Transferrin receptor binding proteins for treatment of 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

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 395,391, filed on August 5, 2022, the entire disclosure of which is incorporated herein by reference.

[0003] Reference Sequence Listing Submitted Electronically

[0004] This application contains a Sequence Listing that has been submitted electronically. The content of the electronic Sequence Listing (JBI6745WOPCT1_Sequence Listing.xml; size: 261,328 bytes; date of creation: July 18, 2023) is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates to novel multispecific antibodies that comprise a first moiety capable of binding to a first antigen and a second moiety capable of binding to a second antigen on a particular tissue (e.g., on the blood-brain barrier). Background Art

[0006] Numerous methods have been investigated to improve the delivery of therapeutic monoclonal antibodies (mAbs). For example, while the blood-brain barrier (BBB) prevents the entry of harmful substances into the brain and is essential for brain homeostasis, it poses a formidable barrier to the effective delivery of drugs to the brain. Macromolecules such as monoclonal antibodies and other biotherapeutics have great therapeutic / diagnostic potential for treating / detecting pathologies in the central nervous system (CNS). However, their entry into the brain is blocked 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, vol. 181, p. 101665, 2019). However, the safety profile and poor pharmacokinetics (PK) of anti-TfR1 monoclonal antibodies have hindered their clinical development as BBB carriers.

[0007] Accordingly, there is a need for a platform that can be used to effectively shuttle drugs into target tissues with improved safety and pharmacokinetics. Summary of the Invention

[0008] In one aspect, provided herein is a multispecific antibody or an antigen-binding fragment thereof that comprises 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).

[0009] In certain embodiments, the first antigen-binding region comprises a first variable heavy chain (VH1) that comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, which comprise the amino acid sequences of SEQ ID NO:5, 6, and 7, respectively; and a first variable light chain (VL1) that comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NO:8, 9, and 10, respectively; the second antigen-binding region comprises a second variable heavy chain (VH2) that comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, which comprise the amino acid sequences of SEQ ID NO:13, 14, and 15, respectively; and a second variable light chain (VL2) that comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NO:16, 17, and 18, respectively; and the third antigen-binding region comprises a first single-chain variable fragment (scFv1) that has: a third variable heavy chain (VH3) that comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and a third variable light chain (VL3) that comprises 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.

[0010] In certain embodiments, VH1 comprises the same amino acid sequence as the VH of the HC sequence as shown in SEQ ID NO:1; and VL1 comprises the same amino acid sequence as the VL of the LC sequence as shown in SEQ ID NO:2; VH2 comprises the same amino acid sequence as the VH of the HC sequence as shown in SEQ ID NO:11; VL2 comprises the same amino acid sequence as the VL of the LC sequence as shown in SEQ ID NO:12; and VH3 and VL3 comprise the same amino acid sequences as the corresponding VH and VL of the scFv as shown in:

[0011] (i) SEQ ID NO:19;

[0012] (ii) SEQ ID NO:26;

[0013] (iii) SEQ ID NO:33;

[0014] (iv) SEQ ID NO:40;

[0015] (v) SEQ ID NO:47;

[0016] (vi) SEQ ID NO:54;

[0017] (vii) SEQ ID NO:59;

[0018] (viii)SEQ ID NO:66;

[0019] (ix) SEQ ID NO:72;

[0020] (x)SEQ ID NO:79;

[0021] (xi) SEQ ID NO:82;

[0022] (xii) SEQ ID NO:85;

[0023] (xiii)SEQ ID NO:87;

[0024] (xiv) SEQ ID NO:94;

[0025] (xv) SEQ ID NO:101;

[0026] (xvi) SEQ ID NO:104;

[0027] (xvii)SEQ ID NO:111;

[0028] (xviii)SEQ ID NO:118;

[0029] (xix) SEQ ID NO:125;

[0030] (xx) SEQ ID NO:132;

[0031] (xxi) SEQ ID NO:138;

[0032] (xxii)SEQ ID NO:144;

[0033] (xxiii)SEQ ID NO:150;

[0034] (xxiv)SEQ ID NO:155;

[0035] (xxv)SEQ ID NO:158;

[0036] (xxvi)SEQ ID NO:164;

[0037] (xxvii)SEQ ID NO:169;

[0038] (xxviii) SEQ ID NO: 175;

[0039] (xxix) SEQ ID NO: 183;

[0040] (xxx) SEQ ID NO: 188;

[0041] (xxxi) SEQ ID NO: 193;

[0042] (xxxii) SEQ ID NO: 198; or

[0043] (xxxiii) SED ID NO: 204.

[0044] 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 containing a first Fc region (Fc1), and scFv1, and (b) a first light chain (LC1) comprising VL1 and a light chain constant region.

[0045] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a second heavy chain (HC2) comprising VH1 and a first heavy chain constant region containing a second Fc region (Fc2), and (b) a second light chain (LC2) comprising VL1 and a light chain constant region.

[0046] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof further comprises a second Fc region (Fc2).

[0047] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising VH2 and a first heavy chain constant region containing a first Fc region (Fc1), (b) a first light chain (LC1) comprising VL2 and a light chain constant region, and (c) a second heavy chain (HC2) comprising scFv1 and a second heavy chain constant region containing a second Fc region (Fc2).

[0048] 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.

[0049] 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 containing a first Fc region (Fc1), and scFv1, (b) a first light chain (LC1) comprising 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 containing a second Fc region (Fc2), wherein scFv2 comprises VH2 and VL2.

[0050] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising VH2, a first heavy chain constant region containing a first Fc region (Fc1), and scFv1; (b) a first light chain (LC1) comprising VL2 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 containing a second Fc region (Fc2), wherein scFv2 comprises VH1 and VL1.

[0051] In certain embodiments, scFv1 and / or scFv2 comprises at least one of the following: (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.

[0052] In certain embodiments, scFv1 and scFv2 each independently comprise a first disulfide bond and a second disulfide bond.

[0053] In certain embodiments, scFv2 comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 3 or 4.

[0054] 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.

[0055] In certain embodiments, Fc1 and Fc2 each comprise one or more heterodimer mutations, or one or more knob and hole mutations.

[0056] In certain embodiments, the heterodimeric 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; 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, and wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0057] 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.

[0058] In certain embodiments, each of Fc1 and Fc2 comprises one or more knob and hole mutations.

[0059] In certain embodiments, the multispecific antibody or an antigen-binding fragment thereof comprises an Fc domain having an amino acid modification that enhances the binding of the multispecific antibody or an antigen-binding fragment thereof to the neonatal Fc receptor (FcRn), preferably the amino acid modification enhances the binding at acidic pH, more preferably the Fc domain has the M252Y / S254T / T256E (YTE) mutation, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0060] In certain embodiments, the multispecific antibody or an antigen-binding fragment thereof comprises an Fc domain having an amino acid modification that reduces or eliminates 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 amino acid modifications of L234A, L235A, D265S, and P331S, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0061] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having one or more amino acid modifications of M252Y, S254T, T256E, L234A, L235A, and D265S, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0062] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having amino acid modifications that do not reduce or eliminate effector function.

[0063] 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 that is 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;

[0064] 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.

[0065] In certain embodiments, the first heavy chain, the light chain, and the second heavy chain each comprise the amino acid sequences 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.

[0066] Another general aspect of the present application relates to an isolated nucleic acid sequence encoding the multispecific antibody or antigen-binding fragment thereof according to the present application. Also provided is 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.

[0067] Another general aspect of the present application relates to a method for producing a multispecific antibody or antigen-binding fragment thereof. The method includes culturing a cell comprising the nucleic acid of the present application under conditions for producing the multispecific antibody or antigen-binding fragment thereof, and recovering the multispecific antibody or antigen-binding fragment thereof.

[0068] Also provided is a pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof according to the present application and a pharmaceutically acceptable carrier.

[0069] Another general aspect of the present invention relates to a method of treating or detecting a disorder (preferably cancer) in a subject in need thereof, the method comprising administering to the subject a multispecific antibody or antigen-binding fragment or pharmaceutical composition of the present application.

[0070] Based on the following disclosure, including the detailed description of the present invention and its preferred embodiments as well as the appended claims, other aspects, features and advantages of the present invention will be apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Illustration showing a bispecific (biparatopic), multispecific antibody fused to a tissue targeting module.

[0072] Figures 2A - 2B Illustration showing the targeting transport mechanism - endocytic trafficking and phagocytosis for target clearance. (A) The antibody binds to a receptor on the cell surface and, by selectively regulating intracellular transport, either enables endocytic trafficking across the BBB or delivers the bound antigen to the lysosome for degradation. The TEM (endocytic trafficking initiation module) is characterized by enhanced antibody exposure in the brain while inducing non-FcγR phagocytosis to eliminate target cells in a robust and safe manner. (B) Illustration of the molecular structure of the TEM mAb, in which a single TfR scFv is fused to the C-terminus of one heavy chain of a bivalent, monovalent or bispecific therapeutic mAb targeting HER2 using a short flexible linker.

[0073] Figures 3A - 3C Binding of HER2-TfR-J-mut, HER2-TfR-K-mut, single HER2-TfR-J-mut and single HER2-TfR-J-mut as well as additional bispecific antibodies to (A) BT474 cells, (B) MBA-MB-361 cells and (C) HCC1954 cells is shown. Binding was compared to trastuzumab, silent trastuzumab, silent pertuzumab and isotype IgG1 (CNTO3930).

[0074] Figures 4A - 4F Kinetics curves of spheroid area formed by (A) MDA-MB-361NR, (B) BT474 NR and (C) BT474 clone 5 co-cultured with iMG at a 1:1 E:T ratio in the presence of anti-HER2 TEM mAb, trastuzumab, silent trastuzumab or isotype IgG1 (all 10 μg / mL) are shown. Each symbol represents the mean + / − SEM of n = 3 wells; values under each condition were normalized to the value at 0 h. Figures 4D - 4FShows the values at day 14 for (D) MDA-MB-361, (E) BT474, and (F) BT474 clone 5, respectively, which are representative for statistical analysis.

[0075] Figures 5A - 5D Shows the kinetic curves of the red fluorescence of BT474 clone 5 co-cultured with human iMG for 72 h at (A) 1:1 E:T ratio or (B) 1:4 E:T ratio in the presence of 1 μg / mL pHrodo Red-labeled and in the presence of trastuzumab, anti-HER2 TEMmAb, or control (all 20 nM). Figures 5C - 5D Also shows the values plotted at 72 h, which show the statistical differences. Each symbol or bar represents the mean + / − SEM of n = 3 wells. Statistical differences determined by one-way ANOVA, * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001, ns = not significant.

[0076] Figures 6A - 6C Shows the spheroid area, which shows the kinetic curves of the spheroid area formed by co-culturing MDA-MB-361 cells with iMG at 1:1 E:T for 192 h in the presence of (A) 80 nM and (B) 8 nM anti-HER2 TEM mAb, trastuzumab, or isotype IgG1. Figure 6C Shows the values plotted at 97 h for 80 nM and 8 nM concentrations.

[0077] Figures 7A - 7B Shows the concentrations of IL-1β, IL-10, and TNFα determined at 20 h and 44 h from co-culturing (A) HCC1954 or (B) MDA-MB-361 and human iMG at 1:1 E:T ratio in conditioned medium in the presence of 20 nM trastuzumab (blue), HER2-TfRk-mut G1 (red), bispecific HER2-TfRk-mut G1 (hollow red), or no antibody treatment (black). Each point is a value from a replicate, and the horizontal line in each column is 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.

[0078] Figures 8A - 8E: TEM promotes NCP in peripheral immune cells. (A) Killing kinetics mediated by trastuzumab and TEM in co - cultures of human PBMCs and the trastuzumab - resistant BT474 clone 5 cell line. (B) Killing kinetics mediated by trastuzumab and TEM in co - cultures of M2a macrophages and the BT474 cell line. (C) Representative photographs of M2a macrophages (blue) and the BT474 cell line (yellow) after 168 h of co - culture with trastuzumab and TEM. (D) pHrodo red phagocytosis kinetics in co - cultures of M2a macrophages (blue) and pHrodo - labeled BT474 clone 5 cells (yellow) with trastuzumab and TEM. (E) Sum of pHrodo red signals in M2a macrophages reflecting the combined number of phagocytosis events within 14 h of co - culture. In all panels, mAb concentration = 80 nM. In panel A, E:T ratio = 10:1, whereas in panels B, C, D, E, E:T ratio = 3:1.

[0079] Figure 9 PK of TEM mAb in non - human primates was 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 at 1 h, 6 h, 24 h, 72 h, and 168 h post - dosing and processed into serum by the test facility laboratory protocol. After the final blood collection, the animals were euthanized at 72 h and 168 h (n = 2 at each time point). Approximately 200 mg of tissue was isolated from pre - determined brain locations (frontal lobe, hippocampus, and temporal lobe).

[0080] Figures 10A - 10C In vivo biodistribution of Zr89 - DFO* - HER2xTfR antibody (HER2xTfR) in C57BL6 (B6) and human TfR knock - in (TfR) mice was shown. Figure 10A SUV of Zr89 - DFO* - HER2xTfR antibody in tissues on day 1 was shown. Figure 10B SUV of Zr89 - DFO* - HER2xCDTfR antibody in tissues on day 5 was shown. Figure 10C SUV of Zr89 - DFO* - HER2xCDTfR antibody in tissues on day 7 was shown.

[0081] Figures 11A - 11D Brain and heart uptake of HER2xTfR and Zr89 - DFO* - HER2 (HER2) antibodies in C57BL6 (BL6) and human TfR knock - in (huTfR KI) mice was shown. Figure 11A SUV in the brain on day 1. Figure 11B SUV in the brain on day 5 was shown.Figure 11C Shows the brain-to-heart ratio of SUV on day 1. Figure 11D Shows the brain-to-heart ratio of SUV on day 5. Detailed implementation

[0082] The present invention relates to multispecific antibodies that comprise 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).

[0083] Definitions

[0084] The techniques and procedures described or cited herein include those commonly known to and / or typically employed by those skilled in the art using conventional methods, such as, for example, the widely utilized methods described in the following documents: Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Current Protocols in Molecular Biology (edited by Ausubel et al., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (edited by An, 2009); Monoclonal Antibodies: Methods and Protocols (edited by Albitar, 2010); and Antibody Engineering, Volumes 1 and 2 (edited by Kontermann and Dübel, 2nd ed., 2010). Unless otherwise defined herein, technical and scientific terms used in this specification have the meanings commonly understood by one of ordinary skill in the art. For the purpose of interpreting this specification, the following terms will be used to describe, and where appropriate, terms used in the singular will also include the plural and vice versa. In the event of any conflict between any description of the terms set forth herein and any document incorporated herein by reference, the description of the terms set forth below shall prevail.

[0085] The terms "antibody", "immunoglobulin", or "Ig" are used interchangeably herein and are used in the broadest sense and specifically encompass, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions having multi-epitope or mono-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, provided they exhibit the desired biological activity), and fragments thereof (e.g., domain antibodies), as described below. Antibodies can be human, humanized, chimeric, and / or affinity matured, as well as antibodies from other species such as mice, rabbits, llamas, etc. The term "antibody" is intended to include the polypeptide products of B cells within the immunoglobulin polypeptide class that are capable of binding to a specific molecular antigen and are composed of two pairs of identical polypeptide chains, where each pair has one heavy chain (about 50 kDa - 70 kDa) and one light chain (about 25 kDa), and each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxyl-terminal portion of each chain includes a constant region. See, for example, Antibody Engineering (edited by Borrebaeck, 2nd edition, 1995); and Kuby, Immunology (3rd edition, 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, including antibodies from camelid species (e.g., llamas or alpacas) or humanized variants thereof, intracellular antibodies, anti-idiotypic (anti-Id) antibodies, and functional fragments of any of the foregoing (e.g., antigen-binding fragments), which 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 Fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab) 2 fragments, F(ab’) 2Fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetra-bodies, and minibodies. Specifically, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen-binding domains or molecules that contain antigen-binding sites that bind an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments are described, e.g., in Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (ed. Myers, 1995); Huston et al., 1993, Cell Biophysics, Vol. 22: pp. 189-224; Plückthun and Skerra, 1989, Meth. Enzymol., Vol. 178: pp. 497-515; and Day, Advanced Immunochemistry (2nd 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 agonistic antibodies or antagonistic antibodies. The antibodies can be neither agonistic nor antagonistic.

[0086] 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., is present on or in a cell.

[0087] A "full-length" antibody is an antibody that comprises an antigen-binding site as well as the CL and at least the heavy-chain constant regions CH1, CH2, and CH3. The constant regions can comprise human constant regions or amino acid sequence variants thereof. In certain embodiments, the full-length antibody has one or more effector functions.

[0088] The terms "binds" or "binding" refer to the interaction between molecules, including, for example, the formation of a complex. The interaction can be, for example, a non-covalent interaction, including hydrogen bonds, ionic bonds, 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 interaction between a single antigen-binding site on 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 ) of a binding molecule (e.g., an antibody) from a monovalent antigen and the association rate (k on ) (k off / k on ) is the dissociation constant K D , which is inversely proportional to the affinity. The lower the K D value, the higher the affinity of the antibody. The K D value varies with different complexes of the antibody and antigen and depends on both k on and k off . The dissociation constant K D of the antibodies provided herein can be determined using any method provided herein or any other method well known to those of skill in the art. The affinity at a single binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When a complex antigen containing multiple repeating antigenic determinants (such as a multivalent antigen) contacts 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 this multiple interaction between a multivalent antibody and an antigen is called avidity.

