al., Protein Eng., 9(7):617-621, 1996). Other heterodimerization mutations can also be used in the present disclosure. In some embodiments, the modified CH3 described herein is used to promote heterodimer formation between two heavy chains.

[0159] In certain embodiments, each of the two heavy chains of the antibody comprises one or more heterodimerization mutations or one or more knob and hole mutations, hi certain embodiments, the one or more heterodimerization mutations are in the CH3 domain.

[0160] In certain embodiments, each of the two heavy chains of the multispecific antibody or antigen-binding fragment thereof comprises a constant heavy chain 3 (CH3) domain that is modified compared to the wild-type CH3 domain to facilitate heterodimer formation between the two heavy chains. Any mutation that promotes heterodimer formation between the two heavy chains can be used. Preferably, the modified CH3 domain of the first heavy chain comprises amino acid modifications at positions T350, L351, F405, and Y407, and the modified CH3 domain of the second heavy chain comprises amino acid modifications at positions T350, T366, K392, and T394. Preferably, the amino acid modification at position T350 is T350V, T350I, T350L, or T350M, the amino acid modification at position L351 is L351Y, the amino acid modification at position F405 is F405A, F405V, F405T, or F405S, the amino acid modification at position Y407 is Y407V, Y407A, or Y407I, the amino acid modification at position T366 is T366L, T366I, T366V, or T366M, the amino acid modification at position K392 is K392F, K392L, or K392M, and the amino acid modification at position T394 is T394W. More preferably, the modified heterodimeric CH3 domain of the first heavy chain comprises the mutations T350V, L351Y, F405A, and Y407V, and the modified heterodimeric CH3 domain of the second heavy chain comprises the mutations T350V, T366L, K392L, and T394W. Throughout this specification, the numbering of amino acid residues in antibodies is done according to the EU index as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), unless expressly specified otherwise. In a specific embodiment, the CH3 domain of one heavy chain comprises the mutations T350V, L351Y, F405A, and Y407V, and the CH3 domain of the other heavy chain comprises the mutations T350V, T366L, K392L, and T394W.

[0161] In addition to heterodimer mutations, other mutations can be introduced. In some embodiments, the Fc region of the antibody further comprises one or more mutations (such as the AAS mutations described herein) that alter (increase or decrease), preferably eliminate, ADCC / CDC, and / or one or more mutations (such as the YTE mutations described herein) that alter (increase or decrease), preferably increase, binding of the antibody to FcRn. In some embodiments, one or more cysteine residues in the antibody are substituted with another amino acid, such as serine.

[0162] In certain embodiments, the crystallizable region (Fc region) of a multispecific antibody or antigen-binding fragment thereof contains substitutions that alter (increase or decrease), preferably eliminate, effector function such as antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Preferably, the Fc region of a multispecific antibody or antigen-binding fragment thereof contains one or more amino acid modifications that reduce or abolish binding of the multispecific antibody or antigen-binding fragment thereof to Fc gamma receptors (FcγRs), avoiding effector function-mediated toxicity. For example, the Fc region of a multispecific antibody or antigen-binding fragment thereof can include one or more amino acid modifications at positions L234, L235, D270, N297, E318, K320, K322, P331, and P329, e.g., one, two, or three of the following mutations: L234A, L235A, and D265S, where the numbering of the amino acid residues is according to the EU index as set forth in Kabat.

[0163] In certain embodiments, the Fc region of the multispecific antibody or antigen-binding fragment thereof contains substitutions that alter (increase or decrease), preferably increase, binding of the multispecific antibody or antigen-binding fragment thereof to the neonatal Fc receptor (FcRn). Preferably, one or more mutations enhance binding at acidic pH, and more preferably the Fc has M252Y / S254T / T256E (YTE) mutations, where the numbering of amino acid residues is according to the EU index as set forth in Kabat.

[0164] In certain embodiments, the Fc region of a multispecific antibody or antigen-binding fragment thereof contains one or more mutations at positions M252Y, S254T, T256E, where the numbering of the amino acid residues is according to the EU index as set forth in Kabat. In certain embodiments, the Fc region of a multispecific antibody or antigen-binding fragment thereof contains one or more mutations at positions M252Y, S254T, T256E, L234A, L235A, and D265S, where the numbering of the amino acid residues is according to the EU index as set forth in Kabat.

[0165] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain that does not have amino acid modifications that reduce or eliminate effector function.

[0166] Also provided is a multispecific antibody comprising a first heavy chain, a light chain, and a second heavy chain, each having an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:211, SEQ ID NO:12, and SEQ ID NO:212, respectively, or SEQ ID NO:213, SEQ ID NO:12, and SEQ ID NO:214, respectively, wherein the first antigen-binding region is capable of specifically binding to a first epitope on HER2, the second antigen-binding region is capable of specifically binding to a second epitope on HER2, and the third antigen-binding region is capable of specifically binding to TfR. Preferably, the first heavy chain, light chain, and second heavy chain each comprise the amino acid sequence of SEQ ID NO:211, SEQ ID NO:12, and SEQ ID NO:212, respectively, or SEQ ID NO:213, SEQ ID NO:12, and SEQ ID NO:214, respectively.

[0167] Polynucleotides In certain embodiments, the present disclosure provides polynucleotides encoding the present antibodies that bind to HER2, and fusion proteins comprising the antibodies that bind to HER2 described herein. The polynucleotides of the present disclosure may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, which may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or non-coding (antisense) strand. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0168] The present disclosure further relates to variants of the polynucleotides described herein, where the variants encode, for example, fragments, analogs, and / or derivatives of the HER2-binding antibodies of the present disclosure. In certain embodiments, the present disclosure provides polynucleotides, including polynucleotides having a nucleotide sequence at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98%, or 99% identical to a polynucleotide encoding a HER2-binding antibody of the present disclosure. As used herein, the phrase "a polynucleotide having a nucleotide sequence at least, e.g., 95% "identical" to a reference nucleotide sequence" is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides of the reference sequence can be deleted or replaced with alternative nucleotides, or up to 5% of the total number of nucleotides in the reference sequence can be inserted into the reference sequence. These variations in the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between these terminal positions, and can be interspersed individually among the nucleotides of the reference sequence or in one or more contiguous groups within the reference sequence.

[0169] Polynucleotide variants can contain alterations in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that result in silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants contain silent substitutions that do not result in changes to the amino acid sequence of a polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be produced for a variety of reasons, such as to optimize codon expression for a particular host (i.e., changing codons in human mRNA to those preferred by a bacterial host, such as E. coli). In some embodiments, polynucleotide variants contain at least one silent mutation in a non-coding or coding region of the sequence.

[0170] In some embodiments, polynucleotide variants are produced to modulate or alter expression (or expression levels) of an encoded polypeptide. In some embodiments, polynucleotide variants are produced to increase expression of an encoded polypeptide. In some embodiments, polynucleotide variants are produced to decrease expression of an encoded polypeptide. In some embodiments, polynucleotide variants increase expression of an encoded polypeptide compared to the parent polynucleotide sequence. In some embodiments, polynucleotide variants decrease expression of an encoded polypeptide compared to the parent polynucleotide sequence.

[0171] Also provided are vectors comprising the nucleic acid molecules described herein. In certain embodiments, the nucleic acid molecules can be incorporated into recombinant expression vectors. The present disclosure provides recombinant expression vectors comprising any of the nucleic acids of the present disclosure. As used herein, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that enables expression of an mRNA, protein, polypeptide, or peptide by a host cell, where the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the cell. The vectors described herein are not naturally occurring in their entirety. However, portions of the vector may be naturally occurring. The described recombinant expression vectors may comprise any type of nucleotide, including, but not limited to, DNA and RNA (which may be single-stranded or double-stranded, synthetic, or derived in part from natural sources, and may contain natural, non-natural, or modified nucleotides). The recombinant expression vectors may comprise naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Non-naturally occurring or modified nucleotides or internucleotide linkages do not inhibit the transcription or replication of the vector.

[0172] In certain embodiments, the recombinant expression vector of the present disclosure may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors include vectors designed for propagation and propagation, or for expression, or both, such as plasmids and viruses. The vector may be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λEMBL4, λNM1149, and λZapII (Stratagene) may also be used. Examples of plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, e.g., a retroviral vector, e.g., a gamma retroviral vector.

[0173] In one embodiment, recombinant expression vectors are prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al. (supra) and Ausubel et al. (supra). Expression vector constructs, either circular or linear, can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from ColE1, SV40, 2μ plasmid, λ, bovine papilloma virus, etc.

[0174] Recombinant expression vectors may, where appropriate, include regulatory sequences, such as transcription and translation initiation and termination codons, specific to the type of host (e.g., bacteria, plants, fungi, or animals) into which the vector will be introduced, taking into account whether the vector is DNA- or RNA-based.

[0175] Recombinant expression vectors may contain one or more marker genes to allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.), complementation to provide prototrophy in auxotrophic hosts, etc. Suitable marker genes for the described expression vectors include, for example, neomycin / G418 resistance genes, histidinol x resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0176] The recombinant expression vector can include a native promoter or a canonical promoter operably linked to the nucleotide sequence of the present disclosure. The selection of a promoter, for example, strong, weak, tissue-specific, inducible, and developmentally specific, is within the skill of one of ordinary skill in the art. Similarly, combining a nucleotide sequence with a promoter is also within the skill of one of ordinary skill in the art. The promoter can be a non-viral promoter or a viral promoter, for example, a cytomegalovirus (CMV) promoter, a RSV promoter, an SV40 promoter, or a promoter found in the long terminal repeat of murine stem cell virus.

[0177] Recombinant expression vectors can be designed for either transient expression, stable expression, or both, and can be made for constitutive or inducible expression.

[0178] Additionally, recombinant expression vectors can be engineered to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes cells expressing the suicide gene to die. A suicide gene can be a gene that confers sensitivity to an agent (e.g., a drug) on a cell expressing the gene, causing the cell to die when contacted with or exposed to the agent. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0179] In certain embodiments, the polynucleotide is isolated. In certain embodiments, the polynucleotide is substantially pure.

[0180] Also provided are host cells containing the nucleic acid molecules described herein. Host cells can be any cell containing heterologous nucleic acid. The heterologous nucleic acid can be a vector (e.g., an expression vector). For example, host cells can be cells from any organism that are selected, modified, transformed, grown, used, or manipulated in any way for the cellular production of a substance, e.g., the cellular expression of a gene, DNA or RNA sequence, protein, or enzyme. An appropriate host can be determined. For example, host cells can be selected based on the vector backbone and the desired result. For example, plasmids or cosmids can be introduced into prokaryotic host cells to replicate some types of vectors. Bacterial cells, such as, but not limited to, DH5α, JM109, and KCB, SURE® competent cells, and SOLOPACK Gold cells can be used as host cells for vector replication and / or expression. Additionally, bacterial cells, such as E. coli LE392, can be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to, yeast (e.g., YPH499, YPH500, and YPH501), insect, and mammalian cells. Examples of mammalian eukaryotic host cells for replication and / or expression of vectors include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, Saos, PC12, SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646), and Ag653 (ATCC CRL-1580) mouse cell lines. An exemplary human myeloma cell line is U266 (ATCC CRL-TIB-196).Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics, Walkersville, MD), CHO-K1 (ATCC CRL-61), or DG44.

