Fully human monoclonal antibody against human HER-2

Abstract

The present invention describes antibodies, particularly monoclonal antibodies, that specifically bind to human HER2 and are useful in treating cancer in patients.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Application No. 63 / 416,270, filed October 24, 2022, which is incorporated by reference in its entirety. Technical Field The present disclosure relates to antibodies, including fully human monoclonal antibodies (mAbs), that specifically bind and internalize human epidermal growth factor-2, also known as HER-2, as well as methods of making the antibodies (e.g., cell lines) and methods of using the antibodies (e.g., treating cancer). [Background technology]

[0002] The human epidermal growth factor receptor (HER) family of receptors plays a pivotal role in the pathogenesis of several human cancers. They mediate cell proliferation and differentiation by acting through multiple signaling pathways. This superfamily consists of four major members: HER1 (also known as epidermal growth factor receptor (EGFR)), HER2, HER3, and HER4 (also known as erbB1, erbB2, ErbB3, and ErbB4). All of these members are characterized by a cysteine-rich extracellular domain containing the ligand-binding site, a transmembrane lipophilic region, and an intracellular domain with tyrosine kinase enzymatic activity. HER receptors exist as monomers on the cell surface. Upon ligand binding, the receptors form homo- or heterodimers with other HER members, leading to phosphorylation. HER2 is a 125-amino acid, 185-kDa transmembrane glycoprotein located on chromosome 17q12. HER2 is overexpressed in many tissues, promoting excessive / uncontrolled proliferation and tumorigenesis. HER2 has no known direct activating ligand and is constitutively activated or activated by heterodimerization with other HER members, leading to autophosphorylation of tyrosine residues within the intracellular domain and initiating various signaling pathways, such as MAP-K and PI3K, which can result in cell proliferation, survival, migration, and angiogenesis.

[0003] HER2 is overexpressed in several cancers. Since its discovery that HER2 induces mammary gland carcinogenesis in vitro and in vivo, most studies have focused on breast cancer. Overexpression, with or without gene amplification, is observed in 15-30% of breast cancers and has both prognostic and predictive implications. HER2 overexpression and amplification are associated with decreased disease-free and overall survival, resistance to certain hormonal agents, and an increased risk of brain metastasis. In addition to overexpression in breast cancer, HER2 overexpression has also been reported in gastric, esophageal, ovarian, and endometrial cancers. In lung cancer, overexpression and HER2 mutations have been reported in 30% of adenocarcinomas. Similar findings have also been reported in bladder cancer. However, clinical trials of HER2 therapy for lung and bladder cancers have so far met with limited success.

[0004] Two categories of treatments targeting HER2 and its biological activity have been developed, approved by the FDA, and used as standard of care: monoclonal antibodies against HER2, and small molecule tyrosine kinase inhibitors that target HER2 and EGFR, such as lapatinib and neratinib, and all members of the HER family, such as afatinib. Trastuzumab, also known as Herceptin, is a humanized anti-HER2 monoclonal antibody that binds to domain IV of the extracellular segment of HER2 and inhibits its signal transduction. Its mechanisms of action include inhibition of HER2-dependent cell signaling, inhibition of the PI3K-AKT pathway, inhibition of angiogenesis, and antibody-dependent cellular cytotoxicity. Trastuzumab has been approved along with a companion diagnostic for patients with Her2-overexpressing breast cancer that is 3+ by immunohistochemistry (IHC) or positive by fluorescence in situ hybridization (FISH). It is administered in combination with standard chemotherapy. Trastuzumab is also approved for the treatment of HER2-overexpressing metastatic gastric cancer or gastroesophageal junction adenocarcinoma. Pertuzumab is a humanized anti-HER2 monoclonal antibody that blocks HER2 receptor activation by inhibiting HER2 receptor dimerization. It acts at a different ligand binding site than trastuzumab. It is approved in combination with trastuzumab and docetaxel for patients with HER2-positive metastatic breast cancer. Based on the fact that the anti-HER2 antibody trastuzumab is internalized, allowing it to deliver a cytotoxic payload to cells and kill them, the antibody-drug conjugate Ado-trastuzumab-emtansine was developed. Ado-trastuzumab-emtansine (Kadcyla) consists of trastuzumab linked to the drug mertansine DM1 (TDM1). TDM1 has been approved as a single agent for the treatment of patients with HER2-positive metastatic breast cancer who have previously received trastuzumab and a taxane.

[0005] The antibody-drug conjugate (fam-trastuzumab-deruxtecan-NHKI (Enhertz)) is another antibody-drug conjugate that uses trastuzumab to deliver the topoisomerase inhibitor deruxtecan. It is approved for unresectable or metastatic HER2-positive breast cancer after prior anti-HER2-based therapy and for locally advanced or metastatic HER2-positive gastric or gastroesophageal junction adenocarcinoma after prior trastuzumab-based therapy. Most anti-HER2 targeted therapies under development are based on trastuzumab and pertuzumab antibodies. Both of these antibodies are derived from mouse monoclonal antibodies that have been humanized by molecular biological recombinant techniques. Therefore, the art has recognized an opportunity to develop a new class of anti-Her2 antibodies, particularly fully human anti-HER2 monoclonal antibodies that can be developed by immunizing fully human mice. Such anti-HER2 antibodies, as well as methods for their production and use, are provided by the present disclosure. Summary of the Invention