[0089] Terms related to the binding molecules described herein, such as "binds", "specifically binds", and similar terms, are also used interchangeably herein and refer to a binding molecule that specifically binds to an antigen-binding domain of an antigen such as a polypeptide. A binding molecule or antigen-binding domain that binds to or specifically binds to an antigen can be, for example, by immunoassay, or other techniques known to those of skill in the art. In some embodiments, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), a binding molecule or antigen-binding domain binds or specifically binds an antigen when it binds the antigen with a higher affinity than to any cross-reactive antigen. Typically, a specific or selective response will be at least twice the background signal or noise and can be more than 10-fold the background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed., 1989) for a discussion of binding specificity. In certain embodiments, the binding of the 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 specific target antigen, e.g., as determined by fluorescence-activated cell sorting (FACS) analysis or RIA. A binding molecule or antigen-binding domain that binds an antigen includes a binding molecule or antigen-binding domain that can bind the antigen with sufficient affinity such that the binding molecule can be used, for example, as a therapeutic and / or diagnostic agent targeting the antigen. In certain embodiments, the binding molecule or antigen-binding domain that binds an antigen has a dissociation constant (K D ) of less than or equal to 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. In certain embodiments, the binding molecule or antigen-binding domain binds an epitope that is conserved among antigens from different species.

[0090] In certain embodiments, the binding molecule or antigen-binding domain may comprise a "chimeric" sequence, where a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass and fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Patent No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, Vol. 81: pp. 6851-6855). Chimeric sequences can include humanized sequences.

[0091] In certain embodiments, the binding molecule or antigen-binding domain may comprise portions of "humanized" forms of non-human (e.g., camelid, murine, non-human primate) antibodies, which antibodies include sequences from human immunoglobulins (e.g., acceptor antibodies), wherein the native CDR residues are replaced with residues of the corresponding CDRs from non-human species such as camelids, mice, rats, rabbits, or non-human primates (e.g., donor antibodies) having the desired specificity, affinity, and capacity. In some instances, one or more FR region residues of the human immunoglobulin sequence are replaced with corresponding non-human residues. Additionally, the humanized antibody may comprise residues not present in the acceptor antibody or the donor antibody. These modifications are made to further improve antibody performance. The humanized heavy or light chain may comprise substantially all of at least one or more variable regions, wherein all or substantially all of the CDRs correspond to those of the non-human immunoglobulin and all or substantially all of the FRs are those of the human immunoglobulin sequence. In certain embodiments, the humanized antibody will comprise at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of a human immunoglobulin. For further details, see Jones et al., Nature, Vol. 321: pp. 522-525, 1986; Riechmann et al., Nature, Vol. 332: pp. 323-329, 1988; Presta, Curr. Op. Struct. Biol., Vol. 2: pp. 593-596, 1992; Carter et al., Proc. Natl. Acad. Sci. USA, Vol. 89: pp. 4285-4289, 1992; U.S. Patent Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

[0092] In certain embodiments, the binding molecule or antigen-binding domain may comprise a portion of a "fully human antibody" or "human antibody", where these terms are used interchangeably herein and refer to an antibody that comprises human variable regions and, for example, human constant regions. The binding molecule may comprise an antibody sequence. In specific embodiments, these terms refer to antibodies that comprise variable and constant regions of human origin. In certain embodiments, "fully human" antibodies may also encompass antibodies that bind polypeptides and are encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence. The term "fully human antibody" includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described, for example, by Kabat et al. (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human and / or prepared using any technique for the production of human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. A variety of techniques known in the art can be used to generate human antibodies, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., Vol. 227: p. 381, 1991; Marks et al., J. Mol. Biol., Vol. 222: p. 581, 1991) and yeast display libraries (Chao et al., Nature Protocols, Vol. 1: pp. 755-768, 2006). Methods that can also be used to prepare human monoclonal antibodies are described in Cole et al., Monoclonal Antibodies and Cancer Therapy, p. 77, 1985; Boerner et al., J. Immunol., Vol. 147, No. 1: pp. 86-95, 1991; and van Dijk and van de Winkel, Curr. Opin. Pharmacol., Vol. 5: pp. 368-374, 2001).Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled, such as a mouse (see, e.g., Jakobovits, Curr. Opin. Biotechnol., Vol. 6, No. 5: pp. 561-566, 1995; Brüggemann and Taussing, Curr. Opin. Biotechnol., Vol. 8, No. 4: pp. 455-458, 1997; and U.S. Patent Nos. 6,075,181 and 6,150,584 regarding the XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, Vol. 103: pp. 3557-3562, 2006 regarding human antibodies generated via human B cell hybridoma technology. TM See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding the XENOMOUSE™ technology.

[0093] In certain embodiments, the binding molecule or antigen-binding domain may comprise a portion of a "recombinant human antibody," where the phrase includes human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from transgenic and / or transchromosomal animals (e.g., mice or cows) of human immunoglobulin genes (see, e.g., Taylor, L.D. et al., Nucl. Acids Res., Vol. 20: pp. 6287-6295, 1992) or antibodies prepared, expressed, produced, or isolated by any other means that involve splicing human immunoglobulin gene sequences to 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, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies are mutagenized in vitro (or, when using transgenic animals for human Ig sequences, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally occur within the in vivo human antibody germline repertoire.

[0094] In certain embodiments, a binding molecule or antigen-binding domain may comprise a portion of a "monoclonal antibody," where the term as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations or well-known post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation, or asparagine or glutamine deamidation that may be present in minor amounts, and each monoclonal antibody typically will recognize a single epitope on an antigen. In specific embodiments, as used herein, a "monoclonal antibody" is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to any particular method for preparing an antibody. For example, monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma method first described by Kohler et al., Nature, Vol. 256: pp. 495, 1975, or may be prepared using recombinant DNA methods in bacteria or eukaryotic animal or plant cells (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, Vol. 352: pp. 624-628, 1991, and Marks et al., J. Mol. Biol., Vol. 222: pp. 581-597, 1991. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed therefrom are well known in the art. See, e.g., Short Protocols in Molecular Biology (edited by Ausubel et al., 5th Edition, 2002).

[0095] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the four-chain unit is typically about 150,000 daltons. Each L chain is linked to an H chain by a covalent disulfide bond, and 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 intra-chain disulfide bonds. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) in each of the α and γ chains and four CH domains in the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at its other end. VL aligns with VH, and CL aligns with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form an interface between the light 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, page 71 (edited by Stites et al., 8th edition, 1994); and Immunobiology (edited by Janeway et al., 5th edition, 2001).

[0096] The term "Fab" or "Fab region" refers to the antibody region that binds antigen. Conventional IgG typically contains two Fab regions, each residing on one of the two arms of the Y-shaped IgG structure. Each Fab region is typically composed of one variable region and one constant region of 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 in the Fab region can be arranged in various ways to confer antigen-binding ability according to the present disclosure. For example, the VH and CH1 regions can be on one polypeptide, and the VL and CL regions can be on separate polypeptides, similar to the Fab region of conventional IgG. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and oriented in a different order, as described in more detail below.

[0097] The terms "variable region", "variable domain", "V region" or "V domain" refer to a part of the light or heavy chain of an antibody, which is usually located at the amino terminus of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and is responsible for the binding and specificity of each particular antibody to its particular antigen. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". The term "variable" refers to the fact that certain segments of the variable regions vary widely in sequence between antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed over the 110 amino acid span of the variable region. Instead, the V region consists of stretches of about 15 to 30 amino acids that are less variable (e.g., relatively invariant), called framework regions (FRs), which are separated by shorter regions of greater variability (e.g., extreme variability), called "hypervariable regions", each of which is about 9 to 12 amino acids long. The variable regions of the heavy and light chains each contain four FRs, which predominantly adopt a β-sheet conformation and are connected by three hypervariable regions, which form loops connecting the β-sheet structures and in some cases form parts of them. The hypervariable regions in each chain are held together by the FRs and in close proximity to the hypervariable regions from the other chain, contributing to the formation of the antigen-binding site of the antibody (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed., 1991)). The constant region does not directly participate in the binding of the antibody to the antigen but exhibits various effector functions, such as the antibody's participation in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary widely in sequence between different antibodies. In a particular embodiment, the variable region is a human variable region.

[0098] The term "Kabat numbering of variable region residues" or "amino acid position numbering as in Kabat" and variations thereof refer to the numbering system for the heavy chain variable region or light chain variable region used in the compilation of antibodies by Kabat et al. (supra). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to deletions or insertions in the FR or CDR of the variable domain. For example, the heavy chain variable domain may include 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, etc.). The Kabat residue numbering of a given antibody can be determined by aligning the homologous regions of the antibody sequence with the "standard" Kabat numbering sequence. When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., supra). When referring to residues in the constant region of the immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (e.g., the EU index reported by 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.

[0099] When used in reference to an antibody, the term "heavy chain" refers to a polypeptide chain of approximately 50-70 kDa, wherein the amino-terminal portion includes a variable region of approximately 120 to 130 or more amino acids, and the carboxyl-terminal portion includes a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types (e.g., isotypes), designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). The different heavy chains vary in size: alpha, delta, and gamma contain approximately 450 amino acids, while mu and epsilon contain approximately 550 amino acids. When combined with a light chain, these different types of heavy chains give rise to five well-known classes (e.g., isotypes) of antibodies, namely IgA, IgD, IgE, IgG, and IgM, including four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4.

[0100] When used in reference to an antibody, the term "light chain" refers to a polypeptide chain of approximately 25 kDa, wherein the amino-terminal portion includes a variable region of approximately 100 to approximately 110 or more amino acids, and the carboxyl-terminal portion includes a constant region. The approximate length of the light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, designated kappa (κ) or lambda (λ).

[0101] As used herein, the terms "hypervariable region", "HVR", "complementary determining region", and "CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework regions of the VH β-sheet framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2, or L3) within the non-framework regions of the VL β-sheet framework of an antibody. CDR1, CDR2, and CDR3 in the VH domain are also referred to as HCDR1, HCDR2, and HCDR3, respectively. CDR1, CDR2, and CDR3 in the VL domain are also referred to as LCDR1, LCDR2, and LCDR3, respectively. Thus, CDRs are variable region sequences interspersed within the framework region sequences.

[0102] CDR regions are well known to those skilled in the art and have been defined by well-known numbering systems. For example, the Kabat complementary determining regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra; Nick Deschacht et al., J Immunol, 2010; Vol. 184: pp. 5696-5704). Chothia, in contrast, refers to the positions of structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol., Vol. 196: pp. 901-917, 1987). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and 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 is present, the loop ends at 32; if only 35A is present, the loop ends at 33; 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 the AbM antibody modeling software from Oxford Molecular (see, e.g., Antibody Engineering, Vol. 2 (edited by Kontermann and Dübel, 2nd ed., 2010)). The "contact" hypervariable regions are based on the analysis of available complex crystal structures. Another commonly used and widely adopted numbering system is ImMunoGeneTics (IMGT) Information (Lafranc et al., Dev. Comp. Immunol., 27(1):55-77 (2003)). IMGT is an integrated information system specialized in immunoglobulins (IG), T cell receptors (TCR) and major histocompatibility complexes (MHC) of humans and other vertebrates. In this article, CDRs are referred to based on both the amino acid sequence and the position within the light or heavy chain. Since the "position" of CDRs within the immunoglobulin variable domain structure is conserved among species and exists within structures called loops, CDRs and framework residues can be readily identified by using a numbering system that aligns variable domain sequences based on structural features. This information can be used to transplant and replace CDR residues of immunoglobulins from one species into a receptor framework that is typically from a human antibody. Honegger and Plückthun developed an additional numbering system (AHon), J. Mol. Biol., Vol. 309: pp. 657-670, 2001. The correspondence between numbering systems, including for example the Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art (see for example, Kabat, ibid.; Chothia and Lesk, ibid.; Martin, ibid.; Lefranc et al., ibid.). Residues from each of these hypervariable regions or CDRs are illustrated in Table 1 below.

[0103] Table 1. Exemplary CDRs According to Various Numbering Systems

[0104]

[0105] The boundaries of a given CDR can vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms "CDR" and "complementary determining region" of a given antibody or its region (such as the variable region), and the individual CDRs of an antibody or its region (e.g., CDR-H1, CDR-H2) should be understood to encompass the complementary determining regions as defined by any known scheme as described above. In some cases, the scheme used to identify a particular CDR or CDRs is specified, such as the CDRs defined by the IMGT, Kabat, Chothia or Contact methods. In other cases, the specific amino acid sequence of the CDR is given. It should be noted that CDR regions can also be defined by a combination of various numbering systems, such as a combination of the Kabat and Chothia numbering systems or a combination of the Kabat and IMGT numbering systems. Thus, terms such as "CDR1 as shown in a particular VH" include any CDR1 defined by the above exemplary CDR numbering systems, but are not limited thereto. Once the variable region (e.g., VH or VL) is given, those skilled in the art should understand that the CDRs within that region can be defined by different numbering systems or combinations thereof.

[0106] The hypervariable regions may include the following "extended hypervariable regions": 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.

[0107] The term "constant region" or "constant domain" refers to the carboxyl-terminal portions of the light and heavy chains, which do not directly participate in antibody-antigen binding but exhibit various effector functions, such as interaction with Fc receptors. The term refers to the portions of the immunoglobulin molecule that have a more conserved amino acid sequence relative to the other parts of the immunoglobulin (the variable regions that contain the antigen-binding sites). The constant region may include the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0108] The term "framework" or "FR" refers to those variable region residues that flank the CDRs. FR residues are present, for example, in chimeric, humanized, human domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than hypervariable region residues or CDR residues.

[0109] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including, for example, a native sequence Fc region, a recombinant Fc region, and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of an antibody or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include a population of antibodies in which all K447 residues have been removed, a population of antibodies in which the K447 residues have not been removed, and a population of antibodies having a mixture of antibodies with and without K447 residues. A "functional Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions generally require the combination of an Fc region with a binding region or binding domain (e.g., an antibody variable region or domain) and can be evaluated using a variety of assays known to those of skill in the art. A "variant Fc region" includes an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, a variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or the Fc region of a parental polypeptide, e.g., from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions, in the native sequence Fc region or the Fc region of a parental polypeptide. A variant Fc region herein can have at least about 80% homology with a native sequence Fc region and / or the Fc region of a parental polypeptide, or at least about 90% homology therewith, e.g., at least about 95% homology therewith.

[0110] As used herein, an "epitope" is a term in the art and refers to a local region of an antigen to which a binding molecule (e.g., an antibody) can specifically bind. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be a continuous amino acid of the polypeptide ("linear" epitope), or the epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide ("conformational", "non-linear", or "discontinuous" epitope). Those of skill in the art will understand that, generally speaking, a linear epitope can depend or not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a set of amino acids regardless of whether they are folded in a native three-dimensional protein structure. In other embodiments, a binding molecule requires the amino acid residues that make up the epitope to exhibit a particular conformation (e.g., bend, twist, flip, or fold) in order to recognize and bind the epitope.

[0111] As used herein, the term "bispecific" antigen-binding molecule or "bispecific" polypeptide shall mean a polypeptide comprising a first immunoglobulin single variable domain and a second immunoglobulin single variable domain as defined herein, wherein the two variable domains are capable of binding to two different epitopes of an antigen.

[0112] The "percent amino acid sequence identity (%)" relative to a peptide, polypeptide or antibody sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific 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. The alignment for the purpose of determining the percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences to be compared.

[0113] The term "specificity" refers to the selective recognition of a particular epitope of an antigen by an antigen-binding protein. For example, a natural antibody is monospecific. As used herein, the term "multispecific" indicates that an antigen-binding protein has two or more antigen-binding sites, wherein at least two antigen-binding sites bind different antigens. As used herein, "bispecific" indicates that an antigen-binding protein has two different antigen-binding specificities. As used herein, the term "monospecific" antibody indicates an antigen-binding protein having one or more binding sites, each binding site binding the same antigen.

[0114] The term "valence" as used herein indicates the specified number of binding sites present 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" indicate the presence of two binding sites, three binding sites, four binding sites, five binding sites and six binding sites, respectively, in an antigen-binding protein.

[0115] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interspersed with non-amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, but not limited to, non-natural amino acids) and other modifications known in the art. It should be understood that since the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, in certain embodiments, the "polypeptide" may occur as a single chain or two or more related chains.

[0116] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may contain modified nucleotides such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to short, generally single-stranded synthetic polynucleotides, which typically but not necessarily have a length of less than about 200 nucleotides. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides applies equally and fully to oligonucleotides. The cells that produce the binding molecules of the present disclosure may include parental hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acids encoding antibodies have been introduced. Unless otherwise specified, the left end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The 5' to 3' addition direction of a nascent RNA transcript is referred to as the transcription direction; the sequence region on a DNA strand that has the same sequence as the RNA transcript and is at the 5' to 5' end of the RNA transcript is referred to as the "upstream sequence"; the sequence region on a DNA strand that has the same sequence as the RNA transcript and is at the 3' to 3' end of the RNA transcript is referred to as the "downstream sequence".

[0117] "Isolated nucleic acid" refers to a nucleic acid that is substantially separated from other genomic DNA sequences that are naturally associated with the native sequence, as well as proteins or complexes such as ribosomes and polymerases, e.g., RNA, DNA, or hybrid nucleic acids. An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding an antibody as described herein are isolated or purified. The term encompasses nucleic acid sequences that have been removed from their natural environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biosynthesized by heterologous systems. A substantially pure molecule can include an isolated form of the molecule. Specifically, an "isolated" nucleic acid molecule encoding an antibody as described herein is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is typically associated in the environment in which it is produced.

[0118] Unless otherwise indicated, the term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of one another and that encode the same amino acid sequence. The phrase "nucleotide sequence" encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may contain introns in some forms.

[0119] The term "control sequence" refers to DNA sequences that are necessary to express an operably linked coding sequence in a particular host organism. For example, suitable control sequences for prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.

[0120] As used herein, when referring to nucleic acids or amino acids, the terms "operably linked" and like phrases (e.g., gene fusion) refer to the operative linking of nucleic acid sequences or amino acid sequences placed in a functional relationship with one another. For example, operably linked promoter, enhancer element, open reading frame, 5' and 3' UTR, and terminator sequences result in the accurate 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, operably linked peptides are peptides in which the functional domains are spaced an appropriate distance from one another to confer the intended function of each domain.