[0181] Antibody preparation and production method Methods for preparing antibodies have been described. See, for example, Els Pardon et al., Nature Protocol, 9(3):674 (2014). Antibodies (e.g., scFv fragments) can be obtained using methods known in the art, for example, by immunizing a Camelidae species (e.g., a camel or a llama) and obtaining hybridomas therefrom, or by cloning a library of antibodies using molecular biology techniques known in the art and then selecting individual clones from the unselected library by ELISA, or by using phage display.

[0182] Antibodies provided herein can be produced by culturing cells transformed or transfected with a vector containing an antibody-encoding nucleic acid. Polynucleotide sequences encoding the polypeptide components of antibodies of the present disclosure can be obtained using standard recombinant techniques. Desired polynucleotide sequences can be isolated and sequenced from antibody-producing cells, such as hybridoma cells or B cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesis agents or PCR technology. Once obtained, the polypeptide-encoding sequence is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in host cells. Many vectors available and known in the art can be used for the purposes of the present disclosure. Selection of an appropriate vector depends primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Suitable host cells for expressing antibodies of the present disclosure include prokaryotes, such as archaebacteria and eubacteria, including gram-negative or gram-positive organisms; eukaryotic microbes, such as filamentous fungi and yeast; invertebrate cells, such as insect cells and plant cells; and vertebrate cells, such as mammalian host cell lines. Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Antibodies produced by the host cells are purified using standard protein purification methods known in the art.

[0183] Methods for antibody production, including vector construction, expression, and purification, are further described in Pluckthun et al., Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation 203-52 (McCafferty et al. eds., 1996), Kwong and Rader, E. coli Expression and Purification of Fab Antibody Fragments, in Current Protocols in Protein Science (2009), Tachibana and Takekoshi, Production of Antibody Fab Fragments in Escherichia coli, in Antibody Expression and Production (Al-Rubeai ed., 2011), and Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed., 2009).

[0184] Of course, alternative methods known in the art are contemplated for preparing anti-HER2 antibodies. For example, the appropriate amino acid sequence or portions thereof can be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis (1969), and Merrifield, J. Am. Chem. Soc. 85:2149-54 (1963)). In vitro protein synthesis can be performed manually or by automation. Various portions of the anti-HER2 antibody can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-HER2 antibody. Alternatively, antibodies can be purified from cells or body fluids (such as milk) of transgenic animals genetically engineered to express the antibody, as disclosed, for example, in U.S. Patent Nos. 5,545,807 and 5,827,690.

[0185] Pharmaceutical Composition In one aspect, the present disclosure further provides a pharmaceutical composition comprising a multispecific antibody or antigen-binding fragment thereof of the present disclosure. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a multispecific antibody or antigen-binding fragment thereof provided herein and a pharmaceutically acceptable excipient.

[0186] Pharmaceutical compositions comprising multispecific antibodies or antigen-binding fragments thereof are prepared by mixing the fusion protein having the desired purity with an optional physiologically acceptable excipient (see, e.g., Remington, Remington's Pharmaceutical Sciences (18th ed. 1980)) in aqueous solution or in lyophilized or other dried form for storage.

[0187] The multispecific antibodies or antigen-binding fragments thereof of the present disclosure can be formulated in any suitable form for delivery to target cells / tissues, for example, as microcapsules or macroemulsions (Remington (supra); Park, et al., Molecules, 2005, 10:146-61; Malik et al., 2007, Curr. Drug. Deliv. 4:141-51), as sustained-release formulations ((Putney and Burke, 1998, Nature Biotechnol. 16:153-57), or in liposomes (Maclean et al., 1997, Int. J. Oncol. 11:325-32; Kontermann, 2006, Curr. Opin. Mol. Ther. 8:39-45).

[0188] The antibodies or antigen-binding fragments thereof provided herein can also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, prepared, for example, by coacervation techniques or by interfacial polymerization, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in Remington (supra).

[0189] Various compositions and delivery systems are known and can be used with the antibodies or antigen-binding fragments thereof described herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the multispecific antibodies or antigen-binding fragments thereof, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-32), construction of nucleic acids as part of retroviral or other vectors, etc. In another embodiment, the compositions can be provided as controlled or sustained release systems. In one embodiment, a pump can be used to achieve controlled or sustained release (see, e.g., Langer (supra); Sefton, 1987, Crit. Ref. Biomed. Eng. 14:201-40; Buchwald et al., 1980, Surgery 88:507-16; and Saudek et al., 1989, N. Engl. J. Med. 321:569-74).In another embodiment, polymeric materials can be used to achieve controlled or sustained release of prophylactic or therapeutic agents (e.g., antibodies or antigen-binding fragments thereof described herein), or compositions provided herein (see, e.g., Medical Applications of Controlled Release (Langer and Wise, eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126; Levy et al., Science 228:190-92 (1985); During et al., 1989, Ann. Neurol. 25:351-56; Howard et al. al., 1989, J. Neurosurg. 71:105-12; U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, and 5,128,326; WO 99 / 15154 and WO 99 / 20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymers used in the sustained release formulations are inert, free of leachable impurities, stable on storage, sterile, and biodegradable.

[0190] In yet another embodiment, a controlled-release or sustained-release system can be placed in close proximity to a specific target tissue, such as the nasal cavity or lungs, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled-release systems are discussed, for example, by Langer, 1990, Science 249:1527-33. Any technique known to those of skill in the art can be used to prepare sustained release formulations comprising one or more antibodies or antigen-binding fragments thereof described herein (see, e.g., U.S. Pat. No. 4,526,938; WO 91 / 05548 and WO 96 / 20698; Ning, et al., Radiotherapy & Oncology, 39:179-89 (1996); Song et al., 1995, PDA J. of Pharma. Sci. & Tech. 50:372-97; Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-60).

[0191] How to use antibodies In one aspect, provided herein is a method of treating or detecting a disorder in a subject in need thereof, the method comprising administering to the subject a multispecific antibody provided herein, or an antibody fragment thereof.

[0192] In one aspect, provided herein is a method for delivering a therapeutic or diagnostic agent to a specific tissue in a subject in need thereof, comprising administering to the subject a multispecific antibody or antibody fragment provided herein.

[0193] In one aspect, provided herein is a method of inducing antibody dependent phagocytosis (ADP) in a subject in need thereof without stimulating the secretion of pro-inflammatory cytokines, the method comprising administering to the subject a multispecific antibody or antigen-binding fragment provided herein.

[0194] In one aspect, provided herein are methods of reducing or eliminating effector function.

[0195] In one aspect, provided herein is a method of attenuating the activity of HER2 on a cell, the method comprising exposing the cell to an effective amount of a multispecific antibody or antigen-binding fragment thereof provided herein.

[0196] In another aspect, provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, the disease or disorder is a HER2-mediated disease or disorder. Also provided herein is a method of treating a disease or disorder, wherein the subject is administered one or more therapeutic agents in combination with an antibody or antigen-binding fragment thereof, or those provided herein.

[0197] The present disclosure also relates to methods of using the antibodies provided herein to inhibit, i.e., antagonize, the function of HER2, thereby inhibiting HER2 activation and resulting in the treatment of pathological disorders.

[0198] The pathological disorder may be cancer. Examples of cancers to be treated include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer (including gastrointestinal cancer and pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0199] In certain embodiments, the disease or disorder is brain metastasis.

[0200] In another aspect, provided herein is the use of a multispecific antibody, or antigen-binding fragment thereof, provided herein in the manufacture of a medicament for treating a disease or disorder in a subject.

[0201] In another aspect, provided herein is the use of a pharmaceutical composition provided herein in the manufacture of a medicament for treating a disease or disorder in a subject.

[0202] In another aspect, provided herein is the use of a multispecific antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament, wherein the medicament is for use in a method for detecting the presence of HER2 in a biological sample, the method comprising contacting the biological sample with the antibody under conditions permissive for binding of the antibody to the HER2 protein, and detecting whether a complex is formed between the antibody and the HER2 protein.

[0203] In other aspects, the antibodies and fragments thereof of the present disclosure are useful for detecting the presence of HER2 in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises a body fluid, cell, or tissue. Diagnostic assays and methods are described in more detail below.

[0204] Methods of Administration and Dosage In certain embodiments, provided herein are compositions for use in the prevention and / or treatment of a disease or condition, comprising an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein are compositions for use in the prevention of a disease or condition, the composition comprising an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein are compositions for use in the treatment of a disease or condition, the composition comprising an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the disease or condition is a HER2-mediated disease. In some embodiments, the disease or disorder is associated with HER2. In some embodiments, the disease or disorder is cancer. Examples of cancers to be treated include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer (including gastrointestinal cancer, pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatic cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer. In certain embodiments, the subject is one in need thereof. In some embodiments, the subject has a disease or condition. In other embodiments, the subject is at risk of having a disease or condition. In some embodiments, the administration results in the prevention, management, treatment, or amelioration of a disease or condition.

[0205] In one embodiment, provided herein is a composition for use in the prevention and / or treatment of symptoms of a disease or condition, the composition comprising an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a composition for use in the prevention of symptoms of a disease or condition, the composition comprising an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a composition for use in the treatment of symptoms of a disease or condition, the composition comprising an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the disease or condition is HER2-mediated and / or a HER2-mediated disease. In some embodiments, the disease or disorder is associated with HER2. In some embodiments, the disease or disorder is cancer. Examples of cancers to be treated include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer (including gastrointestinal cancer, pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatic cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer. In certain embodiments, the subject is one in need thereof. In some embodiments, the subject has a disease or condition. In other embodiments, the subject is at risk of having a disease or condition. In some embodiments, the administration results in prevention or treatment of a symptom of a disease or condition.

[0206] In another embodiment, provided herein is a method for preventing and / or treating a disease or condition in a subject, comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method for preventing a disease or condition in a subject, comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method for treating a disease or condition in a subject, comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the disease or condition is HER2-mediated and / or a HER2-mediated disease. In some embodiments, the disease or disorder is HER2-associated. In some embodiments, the disease or disorder is cancer. Examples of cancers to be treated include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer (including gastrointestinal cancer, pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatic cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer. In certain embodiments, the subject is one in need thereof. In some embodiments, the subject has a disease or condition. In other embodiments, the subject is at risk of having a disease or condition. In some embodiments, the administration results in prevention or treatment of a disease or condition.