[0006] The present invention provides monoclonal antibodies (mAbs), including humanized anti-HER2 antibodies, and antigen-binding fragments, such as polypeptides, comprising complementarity-determining regions (CDRs) that bind to HER-2. In some embodiments, the present disclosure describes isolated antibodies or antigen-binding fragments thereof that bind to human HER2 and compete with antibodies 11D7, 2A2, 12E3, 2D7, 3G7, 8C1, 2A1, 7F9, 9E4, 11C9, or 12A6 for binding; heavy and light chain variable regions thereof; and / or polypeptides comprising the CDRs of such antibodies (see Tables 1 and 2); and / or derivatives thereof (e.g., comprising conservative amino acid substitutions therefor (e.g., see Table 4)), all of which are considered equivalents thereof. In particular, the mAbs herein include anti-human HER2 antibodies having the amino acid sequences of the CDRs listed in Table 1. In some embodiments, the present disclosure provides fully human anti-HER2 antibodies that internalize into cells expressing HER2 and compete with trastuzumab for binding to HER2, as well as methods of using such antibodies for their neutralization and internalization properties. In preferred embodiments, such antibodies include those referred to herein as 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1, and / or CDRs contained in such antibodies (see Table 1); heavy and light chain variable regions thereof (see Table 3); and / or derivatives thereof (e.g., containing conservative amino acid substitutions therein (e.g., see Table 4)). In some embodiments, the present disclosure provides fully human anti-HER2 antibodies that internalize and do not compete with trastuzumab for binding to the HER2 receptor, as well as methods for examining their internalization and biological properties by examining their ability to be cytotoxic (e.g., kill cells) as antibody-drug conjugates. In preferred embodiments, such antibodies include those referred to herein as 2A1, 7F9, 9E4, 11C9, 12A6; those comprising the heavy and light chain variable regions thereof (see Table 3); and / or those comprising the CDRs of Table 2; and / or derivatives thereof (e.g., including conservative amino acid substitutions thereof (see, e.g., Table 4)).These antibodies may further comprise the complete heavy and / or light chains of the antibodies set forth in Table 3, and / or derivatives thereof comprising the CDRs set forth in Tables 1 and 2. In some preferred embodiments, such antibodies may have the amino acid sequence of the variable heavy ("VH") or variable light ("VL") chain polypeptide (VH or VL "chain," respectively) set forth below for that antibody, or their equivalents as set forth in Table 3. In some embodiments, the isolated antibodies, or antigen-binding fragments described herein, have a K sufficient to provide sufficient (e.g., detectable) binding to human HER2, as determined using any suitable method used in the art, such as, but not limited to, a Biolayer-Interferometry scouting assay using an Octet Red 96 instrument (see, e.g., the Examples section herein), or as measured by any assay available to one of skill in the art. D , for example, about 10 -8 Molar (M) ~ approx. 10 -10 Molar (M), preferably about 3.6 x 10 -9 M or any value therebetween.

[0007] In some embodiments, the present disclosure provides nucleotide sequences encoding specific amino acid sequences of the 11D7, 2A2, 12E3, 2D7, 3G7, 8C1, 2A1, 7F9, 9E4, 11C9, or 12A6 antibody (or functional fragments thereof, such as CDRs and / or variable regions), and / or equivalents, which can be readily derived from the amino acid sequence of any of SEQ ID NOs: 62-83 and the information provided in Table 4, and using functional and other assays disclosed herein and / or otherwise available to one of skill in the art. In some embodiments, the disclosure also provides expression vectors comprising isolated nucleic acids comprising and / or consisting of such nucleotide sequences (in preferred embodiments, any of SEQ ID NOs: 62-83 or derivatives thereof), and host cells (e.g., cell lines) comprising such expression vectors. In some embodiments, the present disclosure provides methods of using antibodies and / or fragments thereof (e.g., CDRs) (collectively referred to herein as "antibodies" unless otherwise specified) to treat cancers in which HER2 is implicated. In some embodiments, the present disclosure also provides methods of treating cancer in a patient by identifying a patient having cancer cells that express HER2 and administering to the patient an antibody or antigen-binding fragment as described herein. A variety of human cancers are known to express HER2, including ovarian cancer, breast cancer, multiple myeloma, lung cancer, renal cancer, prostate cancer, hepatocellular carcinoma, uterine cancer, bladder cancer, gallbladder cancer, esophageal cancer, gastric cancer, laryngeal cancer, brain cancer, leukemia, and glioblastoma.