[0121] The term "vector" refers to a substance used to carry or contain a nucleic acid sequence, which includes, for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) as described herein, for introducing the nucleic acid sequence into a host cell. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selectable sequences or markers operable for stable integration into the host cell chromosome. Additionally, a vector may include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that may be included, for example, provide resistance to antibiotics or toxins, complement auxotrophies, or provide key nutrients not present in the medium. Expression control sequences may include constitutive and inducible promoters, transcriptional enhancers, transcriptional terminators, etc., well known in the art. When two or more nucleic acid molecules are co-expressed (e.g., both the antibody heavy chain and light chain or antibody VH and VL), both nucleic acid molecules may be inserted, for example, into a single expression vector or separate expression vectors. For single vector expression, the encoding nucleic acids may be operably linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. Introduction of a nucleic acid molecule into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blotting of mRNA or polymerase chain reaction (PCR) amplification, immunoblotting for gene product expression, or other suitable analytical methods, to test for the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that the nucleic acid molecule is expressed in an amount sufficient to produce the desired product, and further understand that expression levels can be optimized using methods well known in the art to obtain sufficient expression.

[0122] As used herein, the term "host" refers to an animal, such as a mammal (e.g., a human).

[0123] As used herein, the term "host cell" refers to a specific subject cell that can be transfected with a nucleic acid molecule and the progeny or potential progeny of such cells. The progeny of such cells may be different from the parental cell transfected with the nucleic acid molecule, due to mutations that may occur in the progeny or environmental influences, or due to integration of the nucleic acid molecule into the host cell genome.

[0124] The term "transfected" or "transformed" or "transduced" as used herein refers to the process by which an exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0125] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognized pharmacopeias for use in animals and more specifically in humans.

[0126] "Excipient" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, solvent or encapsulating material. Excipients include, for example, encapsulating materials or additives such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, release agents, sterilizing agents, sweetening agents, solubilizers, wetting agents and mixtures thereof. The term "excipient" may also refer to diluents, adjuvants (e.g., Freund's adjuvant (complete or incomplete) or vehicles.

[0127] In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as 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 dextrin; 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 TM , polyethylene glycol (PEG) and PLURONICS TM . Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th Edition, 1990).

[0128] In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation and is suitable for contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th Edition; edited by Rowe et al.; The Pharmaceutical Press and the American Pharmaceutical Association, 2009; Handbook of Pharmaceutical Additives, 3rd Edition; edited by Ash and Ash; Gower Publishing Company, 2007; Pharmaceutical Preformulation and Formulation, 2nd Edition; edited by Gibson; CRC Press LLC, Boca Raton, FL, 2009. In some embodiments, the pharmaceutically acceptable excipients are non-toxic to the cells or mammals exposed thereto at the dosages and concentrations employed. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH-buffered solution.

[0129] In some embodiments, the excipient is a sterile liquid such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary excipient when the composition (e.g., a pharmaceutical composition) is administered intravenously. Aqueous solutions of saline and dextrose and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Excipients can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. If desired, the composition can also contain minor amounts of wetting or emulsifying agents or pH buffering agents. The composition can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral compositions, including formulations, can contain standard excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0130] For example, a composition comprising a pharmaceutical compound can contain a binding molecule (e.g., an antibody), e.g., in isolated or purified form together with a suitable amount of excipient.

[0131] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of an antibody provided herein, or a therapeutic molecule or pharmaceutical composition comprising an agent and an antibody, sufficient to produce a desired result.

[0132] The terms "subject" and "patient" are used interchangeably. As used herein, in certain embodiments, the subject is a mammal, such as a non - primate or a primate (e.g., a human). In a specific embodiment, the subject is a human. In one embodiment, the subject is a mammal, such as a human, diagnosed with a disease or disorder. In another embodiment, the subject is a mammal, such as a human, at risk of developing a disease or disorder.

[0133] "Administering" refers to the act of injecting or otherwise physically delivering a substance that exists in vitro into the body of a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0134] As used herein, the term "treatment" refers to a reduction or improvement in the progression, severity, and / or duration of a disease or condition caused by the administration of one or more therapies. Treatment can be determined by assessing whether there has been a reduction, alleviation, and / or remission of one or more symptoms associated with the underlying disorder, such that, although the patient may still be afflicted with the underlying disorder, an improvement in the patient is observed. The term "treatment" includes the management and amelioration of a disease. The term "management" refers to the beneficial effects obtained by a subject from a therapy that does not necessarily result in a cure of the disease.

[0135] The term "prevention" refers to a decrease in the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms (e.g., diabetes or cancer).

[0136] As used herein, "retarding" the development of cancer means delaying, hindering, slowing, arresting, stabilizing, and / or postponing the development of the disease. Such retardation can be of varying lengths of time, depending on the history of the disease and / or the individual being treated. As will be apparent to those skilled in the art, sufficient or significant retardation can actually encompass prevention, since the individual does not develop the disease. A method for "retarding" cancer development is a method that reduces the likelihood of disease development and / or reduces the degree of the disease within a given time frame as compared to when the method is not used. 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 cancer progression that may initially be undetectable and includes occurrence, recurrence, and onset.

[0137] The term "HER2" (also known as HER2 / neu and ErbB-2) represents "human epidermal growth factor receptor 2". As used herein, it is intended to include variants, isoforms, and species homologs of HER2.

[0138] As used herein, the term "HER2-associated disease or disorder" refers to a disease or disorder that comprises cells or tissues in which HER2 is expressed or overexpressed. In some embodiments, the HER2-associated disease or disorder comprises cells in which HER2 is abnormally expressed. In other embodiments, the HER2-associated disease or disorder comprises cells in which HER2 lacks at least one activity therein or thereon.

[0139] The "blood-brain barrier" or "BBB" refers to the physiological barrier between the peripheral circulation and the brain and spinal cord, which is formed by tight junctions within the endothelial membranes of brain capillaries, thereby creating a tight barrier that restricts molecular transport into the brain. The BBB can restrict the transport of even very small molecules such as urea (60 daltons) into the brain. Examples of the BBB include the BBB within the brain, the blood-spinal cord barrier within the spinal cord, and the blood-retinal barrier within the retina, all of which are continuous capillary barriers within the CNS. The BBB also encompasses the blood-CSF barrier (choroid plexus), where the barrier is formed by ependymal cells rather than capillary endothelial cells.

[0140] The "blood-brain barrier receptor" (abbreviated herein as "R / BBB") is an extracellular membrane-linked receptor protein expressed on brain endothelial cells that is capable of transporting molecules across the BBB or for the transport of exogenously administered molecules. Examples of R / BBB include, but are not limited to, the large neutral amino acid transporter (LAT) complex, including the CD98 component, the transferrin receptor (TfR), the insulin receptor, the insulin-like growth factor receptor (IGF-R), 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 herein is the transferrin receptor (TfR).

[0141] As used herein, the term "transferrin receptor" or "TfR" refers to the cell surface receptor necessary for the uptake of cellular iron by receptor-mediated endocytosis of the carrier protein transferrin. TfR is involved in iron uptake in vertebrates and is regulated in response to intracellular iron concentration. It imports iron by internalizing the transferrin-iron complex through receptor-mediated endocytosis. Two transferrin receptors of humans have been characterized: transferrin receptor 1 and transferrin receptor 2. Both receptors are transmembrane glycoproteins. TfR1 is a high-affinity ubiquitously expressed receptor. TfR2 binds to transferrin with an affinity 25- to 30-fold lower than that of TfR1. The expression of TfR2 is restricted to certain cell types and is not affected by intracellular iron concentration. In one embodiment, the TfR is a human TfR that comprises the amino acid sequence as described, for example, in Schneider et al., Nature, Volume 311: pages 675-678, 1984. It may have a molecular weight of approximately 180,000 daltons and has two subunits each with an apparent molecular weight of approximately 90,000 daltons. Preferably, the TfR is human TfR1.

[0142] The terms "about" and "approximately" mean 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.

[0143] As used in this disclosure and the claims, the singular forms "a", "an", and "the" include the plural forms unless the context clearly indicates otherwise.

[0144] It should be understood that wherever an embodiment is described herein using the term "comprising", other similar embodiments are also provided in accordance with "consisting of" and / or "consisting essentially of". It should also be understood that wherever an embodiment is described herein using the phrase "consisting essentially of", other similar embodiments are also provided in accordance with "consisting of".

[0145] The term "between" as used in phrases such as "between A and B" or "A - B" refers to a range that includes both A and B.

[0146] The term "and / or" as used in phrases such as "A and / or B" is intended herein to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover 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).

[0147] Multispecific Antibodies with Tissue - Targeting Moieties

[0148] In one general aspect, the present application relates to an optimized platform for delivery to a specific tissue. In one aspect, the platform utilizes binding molecules, in particular antibodies or antigen-binding fragments thereof that bind to a target expressed on the specific tissue.

[0149] In one aspect, provided herein is a multispecific antibody that comprises 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).

[0150] HER2 (also known as ErbB2 or Neu; UniProtKB / Swiss-Prot accession number P04626) consists of 1233 amino acids and is structurally similar to EGFR, having an extracellular domain composed 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, vol. 319: pp. 230-234). HER2 is activated by forming heteromeric complexes with other ErbB family members and is thus indirectly regulated by EGFR and HER3 ligands (reviewed in Yarden et al., 2001, Nat Rev Mol Cell Biol., vol. 2: pp. 127-137). HER2 is the preferred heterodimerization partner of three other ErbB receptors (Graus-Porta et al., 1997, EMBO J, vol. 16: pp. 1647-1655; Tzahar et al. 1996, Mol Cell Biol., vol. 16: pp. 5276-5287), enhancing the affinity of other ErbB receptors for their ligands by slowing the rate of ligand-receptor complex dissociation, whereby HER2 enhances and prolongs signaling (Pedersen et al., 2009, Mol Cancer Res., vol. 7: pp. 275-284). Heterodimerization of HER2 and another ligand-binding receptor of the ErbB family induces cross-phosphorylation, leading to phosphorylation of C-terminal amino acids. These in turn serve as scaffolds for signaling molecules (King et al., 1988, EMBO J, vol. 7: pp. 1647-1651). The most active HER2 heterodimer is the HER2-HER3 complex (Pinkas-Kramarski et al., 1996, EMBO J, vol. 15: pp. 2452-2467), in which HER2 complements kinase-deficient HER3 by providing an active kinase (Guy et al., 1994, Proc Natl Acad Sci USA, vol. 91: pp. 8132-8136). In contrast to EGFR, HER2 is resistant to internalization (Hommelgaard et al., 2004, Mol Biol Cell, vol. 15: pp. 1557-1567), avoiding lysosomal degradation and thus remaining on the plasma membrane.

[0151] In certain embodiments, the antigen-binding regions provided herein comprise one or more CDR sequences. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to the IMGT numbering. In some embodiments, the CDRs are numbered according to the Kabat numbering. In some embodiments, the CDRs are numbered according to the AbM numbering. In other embodiments, the CDRs are numbered according to the Chothia numbering. In other embodiments, the CDRs are numbered according to the Contact numbering.

[0152] In some embodiments, the first antigen-binding region provided herein comprises a first variable heavy chain (VH1) and a first variable light chain (VL1), the VH1 comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 as shown in the heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:1, and the VL1 comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 as shown in the light chain (LC) comprising the amino acid sequence of SEQ ID NO:2. In certain embodiments, the first variable heavy chain (VH1) comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, which comprise the amino acid sequences of SEQ ID NOs:5, 6, and 7, respectively; and the first variable light chain (VL1) comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NOs:8, 9, and 10, respectively.

[0153] In certain embodiments, VH1 comprises an amino acid sequence that is 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 as shown in SEQ ID NO:1; and VL1 comprises an amino acid sequence that is 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 as shown in SEQ ID NO:2. In certain embodiments, VH1 comprises an amino acid sequence identical to the VH of the HC sequence as shown in SEQ ID NO:1; and VL1 comprises an amino acid sequence identical to the VL of the LC sequence as shown in SEQ ID NO:2.

[0154] In some embodiments, the second antigen-binding region provided herein comprises a first heavy-chain variable region (VH2) and a second light-chain variable region (VL2), wherein the VH2 comprises heavy-chain complementarity-determining region 1 (HCDR1), HCDR2, and HCDR3 as shown in the HC comprising the amino acid sequence of SEQ ID NO:11, and the VL2 comprises light-chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3 as shown in the 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, which comprise the amino acid sequences of SEQ ID NO:13, 14, and 15, respectively; and the first light-chain variable region (VL1) comprises light-chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NO:16, 17, and 18, respectively.

[0155] In certain embodiments, VH1 comprises an amino acid sequence that is 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 as shown in SEQ ID NO:1; and VL1 comprises an amino acid sequence that is 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 as shown in SEQ ID NO:2. In certain embodiments, VH1 comprises the same amino acid sequence as the VH of the HC sequence as shown in SEQ ID NO:1; and VL1 comprises the same amino acid sequence as the VL of the LC sequence as shown in SEQ ID NO:2.

[0156] The determination of the percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. Non-limiting examples of mathematical algorithms for comparing two sequences are the algorithms of Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A., Vol. 87: pp. 2264-2268, 1990, as Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A., Vol. 90: pp. 5873-5877, 1993. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol., Vol. 215: p. 403, 1990. The BLAST nucleotide search can be performed with the NBLAST nucleotide program parameter set, e.g., for score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. The BLAST protein search can be performed with the XBLAST program parameter set, e.g., for score 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain a gapped alignment for comparison purposes, as in Altschul et al., Nucleic Acids Res. Vol. 25: pp. 3389-3402, 1997. Alternatively, the PSI BLAST can be used to perform an iterative search for detecting the distance relationships (Id.) between molecules. When using the 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, ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, CABIOS, Vol. 4: pp. 11-17, 1998. Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the 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, allowing or not allowing gaps. When calculating the percent identity, generally only exact matches are counted.

[0157] In some embodiments, the antibodies provided herein contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the anti-HER2 antibody comprising the sequence retains the ability to bind HER2. In some embodiments, a total of 1 to 10 amino acids in the reference amino acid sequence have been substituted, inserted, and / or deleted. In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., in the FRs). Optionally, the anti-HER2 antibodies or antigen-binding regions provided herein include post-translational modifications of the reference sequence.

[0158] In some embodiments, functional epitope mapping can be performed, for example, by alanine scanning in combination to identify the amino acids necessary for interaction with the anti-HER2 antibodies provided herein in the HER2 protein. In some embodiments, the conformation and crystal structure of the anti-HER2 antibodies that bind HER2 can be used to identify epitopes. In some embodiments, the present disclosure provides an antibody that specifically binds to the same epitope as any one of the anti-HER2 antibodies provided herein.

[0159] In certain embodiments, the multispecific antibodies of the invention comprise a third antigen-binding portion that binds to a primate TfR, such as human TfR or simian TfR, and the antibody or antigen-binding fragment thereof is optimized for delivering an agent to the brain of a subject in need thereof. The relationship between the binding affinity of anti-TfR antibodies for TfR and the transcytosis efficiency has previously been described as an improvement in transcytosis as the affinity for TfR decreases (Yu, Zhang et al., 2011, Sci Transl Med, Vol. 3, No. 84: 84ra44). Surprisingly, it has been found that the relationship between affinity and endocytic transport efficiency is more subtle than previously described, and the effects from both the association rate and the dissociation rate have an impact on brain concentration. Specifically, in order to achieve optimal brain PK and PD of an agent (such as an mAb) delivered effectively by an anti-TfR antibody or its antigen-binding fragment, a moderate dissociation rate that is neither too fast nor too slow is required. Anti-TfR antibodies and their antigen-binding fragments are described in International Publication No. WO 202105358, which is incorporated herein by reference in its entirety.

[0160] 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 the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are identical to the corresponding CDRs of the scFv shown in Table 2.

[0161] 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 regions 1 (HCDR1), HCDR2, and HCDR3, and a third light-chain variable region (VL3) comprising light-chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each have any one of the amino acid sequences shown in Table 2.

[0162] Table 2. Exemplary Anti - TfR Antibodies or Antigen - Binding Fragments Thereof

[0163]

[0164]

[0165] In certain embodiments, 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 corresponding VH and VL of an scFv as shown in the following sequences: 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 SED ID NO:204.

[0166] In certain embodiments, VH3 and VL3 comprise the same amino acid sequences as the corresponding VH and VL of the scFv shown in the following sequences: 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 SED ID NO:204.

[0167] In certain embodiments, the multispecific antibody or an antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising VH1, a first heavy chain constant region containing a first Fc region (Fc1), and scFv1, and (b) a first light chain (LC1) comprising VL1 and a light chain constant region.

[0168] In certain embodiments, the multispecific antibody or an antigen-binding fragment thereof comprises: (a) a second heavy chain (HC2) comprising VH1 and a first heavy chain constant region containing a second Fc region (Fc2), and (b) a second light chain (LC2) comprising VL1 and a light chain constant region.

[0169] In certain embodiments, the multispecific antibody or an antigen-binding fragment thereof further comprises a second Fc region (Fc2).

[0170] In certain embodiments, the multispecific antibody or an antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising VH2 and a first heavy chain constant region containing a first Fc region (Fc1), (b) a first light chain (LC1) comprising VL2 and a light chain constant region. And (c) a second heavy chain (HC2) comprising scFv1 and a second heavy chain constant region containing a second Fc region (Fc2).

[0171] 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.

[0172] 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 containing a first Fc region (Fc1), and scFv1, (b) a first light chain (LC1) comprising 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 containing a second Fc region (Fc2), wherein scFv2 comprises VH2 and VL2.

[0173] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising VH2, a first heavy chain constant region containing a first Fc region (Fc1), and scFv1, (b) a first light chain (LC1) comprising VL2 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 containing a second Fc region (Fc2), wherein scFv2 comprises VH1 and VL1.