[0207] In another embodiment, provided herein is a method for preventing and / or treating symptoms of a disease or condition in a subject, comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method for preventing symptoms of a disease or condition in a subject, comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method for treating symptoms of a disease or condition in a subject, comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the disease or disorder is a HER2-mediated and / or HER2-associated disease or disorder. In some embodiments, the disease or disorder is associated with HER2. In some embodiments, the disease or disorder is cancer. Examples of cancers to be treated include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer (including gastrointestinal cancer, pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatic cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer. In certain embodiments, the subject is one in need thereof. In some embodiments, the subject has a disease or condition. In other embodiments, the subject is at risk of having a disease or condition. In some embodiments, the administration results in prevention or treatment of a symptom of a disease or condition.

[0208] Also provided herein are methods for preventing and / or treating a disease or condition by administering to a subject an effective amount of an antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof provided herein. In one aspect, the multispecific antibody or antigen-binding fragment thereof is substantially purified (i.e., substantially free from substances that limit its effect or produce undesirable side effects). The subject to which the therapy is administered can be a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey, such as macaque (cynomolgus monkey), or human). In one embodiment, the subject is a human. In another embodiment, the subject is a human with a disease or condition.

[0209] Various delivery systems are known and can be used to administer prophylactic or therapeutic agents (e.g., antibodies or antigen-binding fragments thereof provided herein), including liposomes, microparticles, microcapsules, encapsulation in recombinant cells capable of expressing the multispecific antibodies or antigen-binding fragments thereof, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), and construction of nucleic acids as part of retroviral or other vectors. Methods of administering prophylactic or therapeutic agents (e.g., antibodies or antigen-binding fragments thereof provided herein), or pharmaceutical compositions include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In certain embodiments, prophylactic or therapeutic agents (e.g., antibodies or antigen-binding fragments thereof provided herein), or pharmaceutical compositions are administered intranasally, intramuscularly, intravenously, or subcutaneously. The prophylactic or therapeutic agents or compositions can be administered by any convenient route, e.g., by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, nasal, rectal, and intestinal mucosa), and can be administered in conjunction with other biologically active agents. Administration can be systemic or local. In addition, pulmonary administration can be employed, e.g., by use of an inhaler or nebulizer and formulation with an aerosolizing agent. See, for example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078, and PCT Application Publication Nos. WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated herein by reference in its entirety.

[0210] In certain embodiments, it may be desirable to administer a prophylactic or therapeutic agent or pharmaceutical composition provided herein locally to the area in need of treatment. This may be achieved, for example, but not by way of limitation, by local infusion, by topical administration (e.g., by nasal spray), by injection, or using an implant, which may be a porous, non-porous, or gelatinous material, including a membrane such as a silastic membrane or a fiber. In some embodiments, when administering an antibody or antigen-binding fragment thereof provided herein, care must be taken to use a material to which the antibody or antigen-binding fragment thereof does not absorb.

[0211] In another embodiment, the prophylactic or therapeutic agents or compositions provided herein can be delivered in a vesicle, in particular a liposome (see Langer, Science, 1990, 249:1527-1533; Treat, et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally, ibid.).

[0212] In another embodiment, the prophylactic or therapeutic agents, or compositions provided herein can be delivered in a controlled or sustained release system. In one embodiment, a pump can be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., Surgery, 1980, 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of prophylactic or therapeutic agents (e.g., antibodies provided herein) or compositions provided herein (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; also, Levy, et al., Science, 1985, 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al. al., 1989, J. Neurosurg. 7:1-105), U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, 5,128,326, PCT Application Publication No. WO 99 / 15154, and PCT Application Publication No. WO 99 / 20253.Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In certain embodiments, the polymers used in sustained-release formulations are inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, a controlled-release or sustained-release system can be placed in proximity to the therapeutic target, i.e., the nasal cavity or lungs, such that only a fraction of the systemic dose is required (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Controlled-release systems are discussed in the review by Langer (1990, Science 249:1527-1533). Any technique known to those skilled in the art can be used to prepare sustained-release formulations comprising one or more antibodies or antigen-binding fragments thereof provided herein.See, e.g., U.S. Pat. No. 4,526,938, PCT Application WO 91 / 05548, PCT Application WO 96 / 20698, Ning et al., 1996, "Intratumoral Radioimmunotherapy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy & Oncology 39:179-189, Song et al., 1995, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science & Technology 50:372-397, Cleek et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al., 1997, "Microencapsulation of See "Recombinant Humanized Monoclonal Antibody for Local Delivery," Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in its entirety.

[0213] In certain embodiments where the compositions provided herein are nucleic acids encoding prophylactic or therapeutic agents (e.g., antibodies or antigen-binding fragments thereof provided herein), the nucleic acid is constructed as part of a suitable nucleic acid expression vector and administered such that the nucleic acid becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by microparticle bombardment (e.g., gene gun; Biolistic, Dupont), or by use of a coating with lipids or cell surface receptors or transfection agents, or by administering the nucleic acid in linkage to a homeobox-like peptide known to enter the nucleus (e.g., Biolistic, Dupont), or by use of a coating with lipids or cell surface receptors or transfection agents, or by administering the nucleic acid in linkage to a homeobox-like peptide known to enter the nucleus (e.g., Joliot, et al. The nucleic acid can be administered in vivo to promote expression of its encoded prophylactic or therapeutic agent, such as by transfection with a nucleic acid encoding a prophylactic or therapeutic agent (see, for example, U.S. Pat. No. 6,139,163; see, e.g., J. Immunol. 1999, 14:1864-1868). Alternatively, the nucleic acid can be introduced intracellularly and integrated into host cell DNA for expression by homologous recombination.

[0214] In certain embodiments, the compositions provided herein comprise one, two, or more antibodies or antigen-binding fragments thereof provided herein. In another embodiment, the compositions provided herein comprise one, two, or more antibodies or antigen-binding fragments thereof provided herein and a prophylactic or therapeutic agent other than an antibody or antigen-binding fragment thereof provided herein. In one embodiment, the agent is known to be useful for, is being used for, or is currently being used for, the prevention, management, treatment, and / or amelioration of a disease or condition. In addition to the prophylactic or therapeutic agent, the compositions provided herein may also include an excipient.

[0215] The compositions provided herein include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., compositions suitable for administration to a subject or patient) that can be used to prepare unit dosage forms. In certain embodiments, the compositions provided herein are pharmaceutical compositions. Such compositions comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., an antibody or antigen-binding fragment thereof provided herein, or other prophylactic or therapeutic agent) and a pharmaceutically acceptable excipient. The pharmaceutical composition can be formulated to be suitable for the route of administration to a subject.

[0216] In certain embodiments, the term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle. Pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary excipient when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene, glycol, water, ethanol, and the like. If desired, the composition can further contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral formulations may contain standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Such compositions contain a prophylactically or therapeutically effective amount of a multispecific antibody or antigen-binding fragment thereof provided herein, such as in purified form, together with suitable amounts of excipients to provide a form for proper administration to a patient. The formulation should suit the mode of administration.

[0217] In some embodiments, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile aqueous isotonic buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. However, such compositions may be administered via routes other than the intravenous route.

[0218] The components of the compositions provided herein are supplied individually or mixed together in unit dosage form, for example, as a dry lyophilized powder or a moisture-free concentrate in a sealed container such as an ampoule or sachet indicating the quantity of active agent. When the composition is administered by infusion, the composition can be dispensed using an infusion bottle containing pharmaceutical-grade sterile water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

[0219] The antibodies or antigen-binding fragments thereof provided herein can be packaged in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of antibody. In one embodiment, the multispecific antibodies or antigen-binding fragments thereof are supplied as a dry, sterile, lyophilized powder or water-free concentrate in a hermetically sealed container, and can be reconstituted, for example, with water or saline, to the appropriate concentration for administration to a subject. The lyophilized antibodies or antigen-binding fragments thereof can be stored in their original container at 2-8°C, and the multispecific antibodies or antigen-binding fragments thereof can be administered within 12 hours (e.g., within 6 hours, within 5 hours, within 3 hours, or within 1 hour) after reconstitution. In another embodiment, the antibodies or antigen-binding fragments thereof provided herein are supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the antibody.

[0220] The compositions provided herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0221] The amount of a prophylactic or therapeutic agent (e.g., an antibody or antigen-binding fragment thereof provided herein) or a composition provided herein that will be effective in the prevention and / or treatment of a disease or condition can be determined by standard clinical techniques. In addition, in vitro assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the disease or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances.

[0222] Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0223] In certain embodiments, the route of administration of a dose of an antibody or antigen-binding fragment thereof provided herein to a patient is intranasal, intramuscular, intravenous, subcutaneous, or a combination thereof, although other routes described herein are also acceptable. Each dose may or may not be administered via the same route of administration. In some embodiments, an antibody or antigen-binding fragment thereof provided herein may be administered via multiple routes of administration simultaneously with or after other doses of the same or different antibody or antigen-binding fragment thereof provided herein.

[0224] In certain embodiments, the multispecific antibodies or antigen-binding fragments thereof or fusion constructs provided herein are administered prophylactically or therapeutically to a subject. The multispecific antibodies or antigen-binding fragments thereof provided herein can be administered prophylactically or therapeutically to a subject to prevent, reduce, or ameliorate a disease or symptom.

[0225] Diagnostic Assays and Methods Labeled antibodies, derivatives, and analogs thereof that immunospecifically bind to the HER2 antigen can be used for diagnostic purposes to detect, diagnose, or monitor HER2-mediated disease. Accordingly, provided herein is a method for detecting a HER2-mediated disease, comprising: (a) assaying expression of the HER2 antigen in a cell or tissue sample from a subject using one or more antibodies provided herein that immunospecifically bind to the HER2 antigen; and (b) comparing the level of the HER2 antigen with a control level, e.g., the level in a normal tissue sample (e.g., from a patient without a HER2-mediated disease or from the same patient prior to the onset of the disease), whereby an increase in the assayed level of the HER2 antigen compared to the control level of the HER2 antigen indicates a HER2-mediated disease.

[0226] Also provided herein is a diagnostic assay for diagnosing a HER2-mediated disease, comprising: (a) assaying the level of a HER2 antigen in a cell or tissue sample from an individual using one or more antibodies provided herein that immunospecifically bind to the HER2 antigen; and (b) comparing the level of the HER2 antigen to a control level, e.g., the level in a normal tissue sample, whereby an increase in the assayed level of the HER2 antigen compared to the control level of the HER2 antigen is indicative of a HER2-mediated disease. In certain embodiments, provided herein is a method of treating a HER2-mediated disease in a subject, comprising: (a) assaying the level of a HER2 antigen in a cell or tissue sample from the subject using one or more antibodies provided herein that immunospecifically bind to the HER2 antigen; and (b) comparing the level of the HER2 antigen to a control level, e.g., the level in a normal tissue sample, whereby an increase in the assayed level of the HER2 antigen compared to the control level of the HER2 antigen is indicative of a HER2-mediated disease. In some embodiments, the method further comprises (c) administering an effective amount of an antibody provided herein to a subject identified as having a HER2-mediated disease. A more definitive diagnosis of a HER2-mediated disease may enable medical professionals to employ preventative measures or aggressive treatments earlier, thereby preventing the onset or further progression of the HER2-mediated disease.