[0008] 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. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting. Additional features will be set forth in part in the description that follows, or may be learned by practice as described herein. The foregoing and other features will become apparent to those skilled in the art upon consideration of the following description of exemplary embodiments. The accompanying drawings and photographs, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description serve to explain the principles of the present invention. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows the blocking activity of antibodies 11D7, 8C1, 2A2, 2D7, 12E3, and 3G7 relative to trastuzumab for binding to HER2. [Figure 2] Antibodies 12E3 and 12A6 were each separately conjugated to DM1, and the two resulting ADCs, 12E3-DM1 and 12A6-DM1, were tested in vitro for their ability to inhibit the growth of HER2-overexpressing breast cancer cells alone or in combination with the HER2-overexpressing cell line AU565. [Figure 3]Antibodies 12E3 and 12A6 were each separately conjugated to DM1, and the two resulting ADCs, 12E3-DM1 and 12A6-DM1, were tested in vitro for their ability to inhibit the growth of HER2-overexpressing breast cancer cells alone or in combination with the HER2-overexpressing cell line SKBR3. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present disclosure relates to antibodies, including, but not limited to, fully human monoclonal antibodies, that specifically bind to human HER2 and can be used to treat cancers such as breast cancer. In some embodiments, the present disclosure provides isolated antibodies that bind to human HER2 and compete for binding with antibodies 11D7, 2A2, 12E3, 2D7, 3G7, 8C1, 2A1, 7F9, 9E4, 11C9, or 12A6, one or more antigen-binding fragments thereof; heavy and light chain variable regions thereof (see Table 3); and / or polypeptides comprising the CDRs of such antibodies (see Tables 1 and 2); and / or derivatives thereof (e.g., containing conservative amino acid substitutions therein (e.g., see Table 4)), all of which are considered equivalents thereof. In particular, the mAbs herein include anti-human HER2 antibodies having the amino acid sequences of the CDRs listed in Table 1. In some embodiments, the present disclosure provides fully human anti-HER2 antibodies that internalize into cells expressing HER2 and compete with trastuzumab for binding to HER2, as well as methods for using such antibodies for their neutralization and internalization properties. In preferred embodiments, such antibodies include those referred to herein as 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1, and / or CDRs contained therein (see Table 1); heavy and light chain variable regions thereof (see Table 3); and / or derivatives thereof (e.g., containing conservative amino acid substitutions therein (e.g., see Table 4)). In some embodiments, the present disclosure provides fully human anti-HER2 antibodies that internalize and do not compete with trastuzumab for binding to the HER2 receptor, as well as methods for examining their internalization properties. In preferred embodiments, such antibodies include those referred to herein as 2A1, 7F9, 9E4, 11C9, 12A6; those comprising the heavy and light chain variable regions thereof (see Table 3); and / or those comprising the CDRs of Table 2; and / or derivatives thereof (e.g., comprising conservative amino acid substitutions thereof (see, e.g., Table 4)). These antibodies may further comprise at least a portion of the heavy and / or light chains (most preferably including these CDRs) of the antibodies shown in Table 3 and / or the complete heavy and / or light chains, and / or derivatives thereof comprising the CDRs shown in Tables 1 and 2.In some preferred embodiments, such antibodies may have the amino acid sequence of the variable heavy chain ("VH") or variable light chain ("VL") polypeptide (VH or VL "chain", respectively) set forth below for that antibody, or their equivalents as set forth in Table 3. In some embodiments, the isolated antibodies, or antigen-binding fragments described herein have an IgG antibody activity of 3.6 x 10- as measured by the Octet assay described below (see, e.g., the Examples section herein), or by any assay available to one of skill in the art. 9 K to mole (M) Dand binds to human HER2 at the CDR(s). In some embodiments, the present disclosure provides nucleotide sequences encoding specific amino acid sequences equivalent to the 11D7, 2A2, 12E3, 2D7, 3G7, 8C1, 2A1, 7F9, 9E4, 11C9, or 12A6 antibody, and which can be readily derived from the amino acid sequences of any of SEQ ID NOS: 62-83 and the information set forth in Table 4, and / or equivalents / derivatives thereof. In some embodiments, the present disclosure also provides expression vectors comprising isolated nucleic acids comprising and / or consisting of such nucleotide sequences (in preferred embodiments, any of SEQ ID NOS: 62-83 or derivatives thereof), as well as host cells (e.g., cell lines) comprising such expression vectors. In some embodiments, the present disclosure provides methods for using the present antibodies and / or fragments thereof (e.g., CDRs) (collectively referred to herein as "antibodies" unless otherwise specified) to treat HER2-mediated cancers. In some embodiments, the present disclosure also provides a method of treating cancer in a patient by identifying a patient having cancer cells that express HER2 and administering to the patient an antibody or antigen-binding fragment as described herein. A variety of human cancers are known to express HER2, including ovarian cancer, breast cancer, multiple myeloma, lung cancer, renal cancer, prostate cancer, hepatocellular carcinoma, uterine cancer, bladder cancer, gallbladder cancer, esophageal cancer, gastric cancer, laryngeal cancer, brain cancer, leukemia, and glioblastoma. The use of combinations of antibodies, such as one or more described herein, with others available to those of skill in the art is also contemplated herein. For example, in some embodiments, these combinations can be identified to provide a statistically significant difference from results (e.g., neutralization assays) obtained using one or more antibodies alone but without the other antibodies. In some embodiments, the combinations exhibit additive and / or preferably synergistic activity.In some embodiments, the combination may include the 11D7, 2A2, 12E3, 2D7, 3G7, 8C1, 2A1, 7F9, 9E4, 11C9, or 12A6 antibody (or a derivative thereof) and trastuzumab, pertuzumab, the antibody-drug conjugate Ado-trastuzumab emtansine (KADCYLA, which consists of trastuzumab linked to the drug mertansine DM1 (TDM1)), and / or the antibody-drug conjugate fam-trastuzumab-deruxtecan-nhki (Enhertu). Mertansine (DM1; N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine) is shown below. [ka] DM1 can be conjugated to an antibody ("mab" in the diagram below) using the linker 4-mercaptovaleric acid as shown below. [ka] DM1 can also be linked to the antibody using 4-(3-mercapto-2,5-dioxo-1-pyrrolidinylmethyl)-cyclohexanecarboxylic acid (SMCC), referred to by the term emtansine, as shown below. [ka] The antibodies of the present disclosure can also be combined with chemotherapeutic agents used in standard treatments, some of which are used in combination with anti-HER2 therapy. The antibodies in such compositions can be different entities, such as two or more different monoclonal antibodies or derivatives thereof, or can be present in the same entity, such as a bifunctional antibody (a single antibody or derivative thereof containing multiple binding specificities). Combinations such as those described herein can also be combined with one or more other agents that may affect immune cell function, such as antibodies against CTLA-4. Those skilled in the art will recognize that many such combinations may be suitable for use as described herein.

[0011] The term "antibody," as used herein, refers to whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. Fully human antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-chain connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated as V) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. L The V domain consists of a V-chain constant region (CL) and a V-chain constant region (CL). H Area and V L The region can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or various cells of the immune system (e.g., effector cells) and factors including the first component (Clq) of the classical complement system. The term "isolated antibody" refers to an antibody and / or fragment thereof that is substantially free of other antibodies and / or fragments thereof having different antigen specificities (e.g., an isolated antibody that specifically binds to HER2 is substantially free of antibodies that specifically bind to antigens other than HER2). However, an isolated antibody that specifically binds to HER2 may cross-react with other antigens, e.g., HER2 from species other than human. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals. The term "antigen-binding portion" or "antigen-binding fragment" of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., human granulin). The antigen-binding function of an antibody may be performed by a fragment of the intact antibody. Examples of binding fragments encompassed by the term antigen-binding portion or antigen-binding fragment of an antibody include V L , V H Fab fragment, a monovalent fragment consisting of the CL and CH1 domains; F(ab)2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bond at the hinge region; V H Fd fragment consisting of the CH1 domain and the V domain of a single antibody arm L Domains and V H Fv fragment consisting of domains; V H Domain or V L Single-domain antibody (dAb) fragments comprise the V domain (Ward et al., 1989 Nature 341:544-546); and isolated complementarity-determining regions (CDRs). Additionally, the two domains V of the Fv fragment are L and VH are encoded by separate genes, but V L Area and V H The domains can be linked by artificial peptide linkers, allowing them to be paired and produced as monovalent molecules (known as single-chain Fvs (scFvs); see, e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies contain one or more antigen-binding portions or fragments of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies (intrabodies), diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The antigen-binding portion of an antibody can be grafted onto a polypeptide-based scaffold such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes a fibronectin polypeptide monobody). The antigen-binding fragment comprises a tandem Fv segment (V) that forms a pair of antigen-binding regions with a complementary light chain polypeptide. H -CH1-V HThe term "chimeric antibody" refers to an antibody molecule in which (a) the constant region, or a portion thereof, has been altered, substituted, or replaced so that the antigen-binding site (variable region) is linked to a constant region of a different or altered class, effector function, and / or species, or to an entirely different molecule, such as an enzyme, toxin, hormone, growth factor, drug, etc., that confers novel properties to the chimeric antibody; or (b) the variable region, or a portion thereof, has been altered, substituted, or replaced with a variable region of different or altered antigen specificity. For example, a murine antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. By replacing the constant region with a human constant region, the chimeric antibody can retain its specificity for antigen recognition while reducing its antigenicity in humans compared to the original murine antibody. The terms "monoclonal antibody" or "monoclonal antibody composition," as used herein, refer to the preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. The term "human antibody," as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences or variants of human germline sequences. Human antibodies may contain amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having variable regions in which both the framework and CDR regions are derived from human sequences.In one embodiment, human monoclonal antibodies are produced by hybridomas comprising (i) B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy chain and light chain transgenes, fused to (ii) immortalized cells. A "humanized" antibody is an antibody that retains the reactivity of a non-human antibody but is less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (i.e., the constant and variable region framework portions). See, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984; Morrison and Oi, Adv. Immunol., 44:65-92, 1988; Verhoeyen et al., Science, 239:1534-1536, 1988; Padlan, Molec. Immun., 28:489-498, 1991; and Padlan, Molec. Immun., 31:169-217, 1994. Other examples of human-engineered techniques include, but are not limited to, the Xoma technology disclosed in U.S. Patent No. 5,766,886.