[0174] In certain embodiments, the scFv of the present invention comprises a heavy chain variable region (H V ) covalently linked via a flexible linker to a light chain variable region (L V ). Although the constant regions are removed and a linker is introduced, the scFv can still retain the specificity of the original immunoglobulin. In the scFv, the order of the domains can be H V -linker-L V or L V -linker-H V . The linker can be de novo designed or derived from known protein structures to provide a compatible length and conformation when bridging the variable domains of the scFv without significant steric interference. The linker can have a length of 10 to about 25 amino acids. Preferably, the linker spans between the carboxyl terminus of the variable domain and the amino terminus of the other domain by about A peptide linker that does not affect the ability of the domain to fold and form a complete antigen-binding site (Huston et al., Methods in Enzymology, Vol. 203, pp. 46 - 88, 1991, the full text of which is incorporated herein by reference). The linker preferably contains a hydrophilic sequence to avoid the peptide being embedded within or between the variable domains during the folding of the whole protein (Argos, Journal of Molecular Biology, Vol. 211, No. 4, pp. 943 - 958, 1990). For example, the linker may contain Gly and Ser residues and / or be interspersed with charged residues such as Glu, Thr, and Lys 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). According to the present disclosure, any other suitable linker may also be used.

[0175] In certain embodiments, the scFv of the present invention can 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 an 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. "Stable" means that the scFv is referred to as thermally stable when it retains binding comparable to hK2 compared to an unheated scFv sample. The stapled Fv is described in International Publication No. WO2021 / 030657, the full text of which is incorporated herein by reference.

[0176] 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.

[0177] In certain embodiments, scFv1 and scFv2 each independently comprise a first disulfide bond and a second disulfide bond.

[0178] In certain embodiments, the linker has the amino acid sequence of SEQ ID NO: 210 (GGGSGGSGGCPPCGGSGG).

[0179] In certain embodiments, the scFv also comprises histidine at the N-terminus.

[0180] In certain embodiments, scFv1 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 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. Preferably, 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.

[0181] 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.

[0182] Antibody Variants

[0183] In some embodiments, amino acid sequence modifications of the HER2-binding antibodies described herein are envisioned. 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, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity or solubility. Thus, in addition to the HER2-binding antibodies described herein, variants of the HER2-binding antibodies described herein are envisioned. 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 of understanding amino acid changes can alter the post-translational processes of the antibody.

[0184] Chemical Modifications

[0185] 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 that have been chemically modified, for example, by glycosylation, acetylation, polyethylene glycolylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, conjugation with a cell ligand or other protein, or conjugation with one or more immunoglobulin domains (e.g., Fc or a portion of Fc). Any of a number of chemical modifications can be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis with tunicamycin, etc. Additionally, the antibody can contain one or more non-classical amino acids.

[0186] In some embodiments, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. Addition or deletion of glycosylation sites on the antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0187] When the antibodies provided herein are fused to an Fc region, the attached carbohydrate can be altered. Native antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides that are generally attached by an N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH, Vol. 15: pp. 26-32, 1997. The oligosaccharide can 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, the oligosaccharides in the binding molecules provided herein can be modified to produce variants with certain improved properties.

[0188] In other embodiments, when the antibodies provided herein are fused to an Fc region, the antibody variants provided herein can have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. For example, as described in WO 2008 / 077546, the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures attached to Asn297 (e.g., complex, hybrid, and high-mannose structures) measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants can have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 and US2004 / 0093621. Published examples related to "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US 2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol., Vol. 336: pp. 1239-1249, 2004; Yamane-Ohnuki et al., Biotech. Bioeng., Vol. 87: p. 614, 2004.Examples of cell lines capable of producing afucosylated antibodies include Lec13 CHO cells that lack protein fucosylation (Ripka et al., Arch. Biochem., Biophys., Vol. 249: pp. 533-545, 1986; US Patent Application No. US 2003 / 0157108; and WO 2004 / 056312), as well as knockout cell lines such as the α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng., Vol. 87: p. 614, 2004; Kanda, Y. et al., Biotechnol. Bioeng., Vol. 94, No. 4: pp. 680-688, 2006; and WO2003 / 085107).

[0189] Binding molecules comprising the antibodies provided herein are also provided with aliquots of oligosaccharides, e.g., wherein the biantennary oligosaccharide attached to the Fc region is bisected with GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function. Examples of such variants are described, e.g., in WO 2003 / 011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Variants are also provided in which at least one galactose residue in the oligosaccharide is attached to the Fc region. Such variants may have improved CDC function. Such variants are described, e.g., in WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[0190] In molecules comprising the antibodies of the invention and an Fc region, one or more amino acid modifications may be introduced into the Fc region, thereby generating Fc region variants. The Fc region variants may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0191] In some embodiments, the present application contemplates variants having some but not all effector functions, making it a desirable candidate for applications where the in vivo half-life of the binding molecule is important but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the binding molecule lacks FcγR binding (and thus may lack ADCC activity), but retains the ability to bind FcRn. Non-limiting examples of in vitro assays to evaluate the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA, Vol. 83: pp. 7059-7063, 1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA, Vol. 82: pp. 1499-1502, 1985; 5,821,337 (see Bruggemann, M. et al., J. Exp. Med., Vol. 166: pp. 1351-1361, 1987). Alternatively, non-radioactive assay methods can be employed, see, e.g., ACTI for flow cytometry TM Non-radioactive cytotoxicity assay (CellTechnology, Inc., Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assays (Promega, Madison, WI). Available effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo, for example, in animal models such as those disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA, Vol. 95: pp. 652-656, 1998. A C1q binding assay can also be performed to confirm that the antibody does not bind C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods, Vol. 202: p. 163, 1996; Cragg, M. S. et al., Blood, Vol. 101: pp. 1045-1052, 2003; and Cragg, M. S and M. J. Glennie, Blood, Vol. 103: pp. 2738-2743, 2004). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example, Petkova, S. B. et al., Int'l. Immunol., Vol. 18, No. 12: pp. 1759-1769, 2006).

[0192] Binding molecules with reduced effector function include binding molecules that substitute one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, which has alanine substitutions at residues 265 and 297 (U.S. Patent No. 7,332,581).

[0193] Certain variants with improved or attenuated FcR binding are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem., Vol. 9, No. 2: pp. 6591-6604, 2001.)

[0194] In some embodiments, the variant comprises an Fc region having 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, alterations are made in the Fc region that result in altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol., Vol. 164: pp. 4178-4184, 2000.

[0195] Binding molecules with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol., Vol. 117: p. 587, 1976; and Kim et al., J. Immunol., Vol. 24: p. 249, 1994), are described in US2005 / 0014934A1 (Hinton et al.). Those molecules comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following residues in the Fc region: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, e.g., a substitution at residue 434 of the Fc region (U.S. Patent No. 7,371,826). See also Duncan and Winter, Nature, Vol. 322: pp. 738-740, 1988; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351, which relate to other examples of Fc region variants.

[0196] In some embodiments, it may be desirable to generate cysteine-engineered antibodies in which one or more residues of the antibody are replaced with cysteine residues. In some embodiments, the residues to be replaced are at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as a drug moiety or a linker-drug moiety, to produce an immunoconjugate, as further described herein.

[0197] Substitutions, Deletions or Insertions

[0198] The mutations can be substitutions, deletions or insertions of one or more codons encoding the antibody or polypeptide, which result in a change in the amino acid sequence as compared to the original antibody or polypeptide. The sites of interest for substitution mutagenesis include the CDRs and FRs.

[0199] An amino acid substitution can be the result of replacing one amino acid with another having a similar structure and / or chemical property, such as replacing leucine with serine, e.g., a conservative amino acid substitution. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequences encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that result in amino acid substitutions. The insertions or deletions can optionally be in the range of about 1 to 5 amino acids. In certain embodiments, the substitution, deletion or insertion comprises 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 relative to the original molecule. In a specific embodiment, the substitution is a conservative amino acid substitution at one or more predicted non-essential amino acid residues. The permissible variants can be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for the activity exhibited by the parental antibody.

[0200] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to polypeptides containing multiple residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue.

[0201] 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 described above, families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids having 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), β-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 biological activity of the resulting mutants can be screened to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined. Conservative (e.g., within amino acid groups having similar properties and / or side chains) substitutions can be made in order to maintain or not significantly alter properties. Exemplary substitutions are shown in Table 3 below.

[0202] Table 3. Amino Acid Substitutions

[0203]

[0204] Amino acids can be grouped according to the similarity of their side chain properties (see, e.g., Lehninger, Biochemistry, pp. 73-75 (2nd ed., 1975)): (1) Non-polar: 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 grouped 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 affecting chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe. For example, any cysteine residue that does not participate in maintaining the correct conformation of the antibody can also be replaced, for example, with another amino acid such as alanine or serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Non-conservative substitutions would require exchanging a member of one of these categories for a member of another category.

[0205] One type of substitution variant involves substituting one or more hypervariable region residues of a parental antibody (e.g., a humanized antibody or a human antibody). Typically, the resulting variant selected for further study will have an altered (e.g., improved) (e.g., increased affinity, reduced immunogenicity) and / or will substantially retain certain biological properties of the parental antibody in certain biological properties. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, for example, 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).

[0206] Changes (e.g., substitutions) can be made in the CDRs, e.g., to improve antibody affinity. Such changes can be made in CDR “hot spots,” i.e., residues encoded by codons that undergo mutation at high frequency during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol., Vol. 207: pp. 179-196, 2008) and / or in the SDR (α-CDR), and the binding affinity of the resulting variant antibody or fragment thereof is tested. For example, Hoogenboom et al. have described affinity maturation by constructing secondary libraries and reselecting from the secondary libraries in Methods in Molecular Biology, Vol. 178: pp. 1-37 (edited by O’Brien et al., Human Press, Totowa, NJ, 2001). In some embodiments of affinity maturation, diversity is introduced into the variable gene 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 generated. The library is then screened to identify any antibody variants having the desired affinity. Another method of introducing diversity includes CDR-directed methods, in which several CDR residues (e.g., 4 to 6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, e.g., using alanine-scan mutagenesis or modeling. A more detailed description of affinity maturation is provided below.

[0207] In some embodiments, substitutions, insertions, or deletions can occur within one or more CDRs, provided that such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) can be made in the CDRs that do not substantially reduce binding affinity. In some embodiments of the variant antibody sequences provided herein, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0208] A useful method for identifying residues or regions of an antibody that are amenable to targeted mutagenesis is called “alanine-scan mutagenesis,” as described by Cunningham and Wells, Science, Vol. 244: pp. 1081-1085, 1989. In this method, a residue or a set of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions can be made at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the points of contact between the antibody and the antigen. Such contact residues and adjacent residues can be targeted for substitution or elimination as candidates. Variants can be screened to determine whether they possess the desired properties.

[0209] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to polypeptides containing one hundred or more residues, as well as in-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N- or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide, which extends the serum half-life of the antibody.

[0210] Variations can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter, Biochem J., Vol. 237: pp. 1-7, 1986; and Zoller et al., Nucl. Acids Res., Vol. 10: pp. 6487-6500, 1982), cassette mutagenesis (see, e.g., Wells et al., Gene, Vol. 34: pp. 315-323, 1985), or other known techniques can be performed on the cloned DNA to generate antibody variant DNA.

[0211] Fc Mutations

[0212] To facilitate the formation of heterodimers between two heavy chains, for example, one having a fusion of an anti-HER2 antibody or an antigen-binding fragment thereof, and one not; or one containing an Fc with an anti-HER2 arm and one containing an Fc with a tissue target arm, heterodimer mutations are introduced into the Fc of the two heavy chains. Examples of such Fc mutations include, but are not limited to, Zymeworks mutations (see, e.g., US10,457,742) and "knob-into-hole" mutations (see, e.g., Ridgway et al., Protein Eng., 9(7):617-621, 1996). Other heterodimer mutations can also be used in the present disclosure. In some embodiments, the modified CH3 as described herein is used to facilitate the formation of heterodimers between two heavy chains.

[0213] In a specific embodiment, each of the two heavy chains of the antibody contains one or more heterodimer mutations or one or more knob and hole mutations. In a specific embodiment, the one or more heterodimer mutations are in the CH3 domain.

[0214] In certain embodiments, each of the two heavy chains of a multispecific antibody or an antigen-binding fragment thereof comprises a modified constant heavy chain 3 (CH3) domain compared to the wild-type CH3 domain to promote 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 heterodimer CH3 domain of the first heavy chain comprises the mutations T350V, L351Y, F405A, and Y407V, and the modified heterodimer CH3 domain of the second heavy chain comprises the mutations T350V, T366L, K392L, and T394W. Unless otherwise explicitly stated, throughout the specification, the numbering of amino acid residues in the antibody is performed according to the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991. 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.

[0215] In addition to the heterodimer mutations, other mutations can be introduced. In some embodiments, the Fc region of the antibody further comprises one or more mutations that alter (increase or decrease), preferably eliminate, ADCC / CDC (such as the AAS mutations described herein) and / or one or more mutations that alter (increase or decrease), preferably increase, the binding of the antibody to FcRn (such as the YTE mutations described herein). In some embodiments, one or more cysteine residues in the antibody are replaced with other amino acids (such as serine).

[0216] In certain embodiments, the fragment crystallizable region (Fc region) of a multispecific antibody or antigen-binding fragment thereof contains substitutions that alter (increase or decrease), preferably eliminate effector functions such as antibody-dependent cell cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Preferably, the Fc region of the multispecific antibody or antigen-binding fragment thereof contains one or more amino acid modifications that reduce or eliminate binding of the multispecific antibody or antigen-binding fragment to Fc gamma receptors (FcγR) and avoid effector function-mediated toxicity. For example, the Fc region of the multispecific antibody or antigen-binding fragment thereof may contain one or more amino acid modifications at positions L234, L235, D270, N297, E318, K320, K322, P331, and P329, such as one, two, or three mutations among L234A, L235A, and D265S, wherein amino acid residues are numbered according to the EU index as described in Kabat.

[0217] In certain embodiments, the Fc region of a 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 to the neonatal Fc receptor (FcRn). Preferably, one or more mutations enhance binding at acidic pH, and more preferably, the Fc has the M252Y / S254T / T256E (YTE) mutation, wherein amino acid residues are numbered according to the EU index as described in Kabat.

[0218] In certain embodiments, the Fc region of a multispecific antibody or antigen-binding fragment thereof contains one or more mutations at positions M252Y, S254T, and T256E, and wherein amino acid residues are numbered according to the EU index as described 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, and wherein amino acid residues are numbered according to the EU index as described in Kabat.

[0219] 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 functions.

[0220] Also provided are multispecific antibodies comprising a first heavy chain, a light chain, and a second heavy chain, wherein the first heavy chain, the light chain, and the second heavy chain each have an amino acid sequence that is 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 following sequences: 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 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. Preferably, the first heavy chain, the light chain, and the second heavy chain each comprise the amino acid sequences of the following sequences: 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.

[0221] Polynucleotides

[0222] In certain embodiments, the present disclosure provides polynucleotides encoding the antibodies of the invention that bind to HER2 and fusion proteins comprising the antibodies that bind to HER2 described herein. The polynucleotides of the present disclosure can be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or the non-coding (antisense) strand. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0223] The present disclosure also relates to variants of the polynucleotides described herein, wherein 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 a polynucleotide comprising a polynucleotide having a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% identical and in some embodiments at least about 96%, 97%, 98% or 99% identical to the polynucleotide encoding the HER2-binding antibody of the present disclosure. As used herein, the phrase "a polynucleotide having a nucleotide sequence that is at least, for example, 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 include up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of the nucleotide number of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or at any position between those terminal positions, or may be scattered individually among the nucleotides of the reference sequence, or among one or more contiguous groups within the reference sequence.

[0224] Polynucleotide variants may contain alterations in the coding region, non-coding region, or both. In some embodiments, the polynucleotide variants contain alterations that result in silent substitutions, additions, or deletions without altering the properties or activities of the encoded polypeptide. In some embodiments, the polynucleotide variants contain silent substitutions that do not result in an alteration of the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants may be generated for various reasons, for example, to optimize codon expression for a particular host (i.e., changing codons in a human mRNA to preferred codons of a bacterial host such as Escherichia coli). In some embodiments, the polynucleotide variant contains at least one silent mutation in the non-coding region or the coding region of the sequence.

[0225] In some embodiments, polynucleotide variants are generated to regulate or alter the expression (or expression level) of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to increase the expression of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to decrease the expression of the encoded polypeptide. In some embodiments, the polynucleotide variant has an increased expression of the encoded polypeptide compared to the parental polynucleotide sequence. In some embodiments, the polynucleotide variant has a decreased expression of the encoded polypeptide compared to the parental polynucleotide sequence.

[0226] Also provided are vectors comprising the nucleic acid molecules described herein. In one embodiment, the nucleic acid molecules can be incorporated into recombinant expression vectors. The present disclosure provides recombinant expression vectors comprising any nucleic acid of the present disclosure. As used herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that, when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and the vector is contacted with a host cell under conditions sufficient to allow expression of the mRNA, protein, polypeptide, or peptide in the cell, permits the host cell to express the mRNA, protein, polypeptide, or peptide. The vectors described herein are not naturally occurring as a whole; however, portions of these vectors can be naturally occurring. The recombinant expression vectors can comprise any type of nucleotide, including but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthetic or obtained in part from natural sources, and which can comprise natural, non-natural, or altered nucleotides. The recombinant expression vectors can comprise naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Non-naturally occurring or altered nucleotides or internucleotide linkages do not prevent transcription or replication of the vector.

[0227] In one embodiment, the recombinant expression vectors of the present disclosure can be any suitable recombinant expression vectors and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. Vectors can be selected from the group consisting of: the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, La Jolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Phage vectors can be used, such as λGT10, λGT11, λEMBL4, and λNM1149, λZapII (Stratagene). 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 vectors can be viral vectors, for example retroviral vectors, such as γ-retroviral vectors.

[0228] In one embodiment, recombinant expression vectors are prepared using standard recombinant DNA techniques such as those described in Sambrook et al. (supra) and Ausubel et al. (supra). Circular or linear expression vector constructs can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, ColE1, SV40, 2μ plasmid, λ, bovine papillomavirus, etc.

[0229] The recombinant expression vector can contain regulatory sequences such as transcription and translation start and stop codons that are specific for the type of host (e.g., bacterium, plant, fungus, or animal) into which the vector is to be properly introduced and taking into account whether the vector is DNA-based or RNA-based.