[0227] The antibodies provided herein can be used to assay HER2 antigen levels in biological samples using classical immunohistological methods as described herein or as known to those skilled in the art (see, e.g., Jalkanen et al., 1985, J. Cell. Biol. 101:976-985, and Jalkanen et al., 1987, J. Cell. Biol. 105:3087-3096). Other antibody-based methods useful for detecting protein gene expression include enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include radioisotopes such as glucose oxidase, iodine (I, I), carbon (C), sulfur (S), tritium (H), indium (In), technetium (Tc), luminescent labels such as luminol, and fluorescent labels such as fluorescein and rhodamine, and enzyme labels such as biotin.

[0228] One aspect provided herein is the detection and diagnosis of HER2-mediated disease in humans. In one embodiment, diagnosis involves: a) administering to a subject (e.g., parenterally, subcutaneously, or intraperitoneally) an effective amount of a labeled antibody that immunospecifically binds to a HER2 antigen; b) waiting a time interval after administration to allow the labeled antibody to concentrate at sites in the subject where the HER2 antigen is expressed (and for unbound labeled molecules to be removed to background levels); c) determining the background level; and d) detecting the labeled antibody in the subject, such that detection of the labeled antibody above the background level indicates that the subject has a HER2-mediated disease. The background level can be determined by various methods, including comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

[0229] It will be understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety required to produce a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected is typically in the range of about 5-20 millicuries of 99Tc. The labeled antibody then accumulates at the location of cells containing the specific protein. In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments." (Chapter 13 of Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B.A. Rhodes, eds., Masson Publishing Inc. (1982)).

[0230] Depending on several variables, including the type of label used and the mode of administration, the time interval after administration to allow the labeled antibody to concentrate at the site of interest and for unbound labeled antibody to clear to background levels is 6 to 48 hours, or 6 to 24 hours, or 6 to 12 hours, hi other embodiments, the time interval after administration is 5 to 20 days, or 5 to 10 days.

[0231] In one embodiment, monitoring of the HER2-mediated disease is performed by repeating the method for diagnosing the HER2-mediated disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

[0232] The presence of the labeled molecule can be detected in a subject using methods known in the art for in vivo scanning. These methods depend on the type of label used. Those skilled in the art will be able to determine the appropriate method for detecting a particular label. Methods and devices that can be used in the diagnostic methods provided herein include, but are not limited to, whole-body scans such as computed tomography (CT), position emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound.

[0233] In certain embodiments, the molecule is labeled with a radioisotope and is detected in the patient using a radiation-responsive surgical instrument (Thurston et al., U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence-responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patient using positron emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in the patient using magnetic resonance imaging (MRI).

[0234] kit Also provided herein are kits comprising a multispecific antibody (e.g., an anti-HER2 antibody) or composition (e.g., a pharmaceutical composition) provided herein packaged in suitable packaging material. The kits optionally include a label or package insert containing a description of the components or directions for in vitro, in vivo, or ex vivo use of the components therein.

[0235] The term "packaging material" refers to a physical structure that contains the components of the kit. The packaging material can maintain the sterility of the components and can be made from materials commonly used for such purposes (e.g., paper, cardboard, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0236] The kits provided herein can include a label or insert. A label or insert includes "printed matter," such as paper or cardboard, separate from or attached to a component, kit, or packaging material (e.g., a box), or, for example, attached to an ampoule, tube, or vial containing a kit component. The label or insert can further include a computer-readable medium, such as a disk (e.g., hard disk, card, memory disk), CD or DVD-ROM / RAM, DVD, MP3, optical disk such as magnetic tape, or electronic storage medium such as RAM and ROM, or hybrids thereof, such as magnetic / optical storage medium, FLASH media, or memory-type cards. The label or insert can include information identifying the manufacturer, lot number, manufacturer's address, and date.

[0237] The kits provided herein can further include other components. Each component of the kit can be enclosed in an individual container, and all of the various containers can be in a single package. The kits can also be designed for refrigeration. The kits can further be designed to include cells containing an antibody provided herein or a nucleic acid encoding an antibody provided herein. The cells in the kit can be maintained under appropriate storage conditions until ready for use.

[0238] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0239] As used herein, numerical values are often presented in range format throughout this document. The use of the range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention unless the context clearly dictates otherwise. Thus, the use of a range explicitly includes all possible subranges, all individual numerical values within that range, and all numerical values or ranges, including integers within such ranges and fractions of values or integers within the range, unless the context clearly dictates otherwise. This configuration applies in all contexts throughout this patent document, regardless of the breadth of the range. Thus, for example, a reference to a range of 90 to 100% includes 91 to 99%, 92 to 98%, 93 to 95%, 91 to 98%, 91 to 97%, 91 to 96%, 91 to 95%, 91 to 94%, 91 to 93%, etc. References to the range 90-100% include 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc.

[0240] Furthermore, references to ranges of 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-225, and 225-250 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. In further examples, references to ranges of 25 to 250, 250 to 500, 500 to 1,000, 1,000 to 2,500, 2,500 to 5,000, 5,000 to 25,000, 25,000 to 50,000 include any number or range within or encompassing such values, for example, 25, 26, 27, 28, 29, 250, 251, 252, 253, 254, 500, 501, 502, 503, 504, etc.

[0241] Similarly, as used herein, a series of ranges is disclosed throughout this document. The use of a series of ranges includes combining upper and lower range limits to provide a separate range. This configuration applies in all contexts throughout this patent document, regardless of the breadth of the range. Thus, for example, reference to a series of ranges such as 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 75, 75 to 100, and 100 to 150 includes ranges such as 5 to 20, 5 to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, and 5 to 150, as well as 10 to 30, 10 to 40, 10 to 50, 10 to 75, 10 to 100, and 10 to 150, as well as 20 to 40, 20 to 50, 20 to 75, 20 to 100, and 20 to 150, etc.

[0242] For the sake of brevity, certain abbreviations are used herein. Examples include single letter abbreviations that represent amino acid residues. The amino acids and their corresponding three letter and one letter abbreviations are as follows:

[0243] [Table 4]

[0244] The present invention is generally disclosed herein using affirmative language to describe numerous embodiments. The present invention also specifically includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, procedures, assays or analyses, is excluded in whole or in part. Thus, the present specification generally discloses aspects not explicitly included in the present invention, even if the specification does not expressly state otherwise.

[0245] Many embodiments of the present invention have been described. However, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate, but not limit, the scope of the invention as claimed.