[0012] The term "isotype" refers to the antibody class (e.g., IgM, IgE, IgG, e.g., IgG1 or IgG4) provided by the heavy chain constant region genes. Isotype also includes modified versions of one of these classes that have been modified to alter Fc function, for example, to enhance or reduce effector function or binding to an Fc receptor. Isotype also refers to the antibody class (e.g., kappa, lambda) provided by the light chain constant region. An antibody may comprise an Fc region containing one or more mutations that affect one or more antibody properties, such as stability, pattern of glycosylation or other modifications, effector cell function, pharmacokinetics, etc. In some embodiments, the antibody has reduced or minimized glycosylation. In some embodiments, the antibody has ablated or reduced effector function. Exemplary Fc mutations include, but are not limited to, (i) human IgG1 Fc region mutations L234A, L235A, G237A, and N297A; (ii) human IgG2 Fc region mutations A330S, P331S, and N297A; and (iii) human IgG4 Fc region mutations S228P, E233P, F234V, L235A, delG236, and N297A (EU numbering). In some embodiments, the human IgG2 Fc region comprises the A330S and P331S mutations. In some embodiments, the human IgG4 Fc region comprises the S288P mutation. In some embodiments, the human IgG4 Fc region comprises the S288P and L235E mutations. Antibodies that target cell surface antigens can elicit immunostimulatory and effector functions associated with engagement of Fc receptors (FcRs) on immune cells. There are numerous Fc receptors specific for particular classes of antibodies, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors), and IgM (mu receptors). Binding of the Fc region to a cell surface Fc receptor can elicit many biological responses, including phagocytosis of antibody-coated particles (antibody-dependent cell-mediated phagocytosis, or ADCP), clearance of immune complexes, lysis of antibody-coated cells by killer cells (antibody-dependent cell-mediated cytotoxicity, or ADCC), and release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production.Furthermore, when the C1 component of complement binds to an antibody, it can activate the complement system. Complement activation can be important for lysis of cellular pathogens. However, complement activation can also stimulate inflammatory responses and may be involved in autoimmune hypersensitivity or other immunological disorders. Mutant Fc regions with reduced or eliminated ability to bind to specific Fc receptors are useful for developing therapeutic antibodies and Fc fusion polypeptide constructs that act by targeting, activating, or neutralizing ligand function without damaging or destroying local cells or tissues. An Fc domain monomer refers to a polypeptide chain comprising the second and third antibody constant domains (e.g., CH2 and CH3). In some embodiments, the Fc domain monomer also comprises a hinge domain. In some embodiments, the Fc domain monomer is of any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, and IgD. Furthermore, in some embodiments, the Fc domain monomer is of any IgG subtype (e.g., IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, and IgG4). Additional mutations in the Fc domain and the biological consequences of those mutations are known in the art and can be applied to the antibodies herein, see, e.g., U.S. Patent Application Publication No. 20220002434.

[0013] The term "binding specificity," as used herein, refers to the ability of an individual antibody binding site to react with (e.g., have affinity for) only one antigenic determinant (e.g., epitope). The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes typically consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and typically possess specific three-dimensional structural features as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former is lost in the presence of denaturing solvents, but the binding to the latter is not. The phrase "specifically (or selectively) binds" to an antibody (e.g., a human HER2-binding antibody) refers to a binding reaction that determines the presence of the cognate antigen in a heterogeneous population of proteins and other biologics. The phrases "antibody recognizing an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0014] These antibodies can be used as therapeutic agents for cancer, e.g., breast cancer, including advanced metastatic breast cancer, or other diseases that exhibit elevated expression of HER2. By the term "neutralizing," it is understood that the antibody has the ability to inhibit or block any biological activity of HER2 that leads to tumorigenesis, including the ability to stimulate cell proliferation, survival, or induce tumor growth in experimental animals and humans. An effective amount of an anti-HER2 antibody is administered to a mammal, including a human, by various routes.

[0015] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody "arms" interact with the antigen at multiple sites (e.g., epitopes) through weak non-covalent forces; the more interactions between the antibody and the epitope, the stronger the affinity between them. assoc The terms "K" or "Ka," as used herein, are intended to refer to the association rate of a particular antibody-antigen interaction, while the terms "K" or "Kd," as used herein, are intended to refer to the dissociation rate of a particular antibody-antigen interaction. DThe term "dissociation constant," as used herein, is intended to refer to the dissociation constant obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). D The K value can be determined using methods well established in the art. D Methods for determining K include measuring surface plasmon resonance using a biosensor system such as a Biacore system, or measuring affinity in Octet Red 96 by solution equilibrium titration (SET) or biolayer interferometry. As used herein, the term "high affinity" for an antibody or antigen-binding fragment thereof (e.g., a Fab fragment) generally refers to a K D is 10 -9 This refers to an antibody or antigen-binding fragment of less than M.