[0230] The recombinant expression vector can contain one or more selectable marker genes that allow selection of transformed or transfected hosts. Selectable marker genes include biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.), complementation in auxotrophic hosts to provide prototrophy, and the like. Suitable selectable marker genes for the expression vector include, for example, the neomycin / G418 resistance gene, the histidinol x resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0231] The recombinant expression vector can contain a native or standard promoter operably linked to the nucleotide sequences of the present disclosure. The choice of promoter (e.g., strong promoter, weak promoter, tissue-specific promoter, inducible promoter, and development-specific promoter) is within the skill of the ordinary artisan. Similarly, the combination of nucleotide sequences with a promoter is within the skill of the artisan. The promoter can be a non-viral promoter or a viral promoter such as the cytomegalovirus (CMV) promoter, the RSV promoter, the SV40 promoter, or the promoter found in the long terminal repeat of murine stem cell virus.

[0232] The recombinant expression vector can be designed for transient expression, for stable expression, or for both. Moreover, the recombinant expression vector can be prepared for constitutive expression or for inducible expression.

[0233] Additionally, the recombinant expression vector can be prepared to include a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes death of the cell expressing the suicide gene. A suicide gene can be a gene that confers sensitivity of the cell expressing the gene to an agent such as a drug and causes cell death upon contact or exposure of the cell 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.

[0234] In certain embodiments, the polynucleotide is isolated. In certain embodiments, the polynucleotide is substantially pure.

[0235] Also provided are host cells comprising the nucleic acid molecules described herein. A host cell can be any cell containing a heterologous nucleic acid. The heterologous nucleic acid can be a vector (e.g., an expression vector). For example, a host cell can be a cell from any organism selected, modified, transformed, grown, used, or manipulated in any way for the production of a substance by the cell, such as the expression of a gene, DNA or RNA sequence, protein, or enzyme by the cell. A suitable host can be determined. For example, a host cell can be selected based on the vector backbone and the desired outcome. By way of example, a plasmid or cosmid can be introduced into a prokaryotic host cell for the replication of several types of vectors. Bacterial cells such as, but not limited to, DH5α, JM109, and KCB, competent cells, and SOLOPACK Gold cells can be used as host cells for vector replication and / or expression. Additionally, bacterial cells such as Escherichia coli (E. coli) LE392 can be used as host cells for bacteriophage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to, yeast (e.g., YPH499, YPH500, and YPH501), insects, and mammals. Examples of mammalian eukaryotic host cells for vector replication and / or expression 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) murine cell lines. An exemplary human myeloma cell line is U266 (ATCC CRL-TIB-196). Other available 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.

[0236] Preparation and Preparation Methods of Antibodies

[0237] Methods of preparing antibodies have been described. See, for example, Els Pardon et al., Nature Protocol, Vol. 9, No. 3: pp. 674, 2014. Antibodies, such as scFv fragments, can be obtained using methods known in the art, such as by immunizing Camelid species, such as camels or llamas, and obtaining hybridomas therefrom, or by cloning antibody libraries using molecular biology techniques known in the art and subsequently selecting by ELISA with individual clones of the unselected library or by using phage display.

[0238] The antibodies provided herein can be produced by culturing cells transformed or transfected with a vector containing a nucleic acid encoding the antibody. Polynucleotide sequences encoding the polypeptide components of the antibodies of the present disclosure can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated and sequenced from antibody-producing cells, such as hybridoma cells or B cells. Alternatively, the polynucleotide can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a host cell. Many vectors available and known in the art can be used for the purposes of the present disclosure. The choice of the appropriate vector will depend 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. Host cells suitable for expressing the antibodies of the present disclosure include prokaryotes such as archaebacteria and eubacteria, including Gram-negative or Gram-positive organisms; eukaryotic microorganisms such as filamentous fungi or yeast; invertebrate cells such as insect or plant cells; and vertebrate cells, such as mammalian host cell lines. The host cells are transformed with the above-described expression vectors and cultured in a conventional nutrient medium, which is modified to be suitable for inducing the promoter, selecting transformants, or amplifying the gene encoding the desired sequence. The antibodies produced by the host cells are purified using standard protein purification methods known in the art.

[0239] Antibody production methods including vector construction, expression, and purification are further described in Plückthun et al., Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation, pages 203 - 252 (edited by McCafferty et al., 1996); Kwong and Rader, E. coli Expression and Purification of Fab Antibody Fragments, published in Current Protocols in Protein Science, 2009; Tachibana and Takekoshi, Production of Antibody Fab Fragments in Escherichia coli, published in Antibody Expression and Production (edited by Al - Rubeai, 2011); and Therapeutic Monoclonal Antibodies: From Bench to Clinic (edited by An, 2009).

[0240] Of course, it is contemplated that alternative methods well - known in the art can be employed to prepare anti - HER2 antibodies. For example, solid - phase techniques can be used to generate the appropriate amino acid sequence or portions thereof by direct peptide synthesis (see, e.g., Stewart et al., Solid - Phase Peptide Synthesis, 1969; and Merrifield, J. Am. Chem. Soc., Vol. 85: pp. 2149 - 2154, 1963). In vitro protein synthesis can be carried out using manual techniques or by automation. The 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, the antibody can be purified from the cells or body fluids such as milk of transgenic animals engineered to express the antibody, as disclosed, for example, in U.S. Patents 5,545,807 and 5,827,690.

[0241] Pharmaceutical Compositions

[0242] In one aspect, the present disclosure also provides a pharmaceutical composition comprising the multispecific antibody or antigen - binding fragment thereof of the present disclosure. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the multispecific antibody or antigen - binding fragment thereof provided herein and a pharmaceutically acceptable excipient.

[0243] A pharmaceutical composition comprising a multispecific antibody or an antigen-binding fragment thereof is prepared by mixing the fusion protein having the desired purity with an optional physiologically acceptable excipient for storage in the form of an aqueous solution or in a lyophilized or other dried form (see, e.g., Remington, Remington’s Pharmaceutical Sciences (18th ed., 1980)).

[0244] The multispecific antibodies or antigen-binding fragments thereof of the present disclosure can be formulated into any suitable form for delivery to target cells / tissues, such as, for example, as microcapsules or coarse emulsions (Remington, supra; Park et al., 2005, Molecules, Vol. 10: pp. 146-161; Malik et al., 2007, Curr. Drug Deliv., Vol. 4: pp. 141-151), as sustained-release formulations (Putney and Burke, 1998, Nature Biotechnol., Vol. 16: pp. 153-157), or in liposomes (Maclean et al., 1997, Int. J. Oncol., Vol. 11: pp. 325-332; Kontermann, 2006, Curr. Opin. Mol. Ther., Vol. 8: pp. 39-45).

[0245] The antibodies or antigen-binding fragments thereof provided herein can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, entrapped in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or entrapped in coarse emulsions. Such techniques are disclosed in, for example, Remington, supra.

[0246] A variety of compositions and delivery systems are known and can be used in conjunction with an antibody or antigen-binding fragment thereof as described herein, including but not limited to encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing multispecific antibodies or antigen-binding fragments thereof, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem., Vol. 262: pp. 4429-4432), construction of nucleic acids as part of retroviruses or other vectors, etc. In another embodiment, the composition can be provided as a controlled or sustained release system. 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., Vol. 14: pp. 201-240; Buchwald et al., 1980, Surgery, Vol. 88: pp. 507-516; and Saudek et al., 1989, N. Engl. J. Med., Vol. 321: pp. 569-574). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of prophylactic or therapeutic agents (e.g., an antibody or antigen-binding fragment thereof as described herein) or the compositions provided herein (see, e.g., Medical Applications of Controlled Release (edited by Langer and Wise, 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (edited by Smolen and Ball, 1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem., Vol. 23: pp. 61-126; Levy et al., 1985, Science, Vol. 228: pp. 190-192; During et al., 1989, Ann. Neurol., Vol. 25: pp. 351-356; Howard et al., 1989, J. Neurosurg., Vol. 71: pp. 105-112; U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463 and 5,128,326; PCT Publication Nos. WO 99 / 15154 and WO 99 / 20253).Examples of polymers for 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), poly(lactide-co-glycolide) (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), poly(lactide) (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer for use in a sustained release formulation is inert, free of leachable impurities, storage stable, sterile, and biodegradable.

[0247] In another embodiment, a controlled or sustained release system can be placed near a particular target tissue (e.g., nasal passages or lungs), such that only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, Vol. 2, pp. 115-138, 1984). Controlled release systems are discussed, for example, by Langer, Science, Vol. 249: pp. 1527-1533, 1990. Any technique known to those of skill in the art can be used to produce a sustained release formulation comprising one or more antibodies or antigen-binding fragments thereof as described herein (see, e.g., U.S. Patent No. 4,526,938, PCT Publication Nos. WO 91 / 05548 and WO 96 / 20698, Ning et al., Radiotherapy & Oncology, Vol. 39: pp. 179-189, 1996; Song et al., PDA J. of Pharma. Sci. & Tech., Vol. 50: pp. 372-397, 1995; Cleek et al., Pro. Int’l. Symp. Control. Rel. Bioact. Mater., Vol. 24: pp. 853-854, 1997; and Lam et al., Proc. Int’l. Symp. Control Rel. Bioact. Mater., Vol. 24: pp. 759-760, 1997).

[0248] Methods of Using Antibodies

[0249] 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 or an antibody fragment thereof provided herein.

[0250] In one aspect, provided herein is a method of delivering a therapeutic or diagnostic agent to a particular tissue of a subject in need thereof, the method comprising administering to the subject a multispecific antibody or antibody fragment provided herein.

[0251] In one aspect, provided herein is a method of inducing antibody-dependent phagocytosis (ADP) in a subject in need thereof without stimulating pro-inflammatory cytokine secretion, the method comprising administering to the subject a multispecific antibody or antigen-binding fragment provided herein.

[0252] In one aspect, provided herein is a method of reducing or eliminating effector function.

[0253] In one aspect, provided herein is a method of attenuating HER2 activity on a cell, the method comprising exposing the cell to an effective amount of a multispecific antibody or antigen-binding fragment provided herein.

[0254] In another aspect, provided herein is a method of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment 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 one or more therapeutic agents are administered to the subject in combination with an antibody or antigen-binding fragment provided herein.

[0255] The present disclosure also relates to a method of using an antibody provided herein to inhibit (i.e., antagonize) the function of HER2 in order to inhibit HER2 activation, thereby treating a pathological disorder.

[0256] The pathological disorder can 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 lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer, pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, and head and neck cancer.

[0257] In certain embodiments, the disease or disorder is brain metastasis.

[0258] In another aspect, provided herein is the use of a multispecific antibody or antigen-binding fragment provided herein in the manufacture of a medicament for treating a disease or disorder in a subject.

[0259] 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.

[0260] In another aspect, the present disclosure provides the use of the multispecific antibodies or antigen-binding fragments thereof provided herein in the manufacture of a medicament 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 that permit binding of the antibody to the HER2 protein and detecting whether a complex is formed between the antibody and the HER2 protein.

[0261] In other aspects, the antibodies and fragments thereof of the present disclosure can be used to detect the presence of HER2 in a biological sample. As used herein, the term "detect" includes 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.

[0262] Methods of Administration and Dosing

[0263] In a specific embodiment, the present disclosure provides a composition for preventing and / or treating a disease or disorder comprising the antibody or antigen-binding fragment thereof provided herein. In one embodiment, the present disclosure provides a composition for preventing a disease or disorder, wherein the composition comprises the antibody or antigen-binding fragment thereof provided herein. In one embodiment, the present disclosure provides a composition for treating a disease or disorder, wherein the composition comprises the antibody or antigen-binding fragment thereof provided herein. In some embodiments, the disease or disorder 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 lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer, pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, and head and neck cancer. In certain embodiments, the subject is a subject in need thereof. In some embodiments, the subject has a disease or disorder. In other embodiments, the subject is at risk of developing a disease or disorder. In some embodiments, administration results in prevention, management, treatment, or amelioration of the disease or disorder.

[0264] In one embodiment, provided herein is a composition for preventing and / or treating the symptoms of a disease or disorder, wherein the composition comprises an antibody or an antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a composition for preventing the symptoms of a disease or disorder, wherein the composition comprises an antibody or an antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a composition for treating the symptoms of a disease or disorder, wherein the composition comprises an antibody or an antigen-binding fragment thereof provided herein. In some embodiments, the disease or disorder is a HER2-mediated and / or HER2-associated 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 lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer, pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, and head and neck cancer. In certain embodiments, the subject is a subject in need thereof. In some embodiments, the subject has a disease or disorder. In other embodiments, the subject is at risk of developing a disease or disorder. In some embodiments, administration results in the prevention or treatment of the symptoms of a disease or disorder.

[0265] In another embodiment, provided herein is a method of preventing and / or treating a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or an antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of preventing a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or an antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of treating a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or an antigen-binding fragment thereof provided herein. In some embodiments, the disease or disorder is a HER2-mediated and / or HER2-associated 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 lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer, pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland carcinoma, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, and head and neck cancer. In certain embodiments, the subject is a subject in need thereof. In some embodiments, the subject has a disease or disorder. In other embodiments, the subject is at risk of developing a disease or disorder. In some embodiments, the administration results in the prevention or treatment of the disease or disorder.

[0266] In another embodiment, provided herein is a method of preventing and / or treating the symptoms of a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of preventing the symptoms of a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of treating the symptoms of a disease or disorder in a subject, the method 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 lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer, pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, and head and neck cancer. In certain embodiments, the subject is a subject in need thereof. In some embodiments, the subject has a disease or disorder. In other embodiments, the subject is at risk of developing a disease or disorder. In some embodiments, the administration results in the prevention or treatment of the symptoms of the disease or disorder.

[0267] Also provided herein are methods of preventing and / or treating a disease or disorder 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 of substances that limit its effect or produce unwanted 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 cynomolgus monkey) or human). In one embodiment, the subject is human. In another embodiment, the subject is a human having a disease or disorder.

[0268] A variety of delivery systems are known and can be used to administer prophylactic or therapeutic agents (e.g., the antibodies or antigen-binding fragments thereof provided herein), including but not limited to encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing multispecific antibodies or antigen-binding fragments / receptor-mediated endocytosis thereof (see, e.g., Wu and Wu, J. Biol. Chem., Vol. 262: pp. 4429-4432, 1987), construction of nucleic acids as part of retroviruses or other vectors, etc. Methods of administering prophylactic or therapeutic agents (e.g., the 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 administration, and mucosal administration (e.g., intranasal and oral routes). In a specific embodiment, the prophylactic or therapeutic agent (e.g., the antibody or antigen-binding fragment thereof provided herein) or pharmaceutical composition is administered intranasally, intramuscularly, intravenously, or subcutaneously. The prophylactic or therapeutic agent or composition can be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucosal layers (e.g., oral mucosa, intranasal mucosa, rectal, and intestinal mucosa, etc.) and can be co-administered with other bioactive agents. The administration can be systemic or local. Additionally, pulmonary administration can also be employed, e.g., by using an inhaler or nebulizer and formulating with an aerosol. See, e.g., 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 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.

[0269] In a specific embodiment, it may be desirable to locally administer the prophylactic or therapeutic agent or the pharmaceutical composition provided herein to the area in need of treatment. This can be achieved, for example, but not limited to, by local infusion, by topical application (e.g., by intranasal spray), by injection, or by means of an implant having a porous, non-porous, or gel-like material, including membranes (such as sialastic membranes) or fibers. In some embodiments, when administering the antibody or antigen-binding fragment thereof provided herein, care must be taken to use materials that do not absorb the antibody or antigen-binding fragment.

[0270] In another embodiment, a prophylactic or therapeutic agent or a composition provided herein can be delivered in vesicles, particularly liposomes (see Langer, 1990, Science 249:1527-1533; Treat et al., published 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.).

[0271] In another embodiment, a prophylactic or therapeutic agent or a composition 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., Vol. 14: p. 20; Buchwald et al., 1980, Surgery, Vol. 88: p. 507; Saudek et al., 1989, N. Engl. J. Med., Vol. 321: p. 574). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g., an antibody provided herein) or a composition 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., Vol. 23: p. 61; also see Levy et al., 1985, Science, Vol. 228: p. 190; During et al., 1989, Ann. Neurol., Vol. 25: p. 351; Howard et al., 1989, J. Neurosurg., Vol. 71: p. 105); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253. Examples of polymers for 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), poly(lactide-co-glycolide) (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), poly(lactide) (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained release formulation is inert, free of leachable impurities, storage stable, sterile, and biodegradable.In another embodiment, a controlled or sustained release system can be placed near the therapeutic target (i.e., the nasal passages or the lungs), so that only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, supra, Vol. 2: pp. 115-138, 1984). Controlled release systems are reviewed by Langer (1990, Science 249:1527-1533). Any technique known to those of skill in the art can be used to produce a sustained release formulation containing one or more of the antibodies or antigen-binding fragments thereof provided herein. See, e.g., U.S. Patent No. 4,526,938; PCT Publication WO 91 / 05548; PCT Publication WO 96 / 20698; Ning et al., 1996, “Intratumoral Radioimmunotherapy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel”, Radiotherapy & Oncology, Vol. 39: pp. 179-189; Song et al., 1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulsions”, PDA Journal of Pharmaceutical Science & Technology, Vol. 50: pp. 372-397; Cleek et al., 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application”, Pro. Int’l. Symp. Control. Rel. Bioact. Mater., Vol. 24: pp. 853-854; and Lam et al., 1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery”, Proc. Int’l. Symp. Control Rel. Bioact. Mater., Vol. 24: pp. 759-760, each of which is incorporated herein by reference in its entirety.