[0246] Embodiment The disclosure provided herein also provides the following non-limiting embodiments. 1. A multispecific antibody or antigen-binding fragment thereof, comprising at least one of a first antigen-binding region and a second antigen-binding region, each capable of specifically binding to human epidermal growth factor receptor 2 (HER2), and a third antigen-binding region capable of specifically binding to transferrin receptor (TfR), (1) the first antigen-binding region is a first heavy chain variable region (VH1) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively; and a first light chain variable region (VL1) comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; (2) the second antigen-binding region is a second heavy chain variable region (VH2) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively; and a second light chain variable region (VL2) comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively; (3) A multispecific antibody or antigen-binding fragment thereof, comprising a first single-chain variable fragment (scFv1), wherein the third antigen-binding region comprises a third heavy chain variable region (VH3) comprising heavy chain complementarity-determining region 1 (HCDR1), HCDR2, and HCDR3, and a third light chain variable region (VL3) comprising light chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, and wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 have any of the amino acid sequences in Table 2. 2. (1) VH1 comprises an amino acid sequence identical to the VH of the HC sequence identified as SEQ ID NO: 1, and VL1 comprises an amino acid sequence identical to the VL of the LC sequence identified as SEQ ID NO: 2; (2) VH2 comprises an amino acid sequence identical to the VH of the HC sequence identified as SEQ ID NO: 11, and VL2 comprises an amino acid sequence identical to the VL of the LC sequence identified as SEQ ID NO: 12; (3) VH3 and VL3 are as follows: (i) SEQ ID NO: 19; (ii) SEQ ID NO: 26; (iii) SEQ ID NO: 33; (iv) SEQ ID NO: 40, (v) SEQ ID NO: 47, (vi) SEQ ID NO: 54, (vii) SEQ ID NO: 59, (viii) SEQ ID NO: 66; (ix) SEQ ID NO: 72, (x) SEQ ID NO: 79, (xi) SEQ ID NO: 82, (xii) SEQ ID NO: 85, (xiii) SEQ ID NO: 87, (xiv) SEQ ID NO: 94, (xv) SEQ ID NO: 101, (xvi) SEQ ID NO: 104, (xvii) SEQ ID NO: 111, (xviii) SEQ ID NO: 118, (xix) SEQ ID NO: 125, (xx) SEQ ID NO: 132, (xxi) SEQ ID NO: 138, (xxii) SEQ ID NO: 144, (xxiii) SEQ ID NO: 150, (xxiv) SEQ ID NO: 155, (xxv) SEQ ID NO: 158, (xxvi) SEQ ID NO: 164, (xxvii) SEQ ID NO: 169, (xxviii) SEQ ID NO: 175, (xxix) SEQ ID NO: 183, (xxx) SEQ ID NO: 188, (xxxi) SEQ ID NO: 193, (xxxii) SEQ ID NO: 198, or (xxxiii) SEQ ID NO: 204. The multispecific antibody or antigen-binding fragment thereof of embodiment 1, comprising an amino acid sequence identical to each of the VH and VL of the scFv identified as 3. (a) a first heavy chain (HC1) comprising VH1, a first heavy chain constant region comprising a first Fc region (Fc1), and an scFv1; (b) a first light chain (LC1) comprising a VL1 and a light chain constant region; 3. The multispecific antibody or antigen-binding fragment thereof of embodiment 1 or 2, comprising: 4. (a) a second heavy chain (HC2) comprising a first heavy chain constant region comprising VH1, and a second Fc region (Fc2); (b) a second light chain (LC2) comprising a VL1 and a light chain constant region; and 4. The multispecific antibody or antigen-binding fragment thereof of embodiment 3, further comprising: 5. The multispecific antibody or antigen-binding fragment thereof of embodiment 3, further comprising a second Fc region (Fc2). 6. (a) a first heavy chain (HC1) comprising a first heavy chain constant region comprising VH1, and a first Fc region (Fc1); (b) a first light chain (LC1) comprising a VL1 and a light chain constant region; (c) a second heavy chain (HC2) comprising scFv1 and a second heavy chain constant region comprising a second Fc region (Fc2); 3. The multispecific antibody or antigen-binding fragment thereof of embodiment 1 or 2, comprising: 7. (a) a first heavy chain (HC1) comprising a VH2, a first heavy chain constant region comprising a first Fc region (Fc1), and an scFv1; (b) a first light chain (LC1) comprising a VL2 and a light chain constant region; 3. The multispecific antibody or antigen-binding fragment thereof of embodiment 1 or 2, comprising: 8. (a) a second heavy chain (HC2) comprising a first heavy chain constant region comprising VH2, and a second Fc region (Fc2); (b) a second light chain (LC2) comprising a VL2 and a light chain constant region; and 8. The multispecific antibody or antigen-binding fragment thereof of embodiment 7, further comprising: 9. The multispecific antibody or antigen-binding fragment thereof of embodiment 7, further comprising a second Fc region (Fc2). 10. (a) a first heavy chain (HC1) comprising a first heavy chain constant region comprising VH2, and a first Fc region (Fc1); (b) a first light chain (LC1) comprising a VL2 and a light chain constant region; (c) a second heavy chain (HC2) comprising scFv1 and a second heavy chain constant region comprising a second Fc region (Fc2); 3. The multispecific antibody or antigen-binding fragment thereof of embodiment 1 or 2, comprising: 11. The multispecific antibody or antigen-binding fragment thereof of embodiment 1 or 2, comprising a first antigen-binding region, a second antigen-binding region, and a third antigen-binding region. 12. (a) a first heavy chain (HC1) comprising VH1, a first heavy chain constant region comprising a first Fc region (Fc1), and an scFv1; (b) a first light chain (LC1) comprising a VL1 and a light chain constant region; (c) a second heavy chain (HC2) comprising a first heavy chain constant region comprising a second single chain variable fragment (scFv2) and a second Fc region (Fc2), wherein the scFv2 comprises VH2 and VL2; and 12. The multispecific antibody or antigen-binding fragment thereof of embodiment 11, comprising: 13. (a) a first heavy chain (HC1) comprising a VH2, a first heavy chain constant region comprising a first Fc region (Fc1), and an scFv1; (b) a first light chain (LC1) comprising a VL2 and a light chain constant region; (c) a second heavy chain (HC2) comprising a first heavy chain constant region comprising a second single-chain variable fragment (scFv2) and a second Fc region (Fc2), wherein the scFv2 comprises VH1 and VL1; and 12. The multispecific antibody or antigen-binding fragment thereof of embodiment 11, comprising: 14. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 13, wherein scFv1 and / or scFv2 comprise at least one of: (a) a first disulfide bond between a structurally conserved, surface-exposed VH cysteine (Cys) and a first L Cys; and b) a second disulfide bond between a structurally conserved, surface-exposed VL Cys and a second L Cys. 15. The multispecific antibody or antigen-binding fragment thereof of embodiment 14, wherein scFv1 and scFv2 each independently comprise a first disulfide bond and a second disulfide bond. 16. The multispecific antibody or antigen-binding fragment thereof according to embodiment 13, wherein scFv2 comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 3 or 4. 17. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 16, wherein scFv1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 26, 33, 40, 47, 54, 59, 66, 72, 79, 82, 83, 84, 85, 86, 87, 94, 101, 104, 111, 118, 125, 132, 138, 144, 150, 155, 158, 164, 169, 175, 183, 188, 193, 198, and 204. 18. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 4 to 6, 8 to 10 and 12 to 17, wherein each of Fc1 and Fc2 comprises one or more heterodimer mutations, or one or more knob and hole mutations. 19. The heterodimer mutation comprises amino acid modifications at positions T350, L351, F405, and Y407 in one of Fc1 and Fc2, and amino acid modifications at positions T350, T366, K392, and T394 in the other of Fc1 and Fc2, wherein the amino acid modification at position T350 is T350V, T350I, T350L, or T350M, the amino acid modification at position L351 is L351Y, and the amino acid modification at position F405 is F405A, F405V, F405T, or F405S. 19. The multispecific antibody or antigen-binding fragment thereof according to embodiment 18, wherein the amino acid modification at position Y407 is Y407V, Y407A, or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V, or T366M; the amino acid modification at position K392 is K392F, K392L, or K392M; and the amino acid modification at position T394 is T394W, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat. 20. The multispecific antibody or antigen-binding fragment thereof according to embodiment 19, wherein one of Fc1 and Fc2 comprises the mutations T350V, L351Y, F405A, and Y407V, and the other of Fc1 and Fc2 comprises the mutations T350V, T366L, K392L, and T394W. 21. The multispecific antibody or antigen-binding fragment thereof of embodiment 18, wherein each of Fc1 and Fc2 comprises one or more knob and hole mutations. 22. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 21, comprising an Fc domain with amino acid modifications that enhance binding of the multispecific antibody or antigen-binding fragment thereof to the neonatal Fc receptor (RcRn), preferably the amino acid modifications enhance binding at acidic pH, more preferably the Fc domain has M252Y / S254T / T256E (YTE) mutations, wherein the numbering of the amino acid residues is according to the EU index as set forth in Kabat. 23. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 22, comprising an Fc domain with amino acid modifications that reduce or eliminate effector function, preferably wherein the Fc domain has one or more amino acid modifications at positions L234, L235, D265, D270, N297, E318, K320, K322, P331, and P329, such as one, two, three, or four of L234A, L235A, D265S, and P331S, wherein the numbering of the amino acid residues is according to the EU index as set forth in Kabat. 24. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 23, comprising an Fc domain with one or more of the following amino acid modifications: M252Y, S254T, T256E, L234A, L235A, and D265S, wherein the numbering of the amino acid residues is according to the EU index as set forth in Kabat. 25. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 24, comprising an Fc domain that does not have amino acid modifications that reduce or eliminate effector function. 26. (1) SEQ ID NO: 211, SEQ ID NO: 12, and SEQ ID NO: 212, respectively; or (2) SEQ ID NO: 213, SEQ ID NO: 12, and SEQ ID NO: 214, respectively; a first heavy chain, a light chain, and a second heavy chain, each having an amino acid sequence at least 90% identical to A multispecific antibody, wherein the first antigen-binding region is capable of specifically binding to a first epitope of HER2, the second antigen-binding region is capable of specifically binding to a second epitope of HER2, and the third antigen-binding region is capable of specifically binding to TfR. 27. The first heavy chain, the light chain, and the second heavy chain each comprise: (1) SEQ ID NO: 211, SEQ ID NO: 12, and SEQ ID NO: 212, respectively; or (2) SEQ ID NO: 213, SEQ ID NO: 12, and SEQ ID NO: 214, respectively; 27. The multispecific antibody of embodiment 26, comprising the amino acid sequence of: 28. An isolated nucleic acid encoding a multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 27. 29. A vector comprising the isolated nucleic acid of embodiment 28. 30. A host cell comprising the isolated nucleic acid of embodiment 28 or the vector of embodiment 29. 31. A method for producing a multispecific antibody or antigen-binding fragment thereof, comprising culturing a host cell of embodiment 30 under conditions for producing the multispecific antibody or antigen-binding fragment thereof, and recovering the multispecific antibody or antigen-binding fragment thereof. 32. A pharmaceutical composition comprising a multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 27, and a pharmaceutically acceptable carrier. 33. A method for treating or detecting a disorder, preferably cancer, in a subject in need thereof, comprising administering to the subject a multispecific antibody or antigen-binding fragment according to any one of embodiments 1 to 27, or a pharmaceutical composition according to embodiment 32. 34. The method of embodiment 33, wherein the disease or disorder is a HER2-associated disease or disorder. 35. The method of embodiment 34, wherein the disease or disorder is brain metastasis. 36. A pharmaceutical composition comprising the isolated nucleic acid of embodiment 28, the vector of embodiment 29, or the host cell of embodiment 30, and a pharmaceutically acceptable carrier. 37. A method for treating or detecting a disorder, preferably cancer, in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of embodiment 36. 38. The method of embodiment 37, wherein the disease or disorder is a HER2-associated disease or disorder. 39. The method of embodiment 37, wherein the disease or disorder is brain metastasis. [Example]

[0247] The following is a description of various methods and materials used in testing, and is provided so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure. It is not intended to limit the scope of what the inventors regard as their disclosure, nor is it intended to represent that the experiments that follow are all that can be performed. It should be understood that exemplary descriptions written in the present tense are not necessarily performed, but rather descriptions are those that could be performed to generate data and the like relevant to the teachings of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental error and deviation should be accounted for.

[0248] Example 1. Materials and Methods Generation and screening of TfR antibodies Transgenic rodents were immunized with hTfR protein using a repetitive immunization at multiple sites (RIMMS) protocol. Serum titers were assessed by ELISA to select rodents for fusion. Lymph nodes were harvested from seropositive rodents and fused with myeloma cells to generate hybridomas using standard methods. Supernatant hybridomas were screened by MSD for binding to the protein, TfR-expressing cells, and human brain endothelial cells. Positive clones were further evaluated for internalization with brain endothelial cells, cross-reactivity to human and cynomolgus monkey proteins, and competition with transferrin (data not shown). TfR clones that bound to human and / or cynomolgus monkey TfR and were internalized without competition for transferrin binding were selected for variable sequence recovery and converted to scFv to generate TEM mAbs for further characterization.

[0249] Generation and characterization of bispecific TEM antibodies Bispecific antibodies were generated using anti-HER2 and anti-TfR antibodies using knob-into-hole heterodimerization technology (Ridgway et al., 1996, Protein Eng. 9, 617-621). Anti-HER2 binders were engineered as bivalent Fab, monovalent Fab, or monovalent scFv attached to the N-terminus of Fc, while anti-TfR binders were engineered as monovalent scFvs attached to the C-terminus of one heavy chain Fc via a (G4S)4 linker. In addition to the knob and hole mutations in the Fc, the antibodies also contained mutations in the Fc that abolished effector function [L234A / L235A / D265S (AAS)] and enhanced FcRn binding at acidic pH [M252Y / S254T / T256E (YTE)] for half-life extension (HLE). The constructed TEM mAb was expressed in ExpiCHO-S™ cells by transient transfection with purified plasmid DNA according to the manufacturer's recommendations. Harvested cell culture supernatants were purified by protein A and cation exchange chromatography. The homogeneity and purity of the final antibody were confirmed by SDS-PAGE, analytical size-exclusion chromatography, and mass spectrometry.

[0250] [Table 5]

[0251] Culture and maintenance of HER2-expressing cancer cells. BT-474 (HTB-20), BT-474 clone 5 (CRL-3247), HCC-1954 (CRL-2338), and MDA-MB-361 (HTB-27) were obtained from ATCC. Cells were cultured in RPMI supplemented with 10% low-IgG FBS and 1% non-essential amino acids (BT474 only). For imaging studies, cells were transduced with Incucyte® NucLight Red Lentivirus (Essen Biosciences #4476). Stable cell lines were selected and maintained in medium containing 0.5 μg / mL (BT-474, BT-474 clone 5, MDA-MB-361) or 1 μg / mL puromycin (HCC-1954).