[0016] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0017] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences (see, e.g., Tables 4 and 5). With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, to an essentially identical sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, that codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," as one species of conservatively modified variation. Any nucleic acid sequence of the present invention that encodes a polypeptide represents all possible silent variations of that nucleic acid. Those of skill in the art will recognize that each codon in a nucleic acid (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan) can be altered to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0018] The term "identical" or 100% "identity," with respect to two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences being the same. Two sequences are "substantially identical" if they have a specified percentage of identical amino acid residues or nucleotides (i.e., 60% identity over a specified region, or, if not specified, over the entire sequence) when compared and aligned for maximum correspondence over a comparison window, or designated region, as determined using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region at least about 50 nucleotides (or 10 amino acids) in length, or over a region 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length. For sequence comparison, one sequence generally serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. As used herein, a "comparison window" includes any one segment of a reference sequence, the number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, and more usually about 100 to about 150, within which a sequence can be compared to the reference sequence for the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are well known in the art.Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970, the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou, ed., 2003)). Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BRAST 2.0 algorithms, described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by verifying that short words of length W in the query sequence, when aligned with words of the same length in the database sequences, match or meet a certain positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.Word hits are extended along each sequence in both directions for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction stops when the cumulative alignment score falls below its maximum achieved value by an amount X, when the accumulation of one or more negative-scoring residue alignments causes the cumulative score to fall below zero, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as default a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) with an alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0.001.The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988) as incorporated into the ALIGN program (version 2.0), using a PAM120 weight table, a gap length penalty of 12, and a gap penalty of 4. The percent identity between two amino acid sequences can also be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453, 1970) as incorporated into the GAP program in the GCG software package (available on the world wide web at gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0019] In addition to the percentage of sequence identity discussed above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive (e.g., blocks) with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is generally substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0020] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, natural, and non-natural, have similar binding properties to the reference nucleic acid, and are metabolized similarly to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly set forth, but also conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences. Specifically, as detailed below, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0021] The term "operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Generally, the term refers to the functional relationship of a transcriptional regulatory sequence and a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of that coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous with the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or adjacent to the coding sequences whose transcription they enhance. As used herein, the term "optimized" means that a nucleotide sequence has been modified to encode an amino acid sequence using codons preferred in a production cell or organism, generally a eukaryotic cell, such as a Pichia cell, a Chinese hamster ovary cell (CHO), or a human cell. An optimized nucleotide sequence is engineered to retain, completely or as much as possible, the amino acid sequence originally encoded by the starting nucleotide sequence, also known as the "parent" sequence. As used herein, optimized sequences are engineered to have codons preferred by mammalian cells. However, optimized expression of these sequences in other eukaryotic or prokaryotic cells is also contemplated herein. An amino acid sequence encoded by an optimized nucleotide sequence can also be said to be optimized.

[0022] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof. The term "recombinant human antibody," as used herein, includes any human antibody prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom; antibodies isolated from host cells transformed to express human antibodies, e.g., from transfectants; antibodies isolated from recombinant, combinatorial human antibody libraries; and antibodies prepared, expressed, created, or isolated by any other means, including splicing all or a portion of a human immunoglobulin gene sequence into other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used) to thereby modify the V sequences of the recombinant antibody. H Area and V L The amino acid sequence of the region is human germline V H Sequence and V L These are sequences that are derived from and related to a sequence, but do not naturally occur within the human antibody germline repertoire in vivo.

[0023] The term "recombinant host cell" (or simply "host cell") or "cell line" refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in successive generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" or "cell line" as used herein. Cultured cells containing the vector are also provided. In some embodiments, the cultured cells can be cultured cells transfected with the vector or progeny of such cells, which express an immunogenic polypeptide. Suitable cell lines are known to those of skill in the art and are commercially available, for example, through the American Type Culture Collection (ATCC). The transfected cells can be used in a method for producing antibodies. This method involves culturing cells containing the vector under conditions that allow expression of the antibody, optionally under the control of an expression sequence. The immunogenic polypeptide can be isolated from the cells or culture medium using standard protein purification techniques.

[0024] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals), such as non-human primates (e.g., cynomolgus monkeys), rodents, sheep, dogs, cows, chickens, llamas (no light chains), alpacas (no light chains), camels (no light chains), sharks (no light chains), amphibians, and reptiles. Unless otherwise specified, the terms "patient" and "subject" are used interchangeably herein. As used herein, the term "treatment" of any disease or disorder (e.g., breast cancer) refers, in one embodiment, to ameliorating the disease or disorder (i.e., slowing, halting, or alleviating the progression of the disease or at least one clinical symptom thereof). In another embodiment, "treatment" refers to alleviating or improving at least one physical parameter, including one that may not be discernible to the patient. In yet another embodiment, "treatment" refers to modulating the disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. In yet another embodiment, "treatment" refers to preventing or delaying the onset or development or progression of the disease or disorder. "Prevention" as it relates to the indications described herein, including conditions or disorders associated with HER2-expressing cancers.

[0025] The term "vector" is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As the plasmid is the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably herein. However, it is intended to include other forms of expression vectors that serve equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses). Various viral vectors that have been successfully used to introduce nucleic acids into hosts include retroviruses, adenoviruses, adeno-associated viruses (AAV), herpes viruses, and pox viruses, among others. Vectors can be constructed using standard recombinant techniques widely available to those of skill in the art.Such techniques can be found in general molecular biology references such as Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, D. Goeddel, ed., 1991. Academic Press, San Diego, CA), and PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA). "Non-viral" plasmid vectors may also be suitable in certain embodiments. Preferred plasmid vectors are compatible with bacterial, insect, and / or mammalian host cells. Such vectors include, for example, PCR-ii, PCR3, and pcDNA3.1 (Invitrogen, San Diego, CA), pBSii (Stratagene, La Jolla, CA), pet15 (Novagen, Madison, WI), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFp-n2 (Clontech, Palo Alto, CA), pET1 (Bluebacii, Invitrogen), pDSR-α (PCT Publication WO 90 / 14363), and pFASTBACdual (Gibco-BRL, Grand Island, NY), as well as Bluescript® plasmid derivatives (high copy number COle1-based phagemids, Stratagene Cloning Systems, La Jolla, CA), PCR cloning plasmids designed for cloning TAQ-amplified PCR products (e.g., TOPO™ TA cloning® kit, PCR2.1® plasmid derivatives, Invitrogen, Carlsbad, CA). Bacterial vectors are also available.These vectors include, for example, Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille Calmette-Guerin (BCG), and Streptococcus (see, e.g., WO 88 / 6626; WO 90 / 0594; WO 91 / 13157; WO 92 / 1796; and WO 92 / 21376). Many other non-viral plasmid expression vectors and systems are known in the art and can be used. Other delivery techniques may also be sufficient, including, for example, DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO precipitation, gene gun technology, electroporation, and colloidal dispersion systems. Colloidal dispersion systems include polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system is a liposome, an artificial membrane vesicle useful as a delivery vehicle in vitro and in vivo. RNA, DNA, and intact virions can be encapsulated in the aqueous interior and delivered to cells in a biologically active form (Fraley, R., et al., 1981, Trends Biochem. Sci., 6: 77). Liposomes are typically composed of a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, particularly cholesterol. Other phospholipids or other lipids can also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful for producing liposomes include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, in which the lipid moiety contains 14 to 18 carbon atoms, particularly 16 to 18 carbon atoms, and is saturated. Exemplary phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.