[0272] In a specific embodiment, in the case where the composition provided herein is a nucleic acid encoding a prophylactic or therapeutic agent (e.g., an antibody or an antigen-binding fragment thereof provided herein), the nucleic acid can be administered in vivo to promote the expression of the prophylactic or therapeutic agent it encodes by, for example, using a retroviral vector (see U.S. Patent No. 4,980,286) or by directly injecting or by using particle bombardment (e.g., gene gun; Biolistic, Dupont) to construct the nucleic acid as part of a suitable nucleic acid expression vector and administering it to make it intracellular, or by coating with lipids or cell surface receptors or transfection agents, or by administering the nucleic acid linked to a homeobox-like peptide known to enter the nucleus (see, for example, Joliot et al., 1991, Proc. Natl. Acad. Sci. USA, Vol. 88: pp. 1864-1868), etc. Alternatively, the nucleic acid can be introduced into cells by homologous recombination and incorporated into the host cell DNA for expression.

[0273] In a specific embodiment, the composition provided herein comprises one, two or more antibodies or antigen-binding fragments thereof provided herein. In another embodiment, the composition provided herein comprises one, two or more antibodies or antigen-binding fragments thereof provided herein and a prophylactic or therapeutic agent other than the antibody or antigen-binding fragment thereof provided herein. In one embodiment, the known prophylactic or therapeutic agent can be used for or has been used for or is currently used for preventing, managing, treating and / or ameliorating a disease or disorder. In addition to the prophylactic or therapeutic agent, the composition provided herein may further comprise an excipient.

[0274] The composition provided herein includes a raw material pharmaceutical composition that can be used to prepare a unit dosage form and can be used to manufacture a pharmaceutical composition (e.g., a composition suitable for administration to a subject or patient). In one embodiment, the composition provided herein is a pharmaceutical composition. Such a composition comprises a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., an antibody or an antigen-binding fragment thereof provided herein or other prophylactic or therapeutic agents) and a pharmaceutically acceptable excipient. The pharmaceutical composition can be formulated for a route suitable for administration to a subject.

[0275] In specific embodiments, the term "excipient" may also refer to a diluent, an adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or a vehicle. A pharmaceutical excipient can be a sterile liquid such as water and oils, including those derived from petroleum, animals, plants or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When a pharmaceutical composition is administered intravenously, water is an exemplary excipient. Aqueous saline and aqueous dextrose and glycerol solutions can also be used as liquid excipients, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. If desired, the composition may also contain minor amounts of wetting or emulsifying agents or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Oral formulations may contain standard excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences, 1990, Mack Publishing Co., Easton, PA. Such compositions will contain a prophylactically or therapeutically effective amount of the multispecific antibody or antigen-binding fragment thereof provided herein, such as in purified form, and a suitable amount of excipient so as to provide a form suitable for proper administration to a patient. The formulation should be adapted to the mode of administration.

[0276] In one embodiment, the composition is formulated as a pharmaceutical composition suitable for intravenous administration to humans according to conventional procedures. Generally, a composition for intravenous administration is a sterile isotonic buffered aqueous solution. If necessary, the composition may also contain solubilizing agents and local anesthetics such as lidocaine to alleviate the pain at the injection site. However, such compositions can be administered by routes other than intravenous administration.

[0277] Generally, the components of the compositions provided herein are provided separately or mixed together in unit dosage forms in airtight containers such as ampoules or sachets indicating the amount of the active agent, e.g., as dry lyophilized powders or anhydrous concentrates. In the case where the composition is administered by infusion, an infusion bottle containing sterile pharmaceutical grade water or saline can be used to dispense the composition. In the case where the composition is administered by injection, ampoules of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0278] The antibodies or antigen-binding fragments thereof provided herein can be packaged in an airtight container, such as an ampoule or a sachet, that indicates the amount of the antibody. In one embodiment, the multispecific antibody or antigen-binding fragment thereof is provided as a dry-sterilized lyophilized powder or an anhydrous concentrate in an airtight container and can be reconstituted to an appropriate concentration, for example, with water or saline, for administration to a subject. The lyophilized antibody or antigen-binding fragment thereof can be stored in its original container between 2°C and 8°C, and the multispecific antibody or antigen-binding fragment thereof can be administered within 12 hours after reconstitution, such as within 6 hours, 5 hours, 3 hours, or 1 hour. In an alternative embodiment, the antibodies or antigen-binding fragments thereof provided herein are provided in liquid form in an airtight container that indicates the amount and concentration of the antibody.

[0279] The compositions provided herein can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc.; and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0280] 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 preventing and / or treating a disease or disorder can be determined by standard clinical techniques. Additionally, in vitro assays can optionally be employed to assist in determining the optimal dosage range. The exact dosage to be employed in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be decided according to the judgment of the physician and the circumstances of each patient.

[0281] An effective dose can be extrapolated from a dose-response curve derived from in vitro or animal model test systems.

[0282] 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, but other routes described herein are also acceptable. Each dose may or may not be administered by the same route of administration. In some embodiments, an antibody or antigen-binding fragment thereof provided herein can be administered simultaneously or subsequently via multiple routes of administration to other doses of the same or different antibodies or antigen-binding fragments thereof provided herein.

[0283] In certain embodiments, a multispecific antibody or antigen-binding fragment thereof provided herein is administered prophylactically or therapeutically to a subject. A multispecific antibody or antigen-binding fragment thereof provided herein can be administered prophylactically or therapeutically to a subject in order to prevent, mitigate, or improve a disease or its symptoms.

[0284] Diagnostic Assays and Methods

[0285] Labeled antibodies, their derivatives, and analogs that immunospecifically bind to the HER2 antigen can be used for diagnostic purposes to detect, diagnose, or monitor HER2-mediated diseases. Accordingly, provided herein are methods for detecting HER2-mediated diseases, the methods comprising: (a) assaying for the expression of the HER2 antigen in a cell or tissue sample of 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 to a control level (e.g., the level in a normal tissue sample (e.g., from a patient not suffering from a HER2-mediated disease, or from the same patient prior to the onset of the disease)), whereby an increase in the measured level of the HER2 antigen as compared to the control level of the HER2 antigen indicates a HER2-mediated disease.

[0286] Also provided herein is a diagnostic assay for diagnosing a HER2-mediated disease, the diagnostic assay comprising: (a) assaying for the level of the HER2 antigen in a cell or tissue sample of 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 measured level of the HER2 antigen as compared to the control level of the HER2 antigen indicates a HER2-mediated disease. In certain embodiments, provided herein is a method of treating a HER2-mediated disease in a subject, the method comprising: (a) assaying for the level of the HER2 antigen in a cell or tissue sample of 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 measured level of the HER2 antigen as compared to the control level of the HER2 antigen indicates a HER2-mediated disease. In some embodiments, the method further comprises (c) administering to the subject identified as having a HER2-mediated disease an effective amount of the antibody provided herein. A more definitive diagnosis of a HER2-mediated disease can allow health professionals to institute preventive measures or aggressive treatment earlier, thereby preventing the development or further progression of the HER2-mediated disease.

[0287] The antibodies provided herein can be used to determine the level of HER2 antigen in a biological sample using classical immunohistological methods as described herein or known to those of skill in the art (see, for example, Jalkanen et al., 1985, J. Cell. Biol., Vol. 101: pp. 976-985; and Jalkanen et al., 1987, J. Cell. Biol., Vol. 105: pp. 3087-3096). Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase; radioisotopes such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99Tc); luminescent labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, as well as biotin.

[0288] One aspect provided herein is the detection and diagnosis of HER2-mediated diseases in humans. In one embodiment, the diagnosis comprises: a) administering (e.g., parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled antibody that immunospecifically binds to the HER2 antigen; b) waiting a certain time interval after administration to allow the labeled antibody to accumulate at the site in the subject where the HER2 antigen is expressed (and to clear unbound labeled molecules 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 a variety of methods, including comparing the amount of labeled molecules detected to a standard value previously determined for a particular system.

[0289] It will be understood in the art that the body size of the subject and the imaging system used will determine the amount of the imaging portion required to produce a diagnostic image. In the case of a radioisotope portion, for a human subject, the amount of radioactivity injected is typically in the range of about 5 millicuries to 20 millicuries of 99Tc. The labeled antibody will then accumulate 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, edited by S.W. Burchiel and B.A. Rhodes, Masson Publishing Inc. (1982)).

[0290] Depending on several variables, including the type of label used and the mode of administration, the time interval after administration that allows the labeled antibody to concentrate in the subject's body and the unbound labeled antibody to be cleared to background levels is from 6 hours to 48 hours or from 6 hours to 24 hours or from 6 hours to 12 hours. In another embodiment, the time interval after administration is from 5 days to 20 days or from 5 days to 10 days.

[0291] In one embodiment, the monitoring of HER2-mediated disease is carried out by repeating the method used for diagnosing HER2-mediated disease, such as one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

[0292] The presence of the labeled molecule can be detected in the subject's body 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, computed tomography (CT), whole body scans such as positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasonography.

[0293] In a specific embodiment, the molecule is labeled with a radioisotope and detected in the patient's body 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 detected in the patient's body using a fluorescence-responsive scanning instrument. In another embodiment, the molecule is labeled with a positron-emitting metal and detected in the patient's body using positron emission tomography. In another embodiment, the molecule is labeled with a paramagnetic label and detected in the patient's body using magnetic resonance imaging (MRI).

[0294] Kits

[0295] Also provided herein are kits that contain the multispecific antibodies (e.g., anti-HER2 antibodies) or compositions (e.g., pharmaceutical compositions) provided herein, packaged in a suitable packaging material. The kit optionally includes a label or package insert that includes a description of the components or instructions for the in vitro, in vivo, or ex vivo use of the components therein.

[0296] The term "packaging material" refers to the physical structure that houses the components of the kit. The packaging material can maintain the components aseptically and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0297] The kits provided herein may include a label or instructions for use. The label or instructions for use include "printed matter", such as paper or cardboard, alone or attached to a component, kit or packaging material (such as a box), or attached to, for example, an ampoule, tube or vial containing the components of the kit. The label or instructions for use may additionally include a computer-readable medium, such as a disk (e.g., hard disk, card, storage disk), optical disk (such as a CD or DVD-ROM / RAM, DVD, MP3, magnetic tape) or electrical storage medium (such as RAM and ROM) or a mixture of these (such as a magnetic / optical storage medium, FLASH medium or memory card). The label or instructions for use may include information identifying the manufacturer, lot number, manufacturer location and date.

[0298] The kits provided herein may additionally include other components. Each component of the kit may be encapsulated in a separate container, and all the various containers may be within a single package. The kit may also be designed for refrigeration. The kit may also be designed to contain an antibody provided herein, or a cell containing a nucleic acid encoding an antibody provided herein. The cells in the kit may be maintained under suitable storage conditions until ready for use.

[0299] 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 may be used in the practice or testing of the present invention, the appropriate methods and materials are described herein.

[0300] As used herein, numerical values are generally given in range form throughout this document. The use of range form is merely for convenience and brevity and should not be construed as an absolute limitation on the scope of the present invention unless the context clearly indicates otherwise. Thus, the ranges used herein expressly include all possible sub-ranges, all individual numerical values within the range, and all numerical or numerical ranges (including integers within such ranges and fractions or integers of the numerical values within the range), unless the context clearly indicates otherwise. This construction applies in all contexts throughout this patent document regardless of the width of the range. Thus, for example, 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. Reference to a range of 90% to 100% also includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., and 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc.

[0301] In addition, the ranges mentioned, such as 1 to 3, 3 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, 120 to 130, 130 to 140, 140 to 150, 150 to 160, 160 to 170, 170 to 180, 180 to 190, 190 to 200, 200 to 225, 225 to 250, include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. In another example, the ranges mentioned, such as 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 value or a range within or encompassing such values, such as 25, 26, 27, 28, 29…250, 251, 252, 253, 254…500, 501, 502, 503, 504… etc.

[0302] A series of ranges used herein are disclosed throughout the document. The use of a series of ranges includes combinations of upper and lower limits to provide another range. Regardless of the width of the range, this construction applies in all contexts throughout this patent document. Thus, for example, the mention of 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, 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, 5 to 150 and 10 to 30, 10 to 40, 10 to 50, 10 to 75, 10 to 100, 10 to 150 and 20 to 40, 20 to 50, 20 to 75, 20 to 100, 20 to 150, etc.

[0303] For the sake of brevity, certain abbreviations are used herein. One example is the single-letter abbreviations representing amino acid residues. Amino acids and their corresponding three-letter and single-letter abbreviations are as follows:

[0304] Alanine Ala (A)

[0305] Arginine Arg (R)

[0306] Asparagine Asn (N)

[0307] Aspartic acid Asp (D)

[0308] Cysteine Cys (C)

[0309] Glutamic acid Glu (E)

[0310] Glutamine Gln (Q)

[0311] Glycine Gly (G)

[0312] Histidine His (H)

[0313] Isoleucine Ile (I)

[0314] Leucine Leu (L)

[0315] Lysine Lys (K)

[0316] Methionine Met (M)

[0317] Phenylalanine Phe (F)

[0318] Proline Pro (P)

[0319] Serine Ser (S)

[0320] Threonine Thr (T)

[0321] Tryptophan Trp (W)

[0322] Tyrosine Tyr (Y)

[0323] Valine Val (V)

[0324] This application uses affirmative language to describe multiple embodiments and generally disclose the invention. The invention also specifically includes embodiments in which specific subject matter such as substances or materials, method steps and conditions, schemes, procedures, assays or analyses are excluded in whole or in part. Thus, even though the invention is not generally expressed in terms of what it does not include, aspects that are not expressly included in the invention are still disclosed herein.

[0325] Multiple embodiments of the invention have been described. However, it should 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 rather than limit the scope of the invention described in the claims.

[0326] Embodiments

[0327] The disclosure provided herein also provides the following non-limiting embodiments.

[0328] 1. A multispecific antibody or an antigen-binding fragment thereof, the multispecific antibody or antigen-binding fragment thereof comprising each capable of specifically binding to human epidermal growth factor receptor 2

[0329] At least one of the first antigen-binding region and the second antigen-binding region of (HER2), and a third antigen-binding region capable of specifically binding to the transferrin receptor (TfR), wherein:

[0330] (1) The first antigen-binding region comprises:

[0331] A first heavy chain variable region (VH1), the VH1 comprising heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, which comprise the amino acid sequences of SEQ ID

[0332] NO:5, 6, and 7, respectively; and a first light chain variable region (VL1), the VL1 comprising light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NO:8, 9, and 10, respectively;

[0333] (2) The second antigen-binding region comprises:

[0334] A second heavy chain variable region (VH2), the VH2 comprising heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, which comprise the amino acid sequences of SEQ ID NO:13, 14, and 15, respectively; and a second light chain variable region (VL2), the VL2 comprising light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NO:16, 17, and 18, respectively; and

[0335] (3) The third antigen-binding region comprises a first single-chain variable fragment (scFv1), the scFv1 having: a third heavy chain variable region (VH3) comprising heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and a third light chain variable region (VL3) comprising light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 have any of the amino acid sequences in Table 2.

[0336] 2. The multispecific antibody or antigen-binding fragment thereof according to embodiment 1, wherein:

[0337] (1) The VH1 comprises the same amino acid sequence as the VH of the HC sequence as shown in SEQ ID NO:1; and the VL1 comprises the same amino acid sequence as the VL of the LC sequence as shown in SEQ ID NO:2;

[0338] (2) The VH2 contains the same amino acid sequence as the VH of the HC sequence shown in SEQ ID NO: 11; and the VL2 contains the same amino acid sequence as the VL of the LC sequence shown in SEQ ID NO: 12; and

[0339] (3) The VH3 and the VL3 contain the same amino acid sequences as the corresponding VH and VL of the scFv shown in the following sequences:

[0340] (i) SEQ ID NO: 19;

[0341] (ii) SEQ ID NO: 26;

[0342] (iii) SEQ ID NO: 33;

[0343] (iv) SEQ ID NO: 40;

[0344] (v) SEQ ID NO: 47;

[0345] (vi) SEQ ID NO: 54;

[0346] (vii) SEQ ID NO: 59;

[0347] (viii) SEQ ID NO: 66;

[0348] (ix) SEQ ID NO: 72;

[0349] (x) SEQ ID NO: 79;

[0350] (xi) SEQ ID NO: 82;

[0351] (xii) SEQ ID NO: 85;

[0352] (xiii) SEQ ID NO: 87;

[0353] (xiv) SEQ ID NO: 94;

[0354] (xv) SEQ ID NO: 101;

[0355] (xvi) SEQ ID NO: 104;

[0356] (xvii) SEQ ID NO: 111;

[0357] (xviii) SEQ ID NO: 118;

[0358] (xix) SEQ ID NO: 125;

[0359] (xx)SEQ ID NO:132;

[0360] (xxi)SEQ ID NO:138;

[0361] (xxii)SEQ ID NO:144;

[0362] (xxiii)SEQ ID NO:150;

[0363] (xxiv)SEQ ID NO:155;

[0364] (xxv)SEQ ID NO:158;

[0365] (xxvi)SEQ ID NO:164;

[0366] (xxvii)SEQ ID NO:169;

[0367] (xxviii)SEQ ID NO:175;

[0368] (xxix)SEQ ID NO:183;

[0369] (xxx)SEQ ID NO:188;

[0370] (xxxi)SEQ ID NO:193;

[0371] (xxxii)SEQ ID NO:198; or

[0372] (xxxiii)SED ID NO:204.

[0373] 3. The multispecific antibody or antigen-binding fragment thereof according to embodiment 1 or 2, wherein the multispecific antibody or antigen-binding fragment thereof comprises:

[0374] (a) A first heavy chain (HC1), wherein the HC1 comprises the VH1, a first heavy chain constant region containing a first Fc region (Fc1), and the scFv1; and

[0375] (b) A first light chain (LC1), wherein the LC1 comprises the VL1 and a light chain constant region.

[0376] 4. The multispecific antibody or antigen-binding fragment thereof according to embodiment 3, wherein the multispecific antibody or antigen-binding fragment thereof further comprises:

[0377] (a) A second heavy chain (HC2), said HC2 comprising said VH1 and a first heavy chain constant region containing a second Fc region (Fc2), and

[0378] (b) A second light chain (LC2), said LC2 comprising said VL1 and a light chain constant region.