[0252] Antibody binding to cells. MDA-MB-361, BT474, and HCC-1954 cells were detached with accutase, washed twice with cold DPBS, and diluted to a concentration of 2 × 10 in cold binding buffer (BD binding buffer containing 2 mM EDTA). 6 The antibody solution was adjusted to 100 cells / mL and then added to a 96-well plate at 50 μL / well. 50 μL / well of the antibody solution in cold binding buffer was added, the plate was covered, and incubated at 4°C for 1 hour. The wells were then washed with 100 μL / well of cold DPBS, spun at 300 × g for 5 minutes, and the supernatant removed. The pellet was then resuspended in 100 μL / well of LIVE / DEAD Fixable Dead Cell Dye (Invitrogen, 1 / 1,000 dilution) in cold DPBS and incubated on ice for 30 minutes. The cells were washed two more times in binding buffer as above, resuspended in a final volume of 50 μL / well, and read on an iQue flow cytometer (Sartorius).

[0253] Human PBMC-derived macrophage culture. Human macrophages were differentiated from bulk healthy human PBMCs (Donor 140458, HemaCare). Briefly, PBMCs were thawed and cultured in complete RPMI medium supplemented with 10% heat-inactivated FBS, 1% penicillin-streptomycin, and 50 ng / mL human M-CSF for 6–8 days until M0 macrophages were generated. An additional volume of complete growth medium was added to the PBMC cultures every 2 days. Nonadherent cells were removed on days 6–8. Subsequently, M0 macrophages were polarized to M2a macrophages by administering 20 ng / mL IL-4 and 50 ng / mL M-CSF for an additional 2 days.

[0254] Cytotoxicity assay. NucLight Red-labeled target cells (BT474) were removed, washed, and opsonized with TEM mAb or control mAb at a final concentration of 80 nM for 30 min at 37°C before co-culture with PBMC-derived M2a macrophages. M2a macrophages were removed with Accutase, labeled with CFSE (Invirogen, 34554) according to the manufacturer's instructions, and added to the assay plate at the desired E:T ratio (3:1). Experimental controls included wells without test substance (target and effector only) to control for the cytostatic effect of the molecules (target and antibody only). Co-cultures were monitored by imaging with the Opera Phenix Plus High Content Screening System within 7 days and imaged every 24 h. Target cell loss was determined using Columbus software by quantification of NucLight Red area per image.

[0255] Human iPSC-derived microglia culture. Human microglia were derived from epithelial-derived iPSCs (IPSC0028, male, Sigma) and processed as previously described (Haenseler, 2017, no. 607). Briefly, iPSCs were seeded onto Aggrewell 800 plates and allowed to form embryonic bodies (EBs) to recapitulate embryonic microglial development. Macrophage progenitor cells were cultured in mTESR1 medium for 3 days with bone morphogenetic protein 4 (BMP4, 50 ng / mL), vascular endothelial growth factor (VEGF, 50 ng / mL), and stem cell factor (SCF, 25 ng / mL). EBs were then harvested, transferred to 6-well plates, and cultured in EX-VIVO15 (Lonza) supplemented with Glutamax, penicillin / streptomycin, β-mercaptoethanol, IL-3 (25 ng / mL), and M-CSF (100 ng / mL) for 8 weeks to promote myeloid differentiation. Discharged macrophage precursors were collected from the supernatant, seeded at 20,000 cells per well in 96-well plates, and matured for 14 days in advanced DMEM / F12 supplemented with Glutamax, penicillin / streptomycin, β-mercaptoethanol, IL-34 (100 ng / mL), and GM-SCF (10 ng / mL). Microglia were characterized by positive immunostaining with antibodies against the following proteins: Iba1 (019-19741; 1:500; Wako), P2RY12 (HPA014518; 1:100; Sigma), CX3CR1 (2091; 1:200; ProSci Inc.), CD11b (MAB1699; 1:500, RnD Systems), and CB68 (M078, 1:500, Dako).

[0256] pH-rodo cell phagocytosis. Target cell lines were labeled with pHrodo® Red according to the manufacturer's instructions (Essen Biosciences, 4649). pHrodo®-labeled cells were opsonized with TEM mAb or control mAb for 30 min at 37°C and then co-cultured with either iPSC-derived microglial cells at various E:T ratios. Microglial co-cultures were monitored every 90 min in an Incucyte® SX5 imager, and cell phagocytosis was measured as the total pHrodored area (μm 2 / image) and total pHrodored integrated intensity (RCU × μm 2 / image).

[0257] Co-culture killing assay. NucLight Red-labeled target cell lines were detached, washed, and opsonized with TEM mAb or control mAb for 30 min at 37°C before co-culture with iPSC-derived microglia. Co-cultures were observed by imaging on either an Incucyte® SX5. Target cell loss was determined by quantification of NucLight Red area per image.

[0258] Non-human primate cynomolgus monkey PK study Study Design: TEM and control IgG1 mAb were administered to cynomolgus monkeys at 10 mg / kg via slow bolus IV injection. Blood samples for PK were collected 1, 6, 24, 72, and 168 hours post-dose and processed to serum via the study facility's laboratory protocol. For brain tissue collection, cynomolgus monkeys were placed under deep anesthesia and terminal blood samples were collected. After final blood collection, animals were euthanized at 72 and 168 hours (n = 2 per time point) and subjected to upper body perfusion with cold saline at 250 mL / min for a minimum of 5 minutes, according to the study facility's standard operating procedure (SOP). Approximately 200 mg of tissue was isolated from defined brain locations (frontal lobe, hippocampus, and temporal lobe), snap-frozen in liquid nitrogen, and stored at -70°C until capillary depletion and tissue homogenization.

[0259] Brain Tissue Preparation. The right and left hemispheres were weighed and processed into capillary-depleted brain tissue as previously described, with some modifications. 25 Briefly, brain tissue samples were slowly thawed on wet ice, added to a calculated volume (2.5 μL buffer / 1 mg tissue) of modified DPBS buffer containing protease inhibitors (Pierce; A32955), and transferred to a Lysing Matrix D tube (MP Biomedicals™; 6913-100). A whole cell suspension was generated by homogenizing the tissue at 2.8 m / s for 15 seconds using a Bead Ruptor 24 Elite (Omni International). The whole cell suspension was transferred to a new tube and mixed with an equal volume of dextran buffer (Sigma; 31397) to achieve a final dextran concentration of 13%. The dextran-containing cell suspension was centrifuged at 2000 g for 20 minutes at 4°C. The upper layer (capillary-depleted fraction) was carefully separated from the remaining sample and transferred to a new tube containing 10x radioimmune precipitation assay (RIPA) lysis buffer (Millipore™; 20-188). The capillary-depleted sample plus lysis buffer was vortexed thoroughly and centrifuged at 14,000 rpm for 30 minutes at 4°C, and the supernatant was collected for analysis. The processed brain tissue lysate was tested for protein concentration using a BCA protein assay kit (Pierce™; 23227), and the final sample lysate was normalized to a total protein concentration of 7 mg / mL before immunoassay determination.

[0260] PK assay. The concentrations of TEM and control IgG1 mAbs in NHP brain tissue and plasma were determined by MSD immunoassay on small-spot streptavidin plates. Fresh standard curves were prepared by serial dilution of each mAb in assay diluent containing naive mouse matrix (50% brain tissue lysate or 10% pooled plasma). Frozen quality controls prepared in 100% naive mouse matrix were diluted and tested with each assay. Briefly, plates were blocked with 1% bovine serum albumin in PBS for 30 minutes and washed with 0.05% Tween-20 in PBS. A master mix containing capture and detection reagents (biotinylated and ruthenium-labeled anti-human Fc mAb) was combined with reference standards, quality controls, and samples in a 1:1 volumetric ratio in the assay plate and incubated for 1 hour with shaking. Raw data signals were read on a Meso Sector S 600 imager and analyzed using Watson LIMS software (Thermo Scientific). Data regression is 1 / Y 2 A five-parameter logistic fit with weighting was performed. The quantifiable curve range for the brain tissue lysate assay was 1 to 512 ng / mL, with a minimum required sample dilution of 1:2 for processed tissue. To calculate the total tissue drug concentration in brain tissue, the mAb concentration in the brain lysate was multiplied by the total volume used for processing to a final 7 mg / mL normalized sample. The total tissue drug concentration (ng) was then divided by the brain tissue wet weight to determine the drug:tissue (ng:mg) ratio. The quantifiable curve range for the plasma assay was 2 to 512 ng / mL, with a minimum required sample dilution of 1:10. The assay had a sensitivity limit of 2 ng / mL in brain tissue lysate and 10 ng / mL in plasma.

[0261] Example 2. Antibody binding in HER2-positive cancer cells TfR-based antibody therapies that require Fc activity are well known to result in peripheral reticulocyte depletion. This can be prevented by introducing mutations that impair effector function ("silencing") or by steric blockage of simultaneous TfR and Fc receptor binding. While the use of silent Fc can alleviate safety concerns, the inability to bind to FcγRs may also limit therapeutic efficacy. Therefore, to ensure therapeutic efficacy, it is necessary to replace Fc-mediated effector function with a different, equally potent mechanism. Bispecific antibodies containing one transcytosis receptor-binding arm with optimized affinity for receptor-mediated transport (RMT) and myeloid cell binding, and another high-affinity target antigen-binding arm, were found to effectively cross the BBB and enable antigen clearance by utilizing an Fc-independent mechanism (hereafter referred to as non-classical phagocytosis (NCP)) (Figure 2A).

[0262] To determine the ability of TEM mAbs to mediate phagocytic clearance of cells, we used the anti-human epidermal growth factor receptor 2 (HER2) binding antibody trastuzumab (a bivalent HER2-TfR antibody lacking Fc-mediated effector functions). k -mut), and a biparatopic molecule containing both trastuzumab and pertuzumab (a biparatopic HER2-TfR antibody lacking Fc-mediated effector functions). k -mut G1) was used to generate the TEM construct (Figure 2B). Both trastuzumab and pertuzumab have been extensively characterized both in vitro and in vivo and are FDA-approved drugs.

[0263] Four trastuzumab-based TfR-TEM mAbs using the AAS / YTE Fc scaffold were generated to test the effect that anti-HER2 valency and anti-TfR affinity have on cancer cell clearance by myeloid cells. Binding of the anti-HER2 TEM mAbs to the HER2-expressing breast cancer cell lines BT474 and MDA-MB-361 revealed dose-dependent binding of all mAbs, with the monovalent forms having net increased binding over the bivalent HER2 mAb (Figures 3A-B). Monovalent, bivalent, and biparatopic antibodies were tested for binding in the HCC1954 (HER2-amplified) cell line. All antibodies demonstrated dose-dependent binding, with the monovalent and biparatopic forms having net increased binding over the bivalent HER2 mAb (Figure 3C).

[0264] Example 3. TEM antibodies promote non-classical phagocytosis To further test anti-HER2 TEM, we selected the HER2-expressing breast cancer cell lines BT474 and MDA-MB-361, engineered them to stably express red fluorescent protein (mKATE2) in the nucleus by lentiviral transduction, and used them to generate tumor spheroids, which are widely considered a more physiological model of tumor growth compared to 2D cultures. The spheroids were co-cultured with human induced pluripotent stem cell (iPSC)-derived microglia cells (iMG) in the presence of anti-HER2 TEM or control, and the fluorescent signal was monitored over time for 14 days (Figures 4A-C). For both cell lines, trastuzumab caused a decrease in the red fluorescent area over time, indicating tumor cell death. Interestingly, while the mono-HER2-TFR-J-mut and mono-HER2-TFR-K-mut conditions showed a strong decrease in fluorescence area over time, both bivalent HER2-TFR-mut TEM mAbs had only a moderate cytotoxic effect on the cancer cell population. For both the monovalent and bivalent mAb sets, a correlation between TfR binding affinity and fluorescence area could be established, with the TFR-K-mut form being more potent in reducing fluorescence than the higher affinity counterparts.