[0026] An antibody can be conjugated with a drug to form an antibody-drug conjugate (ADC). Generally, an ADC contains a linker between the drug and the antibody. The linker can be a degradable or non-degradable linker, cleavable or non-cleavable. Degradable linkers are generally readily degraded in the intracellular environment; for example, the linker is degraded at the target site to release the drug from the antibody. Suitable degradable linkers include enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (such as lysosomal or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidase. Peptidyl linkers can include dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that are hydrolyzed at a pH below 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide bond linkers). Non-degradable linkers generally release the drug under conditions where the antibody is hydrolyzed by proteases.

[0027] The linker has a reactive group that can react with a specific amino acid residue before being linked to the antibody, and linkage is achieved through that reactive group. Sulfhydryl-specific reactive groups are preferred, including, for example, maleimide compounds, halogenated amides (such as iodine, bromine, or chloro), halogenated esters (such as iodine, bromine, or chloro), halogenated methyl ketones (such as iodine, bromine, or chloro), benzyl halides (such as iodine, bromine, or chloro), vinyl sulfone, pyridyl disulfide, mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, counterions of acetate, chloride, or nitrate, and polymethylene dimethyl sulfide thiosulfonate. The linker may, for example, comprise a maleimide linked to the antibody via thiosuccinimide. The drug may be any cytotoxic or immunosuppressive drug that inhibits cell proliferation.

[0028] The drug can be any cytotoxic or immunosuppressive drug that inhibits cell proliferation. In some embodiments, a linker connects the antibody and the drug, and the drug has a functional group capable of binding to the linker. For example, the drug can have an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, or a ketone group that can form a bond with the linker. When the drug is directly linked to the linker, the drug has an active group that remains reactive until it is linked to the antibody. The antibodies and / or derivatives thereof disclosed herein can also be adjacent to and / or conjugated to functional agents for in vitro and / or in vivo use. Useful drug categories include, for example, antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Exemplary cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposide, maytansine and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines, and vinca alkaloids). In some embodiments, these antibodies can be adjacent to and / or conjugated to a functional moiety such as a cytotoxic drug or toxin, and / or active fragments thereof, such as diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, among others. Suitable functional moieties can also include radioactive chemicals. The antibodies can be adjacent to and / or conjugated to one or more functional agents using standard techniques in the art.

[0029] As described herein, drug-linkers can be used to form ADCs in a simple, single step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two- or multi-step process. For example, in a first step, a cysteine ​​residue is reacted with a reactive moiety on the linker, and in a second step, a functional group on the linker is reacted with the drug to form the ADC. Many suitable methods for creating drug-linkers used to form ADCs are known to those of skill in the art and are likely to be suitable for use in combination with the reagents disclosed herein (e.g., antibodies and their derivatives) as well as other reagents available to those of skill in the art (e.g., cytotoxic agents not explicitly listed herein). Generally, functional groups on the linker are selected to facilitate specific reaction with appropriate reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently attached to the linker through 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reacting with hydrazides and alkoxyamines), phosphines (suitable for reacting with azides), isocyanates and isothiocyanates (suitable for reacting with amines and alcohols), and activated esters such as N-hydroxysuccinimide esters (suitable for reacting with amines and alcohols). These and other linking strategies, such as those described in "Bioconjugation Technology," 2nd Edition (Elsevier), are well known to those skilled in the art. One skilled in the art will appreciate that when complementary pairs of reactive functional groups are selected for selective reaction between a drug moiety and a linker, each member of the complementary pair can be used in both the linker and the drug.

[0030] Provided herein are monoclonal antibodies, including humanized anti-HER2 antibodies, and antigen-binding fragments, such as polypeptides, comprising complementarity-determining regions (CDRs) that bind to HER-2. In some embodiments, the present disclosure provides fully human anti-HER2 antibodies that internalize into cells expressing HER2 and compete with trastuzumab for binding to HER2, as well as methods of using such antibodies for their neutralizing properties as well as their internalization properties. In some embodiments, the present disclosure provides fully human anti-HER2 antibodies that internalize and do not compete with trastuzumab for binding to the HER2 receptor, as well as methods for investigating their internalization and cell growth inhibitory properties. In some embodiments, the present disclosure provides methods for using antibodies and / or fragments thereof (e.g., CDRs) (collectively referred to herein as "antibodies" unless otherwise specified) to treat HER2-mediated cancers.