[0379] 5. The multispecific antibody or antigen-binding fragment thereof according to embodiment 3, said multispecific antibody or antigen-binding fragment thereof further comprising a second Fc region (Fc2).

[0380] 6. The multispecific antibody or antigen-binding fragment thereof according to embodiment 1 or 2, said

[0381] multispecific antibody or antigen-binding fragment thereof comprises:

[0382] (a) A first heavy chain (HC1), said HC1 comprising said VH1 and a first heavy chain constant region containing a first Fc region (Fc1),

[0383] (b) A first light chain (LC1), said LC1 comprising said VL1 and a light chain constant region; and

[0384] (c) A second heavy chain (HC2), said HC2 comprising said scFv1 and a second heavy chain constant region containing a second Fc region (Fc2).

[0385] 7. The multispecific antibody or antigen-binding fragment thereof according to embodiment 1 or 2, said

[0386] multispecific antibody or antigen-binding fragment thereof comprises:

[0387] (a) A first heavy chain (HC1), said HC1 comprising said VH2, a first heavy chain constant region containing a first Fc region (Fc1) and said scFv1; and

[0388] (b) A first light chain (LC1), said LC1 comprising said VL2 and a light chain constant region.

[0389] 8. The multispecific antibody or antigen-binding fragment thereof according to embodiment 7, said multi-

[0390] specific antibody or antigen-binding fragment thereof further comprises:

[0391] (a) A second heavy chain (HC2), said HC2 comprising said VH2 and a first heavy chain constant region containing a second Fc region (Fc2), and

[0392] (b) A second light chain (LC2), said LC2 comprising said VL2 and a light chain constant region.

[0393] 9. The multispecific antibody or antigen-binding fragment thereof according to embodiment 7, wherein the multispecific antibody or antigen-binding fragment thereof further comprises a second Fc region (Fc2).

[0394] 10. The multispecific antibody or antigen-binding fragment thereof according to embodiment 1 or 2, wherein the

[0395] multispecific antibody or antigen-binding fragment thereof comprises:

[0396] (a) A first heavy chain (HC1), wherein the HC1 comprises the VH2 and a first heavy chain constant region containing a first Fc region (Fc1),

[0397] (b) A first light chain (LC1), wherein the LC1 comprises the VL2 and a light chain constant region; and

[0398] (c) A second heavy chain (HC2), wherein the HC2 comprises the scFv1 and a second heavy chain constant region containing a second Fc region (Fc2).

[0399] 11. The multispecific antibody or antigen-binding fragment thereof according to embodiment 1 or 2, wherein the multispecific antibody or antigen-binding fragment thereof comprises the first antigen-binding region, the second antigen-binding region, and the third antigen-binding region.

[0400] 12. The multispecific antibody or antigen-binding fragment thereof according to embodiment 11, wherein the multi

[0401] specific antibody or antigen-binding fragment thereof comprises:

[0402] (a) A first heavy chain (HC1), wherein the HC1 comprises the VH1, a first heavy chain constant region containing a first Fc region (Fc1), and the scFv1,

[0403] (b) A first light chain (LC1), wherein the LC1 comprises the VL1 and a light chain constant region;

[0404] and

[0405] (c) A second heavy chain (HC2), wherein the HC2 comprises a second single-chain variable fragment (scFv2) and a first heavy chain constant region containing a second Fc region (Fc2), wherein

[0406] the scFv2 comprises the VH2 and the VL2.

[0407] 13. The multispecific antibody or antigen-binding fragment thereof according to embodiment 11, wherein the multi

[0408] specific antibody or antigen-binding fragment thereof comprises:

[0409] (a) A first heavy chain (HC1), said HC1 comprising said VH2, a first heavy chain constant region containing a first Fc region (Fc1), and said scFv1,

[0410] (b) A first light chain (LC1), said LC1 comprising said VL2 and a light chain constant region;

[0411] And

[0412] (c) A second heavy chain (HC2), said HC2 comprising a second single-chain variable fragment (scFv2) and a first heavy chain constant region containing a second Fc region (Fc2), wherein

[0413] Said scFv2 comprises said VH1 and said VL1.

[0414] 14. The multispecific antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, wherein said scFv1 and / or scFv2 comprises at least one of the following: (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.

[0415] 15. The multispecific antibody or antigen-binding fragment thereof according to embodiment 14, wherein said scFv1 and scFv2 each independently comprise said first disulfide bond and said second disulfide bond.

[0416] 16. The multispecific antibody or antigen-binding fragment thereof according to embodiment 13, wherein said scFv2 comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 3 or 4.

[0417] 17. The multispecific antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, wherein said 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.

[0418] 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 said Fc1 and Fc2 each comprise one or more heterodimer mutations, or one or more knob and hole mutations.

[0419] 19. The multispecific antibody or antigen-binding fragment thereof according to embodiment 18, wherein 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; 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, and wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0420] 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.

[0421] 21. The multispecific antibody or antigen-binding fragment thereof according to embodiment 18, wherein each of Fc1 and Fc2 comprises one or more knob and hole mutations.

[0422] 22. The multispecific antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, wherein the multispecific antibody or antigen-binding fragment comprises an Fc domain having an amino acid modification that enhances the binding of the multispecific antibody or antigen-binding fragment to the neonatal Fc receptor (RcRn), preferably the amino acid modification enhances binding at acidic pH, more preferably the Fc domain has the M252Y / S254T / T256E (YTE) mutation, and wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0423] 23. The multispecific antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, wherein the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having an amino acid modification that reduces or eliminates 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 amino acid modifications among L234A, L235A, D265S and P331S, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0424] 24. The multispecific antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, wherein the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having one or more amino acid modifications among M252Y, S254T, T256E, L234A, L235A and D265S, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0425] 25. The multispecific antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, wherein the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having an amino acid modification that does not reduce or eliminate effector function.

[0426] 26. A multispecific antibody, the multispecific antibody comprising a first heavy chain, a light chain and a second heavy chain each having an amino acid sequence that is at least 90% identical to the following:

[0427] (1) SEQ ID NO:211, SEQ ID NO:12 and SEQ ID NO:212, respectively; or

[0428] (2) 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 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.

[0429] 27. The multispecific antibody according to embodiment 26, wherein the first heavy chain, the light chain and the second heavy chain each comprise the amino acid sequence of the following:

[0430] (1) SEQ ID NO:211, SEQ ID NO:12 and SEQ ID NO:212, respectively; or

[0431] (2) are SEQ ID NO:213, SEQ ID NO:12, and SEQ ID NO:214, respectively.

[0432] 28. An isolated nucleic acid encoding a multispecific antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 27.

[0433] 29. A vector comprising the isolated nucleic acid according to embodiment 28.

[0434] 30. A host cell comprising the isolated nucleic acid according to embodiment 28 or the vector according to embodiment 29.

[0435] 31. A method of producing a multispecific antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell according to embodiment 30 under conditions for producing the multispecific antibody or an antigen-binding fragment thereof, and recovering the multispecific antibody or an antigen-binding fragment thereof.

[0436] 32. A pharmaceutical composition comprising a multispecific antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 27 and a pharmaceutically acceptable carrier.

[0437] 33. A method of treating or detecting a disorder, preferably cancer, in a subject in need thereof, the method comprising administering to the subject a multispecific antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 27 or the pharmaceutical composition according to embodiment 32.

[0438] 34. The method according to embodiment 33, wherein the disease or disorder is a HER2-associated disease or disorder.

[0439] 35. The method according to embodiment 34, wherein the disease or disorder is brain metastasis.

[0440] 36. A pharmaceutical composition comprising the isolated nucleic acid according to embodiment 28, the vector according to embodiment 29, or the host cell according to embodiment 30 and a pharmaceutically acceptable carrier.

[0441] 37. A method of treating or detecting a disorder, preferably cancer, in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition according to embodiment 36.

[0442] 38. The method according to embodiment 37, wherein the disease or disorder is a HER2-associated disease or disorder.

[0443] 39. The method according to embodiment 37, wherein the disease or disorder is brain metastasis.

[0444] Examples

[0445] The following is a description of the various methods and materials used in the study, and is provided to enable a person of ordinary skill in the art to make and use the full disclosure and description of the present disclosure, and is not intended to limit the scope that the inventors consider to be their disclosure, nor is it intended to represent all of the experiments that are executable and are performed below. It should be understood that the exemplary descriptions written in the present tense do not necessarily have to be performed, but these descriptions can be performed to generate data and the like associated with the teachings of the present disclosure. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, percentages, etc.), but some experimental errors and deviations should be considered.

[0446] Example 1. Materials and Methods

[0447] TfR Antibody Generation and Screening

[0448] Transgenic rodents were immunized with hTfR protein according to a multi-site repeated immunization (RIMMS) protocol. Serum titers were evaluated by ELISA to select rodents for fusion. Lymph nodes were harvested from serum-positive rodents and fused with myeloma cells using standard methods to generate hybridomas. Supernatant hybridomas were screened by MSD for binding to proteins, TfR-expressing cells, and human brain endothelial cells. Positive clones were further evaluated for internalization using brain endothelial cells, cross-reactivity with human and cynomolgus monkey proteins, and competition with transferrin (data not shown). TfR clones that bind to human and / or cynomolgus monkey TfR, internalize, and do not compete for transferrin binding were selected for variable sequence recovery and converted into scFv to generate TEM mAb for further characterization.

[0449] Bispecific TEM antibody production and characterization

[0450] Anti-HER2 and anti-TfR were used to generate bispecific antibodies using the knob-into-hole heterodimerization technique (Ridgway et al., 1996, Protein Eng., Vol. 9, pp. 617–621). The anti-HER2 conjugate was prepared as a bivalent Fab, monovalent Fab, or monovalent scFv at the N-terminus of Fc, whereas the anti-TfR conjugate was prepared as a monovalent scFv to be linked to the C-terminus of one heavy-chain Fc via a (G4S)4 linker. In addition to the knob and hole mutations in Fc, the antibody also contains mutations in Fc that eliminate effector function [L234A / L235A / D265S (AAS)] and enhance FcRn binding at acidic pH [M252Y / S254T / T256E (YTE)] for half-life extension (HLE). According to the manufacturer's recommendations, the constructed TEM mAb was expressed in ExpiCHO-S cells by transient transfection with purified plasmid DNA. The harvested cell culture supernatant was 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. TM The cells were obtained from ATCC as BT-474 (HTB-20), BT-474 clone 5 (CRL-3247), HCC-1954 (CRL-2338), and MDA-MB-361 (HTB-27). The cells were cultured in RPMI supplemented with 10% low IgG FBS and 1% non-essential amino acids (for BT474 only). For imaging studies, the cells were transduced with

[0451] Table 4. TEM Antibodies

[0452]

[0453] Culture and Maintenance of HER2 - Expressing Cancer Cells 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). MDA-MB-361, BT474, and HCC-1954 cells were detached with Accutase, washed twice with cold DPBS, and resuspended in cold binding buffer (BD binding buffer with 2 mM EDTA) at a concentration of 2x10

[0454] Antibodies Bound to Cells : 6cells / mL, and then 50 μL / well was added in a 96-well plate. 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. Subsequently, the wells were washed with 100 μL / well of cold DPBS and centrifuged at 300 xg for 5 minutes to remove the supernatant. Next, the pellet was resuspended in 100 μL / well of LIVE / DEAD Fixable Dead Cell Stain (Invitrogen, 1 / 1,000 dilution) in cold DPBS and incubated on ice for 30 minutes. The cells were washed two more times in the above binding buffer, resuspended in a final volume of 50 μL / well, and read on an iQue flow cytometer (Sartorius).

[0455] Human PBMC - Derived Macrophage Cultures : Human macrophages were differentiated from a large number of healthy human PBMCs (donor 140458, HemaCare). Briefly, the 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 to 8 days until M0 macrophages were generated. An additional volume of complete growth medium was added to the PBMC culture every two days. Non-adherent cells were removed on days 6 to 8. Subsequently, the M0 macrophages were polarized into M2a macrophages by re-applying 20 ng / mL IL-4 and 50 ng / mL M-CSF for 2 days.

[0456] Cytotoxicity Assays : Before co-culturing with PBMC-derived M2a macrophages, NucLight Red-labeled target cells (BT474) were removed, washed, and incubated with TEM mAb or control mAb at a final concentration of 80 nM at 37 °C for 30 minutes. 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 the test article (target and effector only), and controls for the cytostatic effect of the molecule (target and antibody only). The co-cultures were observed by imaging on an Opera Phenix Plus high-throughput screening system over 7 days and imaged every 24 hours. Target cell loss was determined by quantifying the NucLight Red area of each image in Columbus software.

[0457] Human iPSC - Derived Microglia Cultures: Human microglia were derived from epithelial-derived iPSCs (IPSC0028, male, Sigma) and processed as previously described {Haenseler, 2017#607}. Briefly, iPSCs were plated into Aggrewell 800 plates and allowed to form embryoid bodies (EBs) to recapitulate microglial development in the embryo. Macrophage precursors were generated by culturing EBs in mTESR1 for 3 days, which contained bone morphogenetic protein 4 (BMP4, 50 ng / mL), vascular endothelial growth factor (VEGF, 50 ng / mL), and stem cell factor (SCF, 25 ng / mL). The EBs were then harvested and transferred to six-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. Secreted macrophage precursors were collected from the supernatant and plated at 20,000 cells / well into 96-well plates, where they were allowed to mature 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).

[0458] pH - rodo Cell Phagocytosis : Target cell lines were labeled with red according to the manufacturer's instructions (Essen Biosciences, 4649). Before co-culturing with iPSC-derived microglia at various E:T ratios, the labeled cells were opsonized with TEM mAb or control mAb for 30 minutes at 37 °C. Microglia co-cultures were monitored once every 90 minutes in SX5 imaging, and phagocytosis was measured as the total pHrodoRed area (μm 2 / image) and total pHrodoRed integrated intensity (RCU × μm 2 / image).

[0459] Co - Culture Killing Assays: Prior to co - culturing with iPSC - derived microglia, the NucLight Red - labeled target cell line was isolated, washed and incubated with TEM mAb or control mAb at 37 °C for 30 minutes. Co - cultures were observed by imaging in SX5. Target cell loss was determined by quantification of the NucLight Red area in each image.

[0460] Non - Human Primate Cynomolgus Monkey PK Study

[0461] Study Design : TEM and control IgG1 mAb were administered to cynomolgus monkeys at 10 mg / kg by slow - bolus IV injection. Blood for PK was collected at 1 h, 6 h, 24 h, 72 h, and 168 h post - dosing and processed into serum by the test facility laboratory protocol. To collect brain tissue, cynomolgus monkeys were placed under deep anesthesia and a final blood draw was performed. After the final blood collection, animals were euthanized at 72 h and 168 h (n = 2 at each time point), and upper body perfusion was performed by perfusing cold saline solution at 250 mL / min for at least 5 minutes according to the test facility standard operating procedure (SOP). Approximately 200 mg of tissue was isolated from predetermined brain locations (frontal lobe, hippocampus, and temporal lobe), snap - frozen in liquid nitrogen and stored at - 70 °C until capillary depletion processing and tissue homogenization.

[0462] Brain Tissue Preparation : Each right / left hemisphere was 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 modified DPBS buffer (containing protease inhibitor (Pierce; A32955)) at a calculated volume (2.5 μL buffer / 1 mg tissue) and transferred to Lysing Matrix D tubes (MP Biomedicals TM ; 6913 - 100). Total cell suspensions were generated by homogenizing the tissue at 2.8 m / s for 15 seconds with a Bead Ruptor 24 Elite (OmniInternational). The total cell suspension was transferred to a new tube and mixed with an equal volume of dextran buffer (Sigma; 31397) to a final dextran concentration of 13%. The dextran - containing cell suspension was centrifuged at 2,000 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 radioimmunoprecipitation assay (RIPA) lysis buffer (Millipore TM; in (20–188). The capillary-depleted samples were vortexed thoroughly with lysis buffer and centrifuged at 14,000 rpm for 30 minutes at 4 °C, and the supernatant was collected for analysis. The protein concentration of the treated brain tissue lysates was measured using a BCA Protein Assay Kit (Pierce TM ; 23227), and the final sample lysates were normalized to a total protein concentration of 7 mg / mL prior to immunoassay determination.

[0463] PK Assays : The concentrations of TEM and control IgG1 mAb in NHP brain tissue and plasma were determined using MSD immunoassay on a streptavidin-coated plate. Fresh standard curves were prepared by serially diluting each mAb in assay diluent containing primary mouse matrix (50% brain tissue lysate or 10% pooled plasma). Frozen quality controls prepared in 100% primary mouse matrix were diluted and tested in each assay. Briefly, the plates were blocked with a PBS solution of 1% bovine serum albumin for 30 minutes and washed with a PBS solution of 0.05% Tween-20. The master mixture 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 volume ratio in the assay plates and incubated for 1 hour with shaking. The raw data signals were read on a Meso Sector S 600 imager and analyzed using Watson LIMS software (Thermo Scientific). Data regression was performed using a 1 / Y 2 weighted five-parameter logistic fit. The quantifiable curve range for the brain tissue lysate assay was 1 ng / mL to 512 ng / mL, and the minimum required sample dilution for the treated tissue was 1:2. To calculate the total tissue drug concentration in the brain, the mAb concentration in the brain lysate was multiplied by the total volume used to process to the final 7 mg / mL normalized sample. The total tissue drug concentration (ng) was then divided by the wet weight of the brain tissue to determine the drug:tissue (ng:mg) ratio. The quantifiable curve range for the plasma assay was 2 ng / mL to 512 ng / mL, and the minimum required sample dilution was 1:10. The assay had a sensitivity limit of 2 ng / mL in brain tissue lysates and 10 ng / mL in plasma.