[0265] To determine whether the observed reduction in cancer cell numbers was due to inhibition of non-classical phagocytosis (NCP) or signal transduction, we repeated the experiment using a stable RFP-transduced subline of BT474 clone 5, a breast cancer cell line with high expression of HER2 and mutant PI3K, rendering it resistant to the signal transduction inhibitory effects of trastuzumab. Figure 4C shows that all TEM mAbs caused a similar inhibition of BT474 clone 5 NucLight Red (NR) spheroid area as trastuzumab. Statistical analysis of spheroid area at day 14 demonstrated that mono-HER2-TfR-K-mut was as potent as trastuzumab in achieving total spheroid killing for BT474 NR and BT474 clone 5 NR cells (Figures 4D-F), and had the strongest potency of the TEM mAbs tested, but was slightly less potent than trastuzumab for MDA-MB-361 NR cells (Figure 4D). Taken together, these results demonstrated that anti-HER2 TEMs have potent cytotoxic effects against HER2+ tumor cells that are due to the induction of microglial NCPs and are independent of signaling inhibition.

[0266] Further evidence that TEM mAbs induce NCP in induced pluripotent stem cell (iPSC)-derived microglial cells (iMG) was established when pHrodo red-loaded BT474 clone 5 cells were cocultured with iMG and monitored for the appearance of red fluorescence (Figures 5A-D). In addition to causing ADCC and inhibiting HER2 / HER2 signaling, trastuzumab is also a potent promoter of phagocytosis by iMG. Mono-HER2-TfR-K-mut mAb also had a significant effect in inducing phagocytosis in BT474 clone 5 NR cells, whereas bivalent TEM mAbs had no effect on pHrodo red signal appearance (Figures 5A-B). Notably, the red fluorescence intensity kinetics of trastuzumab differed from that of the mono-HER2-K-mut mAb. Trastuzumab showed peak intensity 4–6 h after the start of the experiment and then plateaued, whereas the mono-HER2-TfR-K-mut mAb slowly increased its red fluorescence intensity over time without signs of saturation, reaching trastuzumab levels toward the end of the experiment. When analyzing the total phagocytosis induced by each mAb, the areas of trastuzumab and mono-HER2-K-mut mAb were not statistically different (Figure 5C–D), demonstrating comparable in vitro potency between the mono-HER2-K-mut mAb and trastuzumab.

[0267] Additional monovalent (BBBB1627), bivalent (BBBB1638), and biparatopic (BBBB1598) TEM antibodies were tested in the MDA-MB-361 cell line. MDA-MB-361 spheroids were cocultured with iMG and monitored for spheroid contraction. TEM was compared with trastuzumab, an isotype control, and untreated cells. Trastuzumab induced phagocytosis of MDA-MB-361 cells at both 80 nM (Figure 6A) and 8 nM (Figure 6B). All TEM mAbs also caused inhibition of MDA-MB-361 cell area in 2D cocultures, similar to trastuzumab (Figure 6C).

[0268] Next, bivalent HER2-TfR k -mut G1 and two paratopic HER2-TfRk We determined whether iMG release proinflammatory cytokines during the -mut G1 NCP. Samples of conditioned medium from iMG and MDA-MB-361 or HCC1954 cocultures were collected at 20 and 44 hours for measurement of IL-10, TNF-α, and IL-1β (Figures 7A-7B). In HCC1954 cocultures, as expected, trastuzumab induced secretion of TNF-α, IL-1β, and IL-10 at significantly higher concentrations than the no-antibody control at both time points. Anti-HER2 TEMS did not induce these cytokines. The same effect was observed for TNF-α and IL-10 concentrations in MDA-MB-361 cocultures. No differences were observed in IL-1β levels, as this cytokine was not produced in this cell line under the conditions tested. These results recapitulated the findings from microglial phagocytosis experiments using TEM, namely, that TEM-induced NCP did not induce the release of pro-inflammatory cytokines, in contrast to the mechanism of action of trastuzumab IgG1.

[0269] Example 4. TEM enables non-classical phagocytosis of live target cells by macrophages Next, we tested the ability of anti-HER2 TEMs to promote the clearance of HER2+ tumor cells by human PBMCs. Over the course of 7 days of co-culture, we observed strong cytotoxicity mediated by TEMs against the trastuzumab-resistant BT474 clone 5 cell line (Figure 8A). Although the TEM molecules acted more slowly compared to trastuzumab, upon completion of the study, we observed strong inhibition of tumor growth by TEMs, demonstrating the efficacy of monovalent HER2-TfR. k -mut G1 expresses monovalent HER2-TfR j demonstrated stronger NCP-mediated cytotoxicity than -mut G1.

[0270] Because the average donor PBMC contains approximately 10-20% CD14+ monocytes with phagocytic capacity, we next tested TEM-mediated cytotoxicity in coculture with purified differentiated macrophages (Figures 8B-8C). Here, differentiated M2a macrophages were used because the M2a subtype is known to exert an anti-inflammatory tumor-promoting profile and promote tumor growth (Yao et al., 2019, Front Immunol 10, 792; Yang et al., 2020, Trends in Pharmacological Sciences 41, 701-714). Significant inhibition of BT474 tumor cell line induced by TEM mAb was observed in coculture with M2a macrophages for 7 days. As captured by the representative images of macrophage and tumor cocultures, monovalent HER2-TfR expression was observed. k -mut G1 TEM demonstrated potent elimination of tumors similar to the effects observed with trastuzumab at a lower ratio of effector cells to target cells (E:T) 3:1.

[0271] To further confirm that the killing mechanism was mediated by phagocytosis, M2a macrophages were cultured with pHrodo-labeled tumor line BT474 clone 5. Upon phagosome formation, pH changes induce a strong pHrodo signal, reflecting active engulfment and digestion of tumor cells by macrophages. Quantification of phagocytic events within macrophages demonstrated robust engulfment in the presence of both trastuzumab and TEM molecules. Surprisingly, monovalent HER2-TfR demonstrated robust killing in cytotoxicity assays. k-mut G1 showed a greater ability to induce phagocytosis compared to the trastuzumab control (Figure 8D). Similar to the co-culture with microglia, trastuzumab and TEM mAb demonstrated different phagocytosis kinetics. Trastuzumab phagocytosis peaked at approximately 2 hours in co-culture and then decreased, whereas TEM mAb showed gradual and sustained phagocytosis. This data suggests that TEM does not induce the macrophage exhaustion observed in the presence of trastuzumab. Further analysis of total phagocytosis, which reflects the sum of phagocytic events across all time points, revealed that monovalent HER2-TfR k Finally, representative images of M2a phagocytosis were obtained with monovalent HER2-TfR compared to minimal phagocytosis in the absence of antibody and maximal phagocytosis in the presence of trastuzumab at 2 h of co-culture (Figure 8E). k We demonstrate the intensity of phagocytic events over time in the presence of -mut G1 TEM (Figure 8F).

[0272] Overall, these data demonstrated that anti-Her2 TEM enabled microglia to eliminate tumor cells in an antigen-dependent, non-Fc effector function-mediated manner. The non-classical phagocytic mechanism proved to be at least (if not more) potent than trastuzumab and was translatable to peripheral immune cells and macrophages.

[0273] Example 5. TEM results of enhanced brain delivery in cynomolgus monkeys NHPs administered TEM mAbs (BBBB1598 and BBBB1627) were compared with a group of NHPs injected with trastuzumab. Brain mAb concentrations at 72 and 168 hours after IV administration (10 mg / kg) were measured in eight perfused and capillary-depleted brain regions. The presence of TEM increased mAb concentrations in the frontal lobe, hippocampus, and temporal lobe compared with trastuzumab, with fold increases ranging from approximately 5- to 7-fold (Figure 9). These data demonstrated that anti-Her2 TEM resulted in increased mAb uptake in the brain relative to trastuzumab.

[0274] Example 6. TEM PET / CT Imaging Studies [Zr89]-DFO in human TfR knock-in female mice (Biocytogen, Cat. No. 110861) (TfR or huTfR KI) and female C57BL6 (Jax) mice (B6 control or BL6) * The in vivo distribution of the -TfRxHER2 (BBBB1598) IgG antibody was evaluated. On the day of the imaging experiment, the tracer precursor was labeled with [Zr89]. Immediately afterward, quality control (QC) measurements were performed on the radiochemical purity and specific activity of the compound.

[0275] Animals were weighed using a Mettler Toledo scale. Then, animals were anesthetized in an anesthesia chamber using isoflurane with oxygen (3.0%-4.5% for induction and 1.0%-3.0% for maintenance) via a Somni AMD-3. After confirming that the animals maintained a stable breathing pattern, the animals were placed on a treatment table for injection, using a heating pad to maintain body temperature.

[0276] Mice were divided into three groups (A, B, and C). Animals in the same group were injected with the PET tracer approximately 2 min apart. At the designated time points, mice were placed in a 4-mouse room of a Sofie GNEXT PET / CT scanner (Sofie, Culver City, CA, USA) equipped with a heating pad to maintain body temperature along with respiratory monitoring. The imaging protocol began with a 30-min static PET scan, followed by a 1-min standard CT scan acquisition protocol.

[0277] Acquisition and reconstruction parameters: PET / CT examinations were performed on days 1, 5, and 7 after tracer injection. To maximize sensitivity and resolution, the whole body was centered in the scanner's axial field of view. CT scans were used for attenuation correction, anatomical imaging, and scatter correction of PET images. The PET scan energy window was set to 350–650 keV with a 3.438 ns timing window. Emission data were collected for 30 minutes in list mode. PET images were reconstructed into a single frame using the iterative 2-dimensional ordered-subsets expectation maximization (OSEM2D) algorithm (4 OSEM2D iterations with Fourier rebinning). The data were reconstructed into a 128 × 128 matrix image.

[0278] result: Zr89-DFO * The HER2xTfR (HER2xTfR) Ab showed enhanced brain uptake in human TfR knock-in mice compared to the B6 control (Figures 10A-C). * -HER2 (HER2) Ab is Zr89-DFO * The HER2xTfR Ab showed higher peripheral organ uptake (liver, kidney, and spleen) compared with the B6 control (data not shown). The HER2xTfR Ab significantly increased the brain-to-heart ratio of normalized uptake values in human TfR knock-in mice compared with the B6 control, whereas no such increase was observed with the HER2 Ab (Figures 11A-11D).