[0031] Those skilled in the art have many suitable techniques for using the antibodies (e.g., antibodies) described herein to identify biological samples containing proteins that bind to them. For example, antibodies can be used to isolate HER2 or HER2-expressing cells and / or cells that express HIV antigens, e.g., using immunoprecipitation or other capture-type assays. This well-known technique is performed by attaching the antibody to a solid support or chromatographic material (e.g., beads coated with Protein A, Protein G, and / or Protein L). The bound antibody is then introduced into a solution that contains or is suspected to contain HER2 antigen (e.g., HER2-expressing cells). The HER2 antigen is then allowed to bind to the antibody, and unbound material is washed away under conditions that allow the HIV antigen to remain bound to the antibody. The bound protein can then be separated from the antibody and analyzed, if desired. Similar methods for isolating proteins using antibodies are well known in the art. Antibodies (e.g., antibodies) can also be used to detect HIV or HIV antigens in biological samples. For example, antibodies may be used in assays such as, for example, flow cytometry analysis, ELISA, immunoblotting (e.g., Western blot), in situ detection, immunocytochemistry, and / or immunohistochemistry. Methods for performing such assays are well known in the art. In some embodiments, the antibodies may be adjacent to and / or conjugated to one or more detectable labels.For example, suitable detectable labels include, for example, fluorosceins (e.g., DyLight, Cy3, Cy5, FITC, HiLyte Fluor 555, HiLyte Fluor 647); 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-HAT (hydroxytryptamine); 5-hydroxytryptamine (HAT); 6-JOE; 6-carboxyfluorescein (6-FAM); FITC; 6-carboxy-1,4-dichloro-2',7'-dichloro-fluorescein (TET); 6-carboxy-1,4-dichloro-2',4',5',7'-tetra-chlorofluorescein (HEX); 6-carboxy-4',5'-dichloro-2',7'-dimethoxy-fluorescein (JOE); Alexa fluors (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY fluorophores (e.g., 492 / 515, 493 / 503, 500 / 510, 505 / 515, 530 / 550, 542 / 563, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650-X, 650 / 665-X, 665 / 676, FL, FL ATP, FI-ceramide, R6G SE, TMR, TMR-X conjugates, TMR-X, SE, TR, TR ATP, TR-X) SE), rhodamine (e.g., 110, 123, B, B 200, BB, BG, B Extra, 5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-carboxyrhodamine 6G, Lissamine, Lissamine rhodamine B, Phallicidine, Phalloidin, Red, Rhodo-2, ROX (6-carboxy-X-rhodamine), 5-ROX (carboxy-X-rhodamine), Sulforhodamine B can C, Sulforhodamine G Extra, TAMRA (6-carboxytetramethyl-rhodamine), Tetramethylrhodamine (TRITC), WT), Texas Red, and / or Texas Red-X. Other detectable labels known in the art may also be suitable for use.Antibodies, such as antibodies, can be adjacent to and / or attached to one or more detectable labels using standard techniques in the art.

[0032] The antibodies described herein can also be used to determine the presence of a pathological condition in a patient, to predict prognosis, or to determine the effectiveness of a chemotherapy or other treatment regimen. Expression profile assays performed as described herein or otherwise known in the art can be used, for example, to determine the relative expression level of HER2 in cells. Expression levels can then be correlated with basal (e.g., control) levels to determine whether a particular disease is present in a patient, the patient's prognosis, or whether a particular treatment regimen is effective. For example, if a patient is being treated with a particular anti-infective regimen, an increase or decrease in the expression level of HER2 in the patient's tissues (e.g., in plasma) can indicate that the treatment regimen is worsening or ameliorating the burden of HER2-expressing cells (e.g., cancer) in the host. Increased or decreased expression can indicate that the treatment regimen is or is not having the desired effect, and an alternative treatment may therefore be selected.

[0033] The antibodies described herein can also be used as reagents in drug screening assays, for example, to test new drug candidates. The reagents can be used to confirm the effect of drug candidates on the expression of immunogenic targets in cell lines or in patient cells or tissues. Expression profiling techniques, combined with high-throughput screening techniques, enable the rapid identification of useful compounds and the monitoring of the effectiveness of treatment with drug candidates (see, e.g., Zlokarnik, et al., Science 279, 84-8 (1998)). Drug candidates can be naturally occurring or synthetically obtained chemical compounds, nucleic acids, proteins, antibodies, or derivatives thereof. Drug candidates identified in this manner can be utilized, among other uses, as pharmaceutical compositions for administration to patients or for use in further screening assays.

[0034] The antibodies (e.g., polypeptides) and nucleic acids described herein may also be combined with one or more pharmaceutically acceptable carriers prior to administration to a host. A pharmaceutically acceptable carrier is a material that is not biologically or otherwise undesirable; e.g., the material may be administered to a subject without causing any undesired biological effects or adversely interacting with any of the other components of the pharmaceutical composition in which it is contained. As is well known to those skilled in the art, the carrier will naturally be selected to minimize degradation of the active ingredient and minimize adverse side effects in the subject. Suitable pharmaceutical carriers and their formulations are described, for example, in Remington's: The Science and Practice of Pharmacy, 21st ed., edited by David B. Troy, Lippicott Williams & Wilkins (2005). Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions such as Ringer's solution, and dextrose solution. The pH of the solution is generally about 5 to about 8, or about 7 to about 7.5. Other carriers include sustained-release preparations, such as semipermeable matrices of solid hydrophobic polymers containing the polypeptide or fragment thereof. The matrices may be in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and concentration of the composition being administered. The carrier is one suitable for administration of the polypeptide and / or fragment thereof to humans or other subjects. In addition to the immunogenic polypeptide, pharmaceutical compositions may also contain carriers, thickeners, diluents, buffers, preservatives, surfactants, adjuvants, and immunostimulants. Pharmaceutical compositions may also contain one or more active ingredients, such as antibacterial agents, anti-inflammatory agents, and anesthetics. Pharmaceutical compositions can be administered orally, parenterally, by inhalation spray, rectally, intranodally, or topically in unit dosage forms containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier," as used herein, refers to one or more formulation materials suitable for achieving or facilitating delivery of a nucleic acid, polypeptide, or peptide as a pharmaceutical composition. A "pharmaceutical composition" is a composition containing a therapeutically effective amount of a nucleic acid or polypeptide. The terms "effective amount" and "therapeutically effective amount," respectively, refer to the amount of an antibody, nucleic acid, etc. used to achieve a desired therapeutic effect (e.g., elimination of HER2-expressing cells, e.g., cancerous HER2-expressing cells).