[0464] Example 2. Antibody Binding in HER2 - Positive Cancer Cells

[0465] It is well known that TfR-based antibody therapies that require Fc activity result in the depletion of reticulocytes in the periphery. This can be prevented by introducing mutations that impair effector function ("silencing") or by spatial blockade of simultaneous TfR and Fc receptor engagement. While the use of silent Fc can mitigate safety concerns, the inability to bind FcγR may also limit therapeutic efficacy. Therefore, it is necessary to replace Fc-mediated effector function with a different and equally effective mechanism to ensure therapeutic efficacy. It has been found that bispecific antibodies containing an endocytic trafficking receptor-binding arm with optimized affinity for receptor-mediated transport (RMT) and myeloid cell engagement and another high-affinity target antigen-binding arm will effectively cross the BBB and achieve antigen clearance by utilizing an Fc-independent mechanism (hereinafter referred to as non-classical phagocytosis (NCP))( Figure 2A ).

[0466] To determine the ability of TEM mAbs to mediate the phagocytic clearance of cells, the anti-human epidermal growth factor receptor 2 (HER2)-binding antibody trastuzumab (a bivalent HER2-TfR k -mut) without Fc-mediated effector function and a bispecific molecule containing both trastuzumab and pertuzumab (a bispecific HER2-TfR k -mut G1) without Fc-mediated effector function were used to generate TEM constructs( Figure 2B ). Both trastuzumab and pertuzumab have been extensively characterized in vitro and in vivo and are FDA-approved drugs.

[0467] Four trastuzumab-based TfR-TEM mAbs using the AAS / YTE Fc backbone were generated to test the effect of anti-HER2 valence and anti-TfR affinity on cancer cell clearance in myeloid cells. Binding of 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 form having a net increase in binding compared to the bivalent HER2 mAb( Figures 3A - 3B ). Binding of monovalent, bivalent, and bispecific antibodies was tested in the HCC1954 (HER2-amplified) cell line. All antibodies showed dose-dependent binding, with the monovalent and bispecific forms having a net increase in binding over the bivalent HER2 mAb( Figure 3C ).

[0468] Example 3. TEM Antibodies Promote Non - Classical Phagocytosis

[0469] To further test anti-HER2 TEM, breast cancer cell lines BT474 and MDA-MB-361 expressing HER2 were selected and engineered to stably express red fluorescent protein (mKATE2) in the nucleus by lentiviral transduction and used to generate tumor spheroids, which are widely considered a more physiological model of tumor growth compared to 2D cultures. In the presence of anti-HER2 TEM or control, the spheroids were co-cultured with human induced pluripotent stem cell (iPSC)-derived microglia (iMG), and the fluorescence signal was monitored over time for 14 days ( Figures 4A - 4C ). For both cell lines, trastuzumab led to a decrease in the red fluorescence area over time, indicating tumor cell killing. Interestingly, the single-HER2-TFR-J-mut and single-HER2-TFR-K-mut conditions showed a strong decrease in the fluorescence area over time, while the two bivalent HER2-TFR-mut TEM mAbs had only moderate cytotoxic effects on the cancer cell population. For both the monovalent mAb and bivalent mAb groups, a relationship could be established between the TfR binding affinity and the fluorescence area, where the TFR-K-mut form was more effective in terms of fluorescence reduction than the higher affinity form.

[0470] To determine whether the observed decrease in cancer cell number was due to non-classical phagocytosis (NCP) or due to inhibition of signaling, the experiment was repeated using a stable RFP-transduced subline of BT474 clone 5, a breast cancer cell line with high expression of HER2 and mutant PI3K, which renders it resistant to the signaling inhibitory effects of trastuzumab. Figure 4C All TEM mAbs were shown to cause a similar inhibition of the BT474 clone 5 NucLight Red (NR) spheroid area as trastuzumab. Statistical analysis of the spheroid area on day 14 demonstrated that for both BT474 NR and BT474 clone 5 NR cells, single-HER2-TfR-K-mut was as effective as trastuzumab in achieving complete killing of the spheroids ( Figures 4D - 4F ), and for MDA-MB-361 NR cells, the tested TEM mAbs had the strongest effect, although slightly less effective than trastuzumab ( Figure 4D ). In summary, these results demonstrate that anti-HER2 TEM has a strong cytotoxic effect on HER2+ tumor cells due to induction of microglial NCP and is independent of signaling inhibition.

[0471] Further evidence for the induction of NCP by TEM mAbs in human induced pluripotent stem cell (iPSC)-derived microglia (iMG) was established when BT474 clone 5 cells loaded with pHrodo Red were co-cultured with iMG and the appearance of red fluorescence was monitored.Figures 5A - 5D )。In addition to causing ADCC and HER2 / HER2 signal transduction inhibition, trastuzumab is also a strong promoter of phagocytosis by iMG. The single-HER2-TfR-K-mut mAb also had a significant effect in inducing phagocytosis of BT474 clone 5NR cells, while the bivalent TEM mAb had no effect on the appearance of the pHrodo red signal ( Figures 5A - 5B ). Notably, the red fluorescence intensity kinetics of trastuzumab differed from those of the single-HER2-K-mut mAb. Trastuzumab showed peak intensity 4 to 6 hours after the start of the experiment and then decreased to a plateau, while the single-HER2-TfR-K-mut mAb slowly increased the red fluorescence intensity over time, showed no signs of saturation, and reached the trastuzumab level near the end of the experiment. When analyzing the total phagocytosis induced by each mAb, the areas under the curve of trastuzumab and the single-HER2-K-mut mAb were not statistically different ( Figures 5C - 5D ), demonstrating comparable in vitro potency between the single-HER2-K-mut mAb and trastuzumab.

[0472] Additional monovalent (BBBB1627), bivalent (BBBB1638), and bispecific (BBBB1598) TEM antibodies were tested in the MDA-MB-361 cell line. MDA-MB-361 spheroids were co-cultured with iMG and the contraction of the spheroids was monitored. The TEMs were compared to trastuzumab, isotype control, and untreated cells. Trastuzumab induced phagocytosis of MDA-MB-361 cells at both 80 nM ( Figure 6A ) and 8 nM ( Figure 6B ). In 2D co-cultures, all TEM mAbs also caused inhibition of the MDA-MB-361 cell area, similar to that of trastuzumab ( Figure 6C ).

[0473] Next, it was determined whether iMG released pro-inflammatory cytokines during bivalent HER2-TfR k -mut G1 and bispecific HER2-TfR k -mut G1 NCP. Samples of conditioned medium were taken from co-cultures of iMG and MDA-MB-361 or HCC1954 at 20 hours and 44 hours for measurement of IL-10, TNF-α, and IL-1β ( Figures 7A - 7B)。In the HCC1954 co-cultures, as expected, the concentrations of trastuzumab-induced TNF-α, IL-1β, and IL-10 secretion were significantly higher at both time points than those of the antibody-free control. Anti-HER2 TEM did not induce these cytokines. Similar effects were observed for TNF-α and IL-10 concentrations in the MDA-MB-361 co-cultures; no differences were observed in IL-1β levels because this cytokine was not produced under the tested conditions in this cell line. These results recapitulate the findings from the microglial phagocytosis experiments using TEM, namely that, in contrast to the mechanism of action of trastuzumab IgG1, TEM-induced NCP does not trigger the release of pro-inflammatory cytokines.

[0474] Example 4. TEM Enables Non - Classical Phagocytosis of Live Target Cells by Macrophages

[0475] Next, the ability of anti-HER2 TEM to promote the clearance of HER2+ tumor cells by human PBMCs was tested. During the 7-day co-culture, strong cytotoxicity mediated by TEM against the trastuzumab-resistant BT474 clone 5 cell line was observed ( Figure 8A ). Although the TEM molecule acts slower compared to trastuzumab, effective inhibition of tumor growth by TEM was observed at the end of the study. Monovalent HER2-TfR k -mut G1 demonstrated stronger NCP-mediated cytotoxicity than monovalent HER2-TfR j -mut G1.

[0476] Since on average donor PBMCs contain approximately 10%-20% CD14+ monocytes capable of phagocytosis, TEM-mediated cytotoxicity in co-culture with purified differentiated macrophages was next tested ( Figures 8B - 8C ). Here, differentiated M2a macrophages were used because the M2a subtype is known to exert anti-inflammatory pro-tumorigenic characteristics and promote tumor growth (Yao et al., 2019, Front Immunol, Vol. 10, p. 792; Yang et al., 2020, Trends in Pharmacological Sciences, Vol. 41, pp. 701–714). Significant inhibition of the BT474 tumor cell line triggered by TEMmAb was observed during the 7-day co-culture with M2a macrophages. As captured by representative images of macrophage and tumor co-cultures, monovalent HER2-TfR k -mut G1 TEM demonstrated effective elimination of the tumor, similar to that observed with trastuzumab at a lower effector-to-target ratio (E:T) of 3:1.

[0477] To further confirm that the killing mechanism is mediated by phagocytosis, M2a macrophages were co-cultured with the pHrodo-labeled tumor line BT474 clone 5. After phagosome formation, pH changes will trigger a strong pHrodo signal, which reflects the active ingestion and digestion of tumor cells by macrophages. Quantification of phagocytic events within macrophages demonstrated efficient phagocytosis in the presence of both trastuzumab and TEM molecules. Notably, the monovalent HER2-TfR k -mut G1 that demonstrated robust killing in the cytotoxicity assay showed an even higher ability to trigger phagocytosis ( Figure 8D ). Similar to co-culture with microglia, trastuzumab and TEM mAb demonstrated different phagocytosis kinetics. A peak in trastuzumab phagocytosis was observed around 2 hours in co-culture and then declined, while TEM mAb exhibited a gradual and sustained phagocytosis. This data indicates that TEM does not trigger macrophage depletion observed in the presence of trastuzumab. Further analysis of total phagocytosis, which reflects the sum of phagocytic events at all time points, showed that phagocytosis triggered by the monovalent HER2-TfR k -mut G1 was comparable to trastuzumab ( Figure 8E ). Finally, representative images of M2a phagocytosis demonstrated enhanced phagocytic events over time in the presence of monovalent HER2-TfR k -mut G1 TEM compared to minimal phagocytosis in the absence of antibody and onset of maximal phagocytosis after 2 hours of co-culture in the presence of trastuzumab (Figure 8F).

[0478] In summary, these data demonstrate that anti-HER2 TEM enables microglia to clear tumor cells in an antigen-dependent, non-Fc effector function-mediated manner. The non-classical phagocytic mechanism was shown to be at least (if not more) effective than trastuzumab and can be transferred to peripheral immune cells and macrophages.

[0479] Example 5. TEM Results in Enhanced Brain Delivery in Cynomolgus Monkeys

[0480] NHPs administered with TEM mAb (BBBB1598 and BBBB1627) were compared to the group of NHPs injected with trastuzumab. Brain mAb concentrations were measured at 72 hours and 168 hours after IV administration (10 mg / kg) in eight perfused and capillary-depleted brain regions. Compared to trastuzumab, the presence of TEM increased the mAb concentration in the frontal lobe, hippocampus, and temporal lobe by a fold increase ranging from approximately 5x to 7x ( Figure 9 ). These data demonstrate that anti-HER2 TEM results in increased mAb uptake in the brain compared to trastuzumab.

[0481] Example 6. TEM PET / CT Imaging Study

[0482] The in vivo distribution of [Zr89]-DFO*-TfRxHER2(BBBB1598)IgG antibody was evaluated in human TfR knock-in female mice (Biocytogen, catalog #110861) (TfR or huTfR KI) and female C57BL6 mice (Jax) mice (B6 control or BL6). On the day of the imaging experiment, the tracer precursor was labeled with [Zr89]. Shortly thereafter, quality control (QC) of the radiochemical purity and specific activity of the compound was measured.

[0483] Animal body weights were measured using a Mettler Toledo balance. The animals were then anesthetized in an anesthesia chamber using isoflurane with oxygen (3.0% - 4.5% for induction and 1.0% - 3.0% for maintenance) via Somni AMD-3. After ensuring that the animals maintained a stable breathing pattern, the animals were placed on an operating table for injection, and a heating pad was used to maintain body temperature.

[0484] The mice were divided into three groups (A, B, and C). Animals in the same group were injected with the PET tracer at approximately 2-minute intervals. At the designated time points, the mice were placed on a 4-mouse stage of a Sofie GNEXT PET / CT scanner (Sofie, Culver City, CA, USA), and a heating pad was used to maintain body temperature and for respiratory monitoring. The imaging protocol started with a 30-minute static PET scan, followed by a 1-minute standard CT scan acquisition protocol.

[0485] Acquisition and Reconstruction Parameters :

[0486] PET / CT studies were performed on days 1, 5, and 7 after tracer injection. The entire body was centered on the axial FOV of the scanner to maximize sensitivity and resolution. The CT scan was used for attenuation correction, anatomical imaging, and scatter correction of the PET image. The PET scan energy window was set between 350 KeV and 650 KeV, and the timing window was 3.438 ns. Emission data were collected in list mode for 30 minutes. The PET images were reconstructed into a single frame using the iterative 2-dimensional ordered subset expectation maximization (OSEM2D) algorithm (4 OSEM2D – iterations, with Fourier rebinning). The data were reconstructed into images with a 128×128 matrix size.

[0487] Results :

[0488] Zr89-DFO*-HER2xTfR (HER2xTfR) Ab showed enhanced brain uptake in human TfR knock-in mice compared to the B6 control (Figures 10A - 10C )。The Zr89-DFO*-HER2 (HER2) Ab showed higher peripheral organ uptake (liver, kidney, and spleen) compared to Zr89-DFO*-HER2xTfR (data not shown). The brain-to-heart ratio of the standardized uptake value was significantly increased for HER2xTfR Ab in human TfR knock-in mice compared to that in B6 controls, and this increase was not observed with the use of HER2 Ab ( Figures 11A - 11D ).

Claims

1. A multispecific antibody or an antigen-binding fragment thereof, said 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), wherein: (4) The first antigen-binding region comprises: A first heavy-chain variable region (VH1), said VH1 comprising heavy-chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, which comprise the amino acid sequences of SEQ ID NO:5, 6, and 7 respectively; and a first light-chain variable region (VL1), said VL1 comprising light-chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NO:8, 9, and 10 respectively; (5) The second antigen-binding region comprises: A second heavy-chain variable region (VH2), said VH2 comprising heavy-chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, which comprise the amino acid sequences of SEQ ID NO:13, 14, and 15 respectively; and a second light-chain variable region (VL2), said VL2 comprising light-chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NO:16, 17, and 18 respectively; and (6) The third antigen-binding region comprises a first single-chain variable fragment (scFv1), said scFv1 having: a third heavy-chain variable region (VH3) comprising heavy-chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and a third light-chain variable region (VL3) comprising light-chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 have any of the amino acid sequences in Table 2.

2. The multispecific antibody or antigen-binding fragment thereof according to claim 2, wherein: (4) said VH1 comprises the same amino acid sequence as the VH of the HC sequence as shown in SEQ ID NO:1; and said VL1 comprises the same amino acid sequence as the VL of the LC sequence as shown in SEQ ID NO:2; (5) said VH2 comprises the same amino acid sequence as the VH of the HC sequence as shown in SEQ ID NO:11; and said VL2 comprises the same amino acid sequence as the VL of the LC sequence as shown in SEQ ID NO:12; and (6) said VH3 and said VL3 comprise the same amino acid sequences as the corresponding VH and VL of the scFv as shown in the following sequences: (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)SED ID NO:

204.

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

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

5. The multispecific antibody or antigen-binding fragment thereof according to claim 3, wherein the multispecific antibody or antigen-binding fragment thereof further comprises a second Fc region (Fc2).

6. The multispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the multispecific antibody or antigen-binding fragment thereof comprises: (d) A first heavy chain (HC1) comprising the VH1 and a first heavy chain constant region containing 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), wherein the HC2 comprises the scFv1 and a second heavy chain constant region containing a second Fc region (Fc2).

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

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

9. The multispecific antibody or antigen-binding fragment thereof according to claim 7, wherein the multispecific antibody or antigen-binding fragment thereof further comprises a second Fc region (Fc2).

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

11. The multispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the multispecific antibody or antigen-binding fragment thereof comprises the first antigen-binding region, the second antigen-binding region, and the third antigen-binding region.

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

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

14. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein said scFv1 and / or scFv2 comprises at least one of the following: (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 according to claim 14, wherein said scFv1 and scFv2 each independently comprise said first disulfide bond and said second disulfide bond.

16. The multispecific antibody or antigen-binding fragment thereof according to claim 13, wherein said 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 the preceding claims, wherein said 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.

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

19. The multispecific antibody or antigen-binding fragment thereof according to claim 18, wherein said 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; 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, and wherein the amino acid residues are numbered according to the EU index as described in Kabat.

20. The multispecific antibody or antigen-binding fragment thereof according to 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. The multispecific antibody or antigen-binding fragment thereof according to claim 18, wherein each of said Fc1 and said Fc2 comprises one or more knob and hole mutations.

22. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having an amino acid modification that enhances the binding of the multispecific antibody or antigen-binding fragment thereof to the neonatal Fc receptor (FcRn), preferably the amino acid modification enhances the binding at acidic pH, more preferably the Fc domain has the M252Y / S254T / T256E (YTE) mutation, and wherein the amino acid residues are numbered according to the EU index as described in Kabat.

23. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having an amino acid modification that reduces or eliminates 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 amino acid modifications among L234A, L235A, D265S and P331S, and wherein the amino acid residues are numbered according to the EU index as described in Kabat.

24. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having one or more amino acid modifications among M252Y, S254T, T256E, L234A, L235A and D265S, and wherein the amino acid residues are numbered according to the EU index as described in Kabat.

25. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having an amino acid modification that does not reduce or eliminate effector function.

26. A multispecific antibody, the multispecific antibody comprising a first heavy chain, a light chain, and a second heavy chain each having an amino acid sequence that is at least 90% identical to the following: (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; 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 multispecific antibody according to claim 26, wherein the first heavy chain, the light chain, and the second heavy chain each comprise the amino acid sequence of the following: (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.

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

29. A vector comprising the isolated nucleic acid according to claim 28.

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

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

32. A pharmaceutical composition comprising the multispecific antibody or an antigen-binding fragment thereof according to any one of claims 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, the method comprising administering to the subject the multispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 27 or the pharmaceutical composition according to claim 32.

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

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

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