Claims

1. A multispecific antibody or antigen-binding fragment thereof, comprising at least one of a first antigen-binding region and a second antigen-binding region, each capable of specifically binding to human epidermal growth factor receptor 2 (HER2), and a third antigen-binding region capable of specifically binding to transferrin receptor (TfR), (4) The first antigen-binding region is a first heavy chain variable region (VH1) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively; and a first light chain variable region (VL1) comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; (5) The second antigen-binding region is a second heavy chain variable region (VH2) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively; and a second light chain variable region (VL2) comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively; (6) A multispecific antibody or antigen-binding fragment thereof comprising a first single-chain variable fragment (scFv1) having the third antigen-binding region: a third heavy chain variable region (VH3) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3; and a third light chain variable region (VL3) comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3; wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 have any of the amino acid sequences in Table 2.

2. (4) The VH1 comprises an amino acid sequence identical to the VH of the HC sequence identified as SEQ ID NO: 1, and the VL1 comprises an amino acid sequence identical to the VL of the LC sequence identified as SEQ ID NO: 2; (5) The VH2 comprises an amino acid sequence identical to the VH of the HC sequence identified as SEQ ID NO: 11, and the VL2 comprises an amino acid sequence identical to the VL of the LC sequence identified as SEQ ID NO: 12; (6) The VH3 and the VL3 are the following: (xxxiv) SEQ ID NO: 19, (xxxv) SEQ ID NO: 26, (xxxvi) SEQ ID NO: 33, (xxxvii) SEQ ID NO: 40, (xxxviii) SEQ ID NO: 47, (xxxix) SEQ ID NO: 54, (xl) SEQ ID NO: 59, (xli) SEQ ID NO: 66, (xlii) SEQ ID NO: 72, (xliii) SEQ ID NO: 79, (xliv) SEQ ID NO: 82, (xlv) SEQ ID NO: 85, (xlvi) SEQ ID NO: 87, (xlvii) SEQ ID NO: 94, (xlviii) SEQ ID NO: 101, (xlix) SEQ ID NO: 104, (l) SEQ ID NO: 111, (li) SEQ ID NO: 118, (lii) SEQ ID NO: 125, (liii) SEQ ID NO: 132, (liv) SEQ ID NO: 138, (lv) SEQ ID NO: 144, (lvi) SEQ ID NO: 150, (lvii) SEQ ID NO: 155, (lviii) SEQ ID NO: 158, (lix) SEQ ID NO: 164, (lx) SEQ ID NO: 169, (lxi) SEQ ID NO: 175, (lxii) SEQ ID NO: 183, (lxiii) SEQ ID NO: 188, (lxiv) SEQ ID NO: 193, (lxv) SEQ ID NO: 198, or (lxvi) SEQ ID NO: 204, 3. The multispecific antibody or antigen-binding fragment thereof of claim 2, comprising an amino acid sequence identical to the VH and VL of each of the scFvs identified as:

3. (c) a first heavy chain (HC1) comprising the VH1, a first heavy chain constant region comprising a first Fc region (Fc1), and the scFv1; (d) a first light chain (LC1) comprising the VL1 and a light chain constant region; and 3. The multispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising:

4. (c) a second heavy chain (HC2) comprising the VH1 and a first heavy chain constant region comprising a second Fc region (Fc2); and (d) a second light chain (LC2) comprising the VL1 and a light chain constant region; and The multispecific antibody or antigen-binding fragment thereof of claim 3, further comprising:

5. 4. The multispecific antibody or antigen-binding fragment thereof of claim 3, further comprising a second Fc region (Fc2).

6. (d) a first heavy chain (HC1) comprising the VH1 and a first heavy chain constant region comprising a first Fc region (Fc1); (e) a first light chain (LC1) comprising the VL1 and a light chain constant region; and (f) a second heavy chain (HC2) comprising the scFv1 and a second heavy chain constant region comprising a second Fc region (Fc2); 3. The multispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising:

7. (c) a first heavy chain (HC1) comprising the VH2, a first heavy chain constant region comprising a first Fc region (Fc1), and the scFv1; (d) a first light chain (LC1) comprising the VL2 and a light chain constant region; and 3. The multispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising:

8. (c) a second heavy chain (HC2) comprising the VH2 and a first heavy chain constant region comprising a second Fc region (Fc2); and (d) a second light chain (LC2) comprising the VL2 and a light chain constant region; and 8. The multispecific antibody or antigen-binding fragment thereof of claim 7, further comprising:

9. 8. The multispecific antibody or antigen-binding fragment thereof of claim 7, further comprising a second Fc region (Fc2).

10. (d) a first heavy chain (HC1) comprising the VH2 and a first heavy chain constant region comprising a first Fc region (Fc1); (e) a first light chain (LC1) comprising the VL2 and a light chain constant region; and (f) a second heavy chain (HC2) comprising the scFv1 and a second heavy chain constant region comprising a second Fc region (Fc2); 3. The multispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising:

11. 3. The multispecific antibody or antigen-binding fragment thereof of claim 1, comprising the first antigen-binding region, the second antigen-binding region, and the third antigen-binding region.

12. (d) a first heavy chain (HC1) comprising the VH1, a first heavy chain constant region comprising a first Fc region (Fc1), and the scFv1; (e) a first light chain (LC1) comprising the VL1 and a light chain constant region; and (f) a second heavy chain (HC2) comprising a first heavy chain constant region comprising a second single chain variable fragment (scFv2) and a second Fc region (Fc2), wherein the scFv2 comprises the VH2 and VL2; 12. The multispecific antibody or antigen-binding fragment thereof of claim 11, comprising:

13. (d) a first heavy chain (HC1) comprising the VH2, a first heavy chain constant region comprising a first Fc region (Fc1), and the scFv1; (e) a first light chain (LC1) comprising the VL2 and a light chain constant region; and (f) a second heavy chain (HC2) comprising a first heavy chain constant region comprising a second single-chain variable fragment (scFv2) and a second Fc region (Fc2), wherein the scFv2 comprises the VH1 and VL1; 12. The multispecific antibody or antigen-binding fragment thereof of claim 11, comprising:

14. 14. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the scFv1 and / or scFv2 comprises at least one of (a) a first disulfide bond between a structurally conserved, surface-exposed VH cysteine (Cys) and a first L Cys, and b) a second disulfide bond between a structurally conserved, surface-exposed VL Cys and a second L Cys.

15. The multispecific antibody or antigen-binding fragment thereof of claim 14, wherein the scFv1 and scFv2 each independently comprise the first disulfide bond and the second disulfide bond.

16. The multispecific antibody or antigen-binding fragment thereof of claim 13, wherein the scFv2 comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 3 or 4.

17. 17. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the scFv1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 26, 33, 40, 47, 54, 59, 66, 72, 79, 82, 83, 84, 85, 86, 87, 94, 101, 104, 111, 118, 125, 132, 138, 144, 150, 155, 158, 164, 169, 175, 183, 188, 193, 198, and 204.

18. 18. The multispecific antibody or antigen-binding fragment thereof of any one of claims 4 to 6, 8 to 10, and 12 to 17, wherein each of Fc1 and Fc2 comprises one or more heterodimer mutations, or one or more knob and hole mutations.

19. The heterodimer mutations include amino acid modifications at positions T350, L351, F405, and Y407 in one of Fc1 and Fc2, and amino acid modifications at positions T350, T366, K392, and T394 in the other of Fc1 and Fc2, wherein the amino acid modification at position T350 is T350V, T350I, T350L, or T350M, the amino acid modification at position L351 is L351Y, and the amino acid modification at position F405 is F405A, F405V, F405T, or F405S.

19. The multispecific antibody or antigen-binding fragment thereof according to claim 18, wherein the amino acid modification at position Y407 is Y407V, Y407A, or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V, or T366M; the amino acid modification at position K392 is K392F, K392L, or K392M; and the amino acid modification at position T394 is T394W, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.

20. 20. The multispecific antibody or antigen-binding fragment thereof of claim 19, wherein one of Fc1 and Fc2 comprises the mutations T350V, L351Y, F405A, and Y407V, and the other of Fc1 and Fc2 comprises the mutations T350V, T366L, K392L, and T394W.

21. 19. The multispecific antibody or antigen-binding fragment thereof of claim 18, wherein each of the Fc1 and Fc2 comprises one or more knob and hole mutations.

22. 22. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, comprising an Fc domain with amino acid modifications that enhance binding of the multispecific antibody or antigen-binding fragment thereof to the neonatal Fc receptor (RcRn), preferably wherein said amino acid modifications enhance said binding at acidic pH, more preferably wherein the Fc domain has M252Y / S254T / T256E (YTE) mutations, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.

23. 23. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, comprising an Fc domain with amino acid modifications that reduce or eliminate effector function, preferably said Fc domain with one or more amino acid modifications at positions L234, L235, D265, D270, N297, E318, K320, K322, P331 and P329, such as one, two, three or four of L234A, L235A, D265S and P331S, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.

24. 24. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, comprising an Fc domain with one or more of the following amino acid modifications: M252Y, S254T, T256E, L234A, L235A, and D265S, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.

25. 25. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 24, comprising an Fc domain that does not have amino acid modifications that reduce or eliminate the effector function.

26. (3) SEQ ID NO: 211, SEQ ID NO: 12, and SEQ ID NO: 212, respectively; or (4) SEQ ID NO: 213, SEQ ID NO: 12, and SEQ ID NO: 214, respectively; a first heavy chain, a light chain, and a second heavy chain, each having an amino acid sequence at least 90% identical to A multispecific antibody, wherein a first antigen-binding region is capable of specifically binding to a first epitope of HER2, a second antigen-binding region is capable of specifically binding to a second epitope of HER2, and a third antigen-binding region is capable of specifically binding to TfR.

27. the first heavy chain, the light chain, and the second heavy chain each comprise: (3) SEQ ID NO: 211, SEQ ID NO: 12, and SEQ ID NO: 212, respectively; or (4) SEQ ID NO: 213, SEQ ID NO: 12, and SEQ ID NO: 214, respectively; 27. The multispecific antibody of claim 26, comprising the amino acid sequence:

28. 28. An isolated nucleic acid sequence encoding the multispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 27.

29. 29. A vector comprising the isolated nucleic acid of claim 28.

30. 30. A host cell comprising the isolated nucleic acid of claim 28 or the vector of claim 29.

31. 31. A method of producing a multispecific antibody or antigen-binding fragment thereof, comprising culturing the host cell of claim 30 under conditions to produce the multispecific antibody or antigen-binding fragment thereof, and recovering the multispecific antibody or antigen-binding fragment thereof.

32. A pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 27 and a pharmaceutically acceptable carrier.

33. 32. A method for treating or detecting a disorder, preferably cancer, in a subject in need thereof, comprising administering to said subject a multispecific antibody or antigen-binding fragment according to any one of claims 1 to 27, or a pharmaceutical composition according to claim 32.

34. 34. The method of claim 33, wherein the disease or disorder is a HER2-associated disease or disorder.

35. 35. The method of claim 34, wherein the disease or disorder is brain metastasis.

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