[0035] Also provided are methods for treating one or more conditions (e.g., cancer) in a mammalian host, comprising administering to the mammal at least one effective dose of an antibody (and / or derivative thereof) described herein. In some embodiments, the antibody is a monoclonal antibody comprising an amino acid sequence set forth in one or more of SEQ ID NOs: 1-61, Tables 1 and 2, and / or encoded by a nucleotide sequence in Table 3, or a fragment or derivative thereof, and / or a substituted derivative and / or fragment thereof; and in some embodiments, a conservative substitution variant thereof. The one or more antibodies may be administered at a dosage of about 1 to about 50 mg / kg, about 1 to about 30 mg / kg, or about 5 to about 30 mg / kg (e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 mg / kg). In certain embodiments, the one or more antibodies may be administered to a mammal at about 10 mg / kg one or more times (e.g., intradermally, intravenously, orally, rectally). When multiple doses are administered, the doses may contain approximately the same or different amounts of antibody in each administration. The administrations may also be separated in time from each other by equal or different intervals. For example, administrations can be separated by about 6, 12, 24, 36, 48, 60, 72, 84, or 96 hours, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 years, 2 years, 3 years, 4 years, 5 years, or any time period before, after, and / or between any of these time periods. In some embodiments, the antibody may be administered along with other agents (e.g., anti-infectives and / or chemotherapeutic agents). Such other agents may be administered at about the same time as the antibody or at different times and / or frequencies. Other embodiments of such methods may also be suitable, as can be readily determined by one of skill in the art.

[0036] To assist those skilled in the art in using antibodies, such as those described herein, they can be provided in kit format. Kits containing one or more such antibodies and optional other components necessary for their use to detect HIV-expressing cells are also provided. The antibodies of the kit can be provided in any suitable form, such as frozen, lyophilized, or in a pharmaceutically acceptable buffer such as TBS or PBS. The kit may also include other reagents necessary for utilizing the antibody in vitro or in vivo, such as buffers (e.g., TBS, PBS), blocking agents (solutions containing nonfat dry milk, normal serum, Tween-20 detergent, BSA, or casein), and / or detection reagents (e.g., goat anti-mouse IgG biotin, streptavidin-HRP conjugate, allophycocyanin, B-phycoerythrin, R-phycoerythrin, peroxidase, detectable labels, and other labeling and / or staining kits (e.g., ABC staining kit, Pierce)). The kit may also include other reagents and / or instructions for using the antibody in commonly used assays such as those described above, such as flow cytometry analysis, ELISA, immunoblotting (e.g., Western blot), in situ detection, immunocytochemistry, and / or immunohistochemistry. In one embodiment, the kit provides the antibody in purified form. In another embodiment, the antibody may be provided in biotinylated form, either alone or in combination with an avidin-conjugated detection reagent (e.g., antibody). In another embodiment, the kit includes an antibody containing one or more detectable labels that can be used to directly detect HER2. Buffers and the like required for using any of these systems are well known in the art and / or may be prepared by the end user or provided as a component of the kit. The kit may also include a solid support containing positive and negative control proteins and / or tissue samples. For example, a kit for performing a spotting or Western blot-type assay may include control cell or tissue lysates for use in SDS-PAGE, or a pre-fixed control sample, plus a nylon or other membrane with additional space for the test sample.A kit for visualizing HIV in cells on a slide may include a pre-formatted slide containing a control cell or tissue sample, plus space for the test sample. As will be appreciated by those skilled in the art, other embodiments of the kit are contemplated herein.

[0037] The isolated antibody or antigen-binding fragment described herein binds to human HER2 at a concentration of 3.6 x 10 -9 K to mole (M) D This is similar to commercially available trastuzumab (3.1 × 10 in our experiments). -10 M) and reported-manufactured trastuzumab (5 x 10 as measured by the Octet assay described below (see, e.g., the Examples section herein) or by any assay available to one of skill in the art). -9 M. In some embodiments, the present disclosure provides fully human anti-HER2 antibodies that internalize into cells that express HER2 and compete with trastuzumab for binding to HER2, as well as methods of using such antibodies for their neutralizing properties as well as their internalizing properties. In preferred embodiments, such antibodies include those referred to herein as 11D7, 2A2, 12E3, 2D7, 3G7, 8C1, and / or the CDRs contained by such antibodies (see Table 1); their heavy and light chain variable regions (see Table 3); and / or derivatives thereof (e.g., containing conservative amino acid substitutions therefor (see, e.g., Table 4)). In some embodiments, the present disclosure provides fully human anti-HER2 antibodies that internalize into cells expressing HER2 and do not compete with trastuzumab for binding to the HER2 receptor, and methods for studying their internalization and biological properties, including ADCs. In preferred embodiments, such antibodies include those referred to herein as 2A1, 7F9, 9E4, 11C9, and 12A6; those comprising the heavy and light chain variable regions thereof (see Table 3); and / or those comprising the CDRs of Table 2; and / or derivatives thereof (e.g., containing conservative amino acid substitutions therefor (see, e.g., Table 4)).

[0038] In some embodiments, the disclosure described herein relates to isolated antibodies, or antigen-binding fragments thereof, that bind to human HER2 and compete for binding with antibody 11D7, 2A2, 12E3, 2D7, 3G7, 8C1, 2A1, 7F9, 9E4, 11C9, or 12A6; heavy and light chain variable regions thereof; and / or polypeptides comprising the CDRs of such antibodies (see Tables 1 and 2); and / or derivatives thereof (e.g., containing conservative amino acid substitutions therefor (see, e.g., Table 4)), all of which are considered equivalents thereof. Antibodies can also be compared to antibodies 11D7, 2A2, 12E3, 2D7, 3G7, 8C1, 2A1, 7F9, 9E4, 11C9, or 12A6 and identified as equivalents by ELISA for binding to HER-ECD (HER2 extracellular domain), flow cytometry for binding to SKBRIII, competition with trastuzumab for binding to HER2 (e.g., demonstrating differences in epitope specificity of antibodies and / or combinations of their CDRs), internalization assays, Octet scouting and epitope binning, killing assays, cytotoxic payload, and / or amino acid and / or nucleotide sequencing. For example, antibodies of the present disclosure can be identified by reference to the amino acid and / or nucleic acid sequences corresponding to their variable and / or complementarity determining regions ("CDRs") and / or their activities (e.g., that one antibody blocks binding of another antibody to HER2). CDRs comprise amino acid residues in the variable regions identified according to Kabat, Chothia, the cumulative sum of both Kabat and Chothia, AbM, contact definitions, and / or conformational definitions, or any method of CDR determination known in the art, antibody modeling software (now Accelrys®), or the "contact definitions" of CDRs based on observed antigen contacts as described in MacCallum et al., 1996, J. Mol. Biol., 262:732-745.A "conformational definition" of a CDR can identify the position of the CDR as a residue that contributes enthalpic- ically to antigen binding (Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166). Still other CDR boundary definitions may not strictly follow one of the above approaches, but may still overlap with at least a portion of the Kabat CDR, and may be shorter or longer in light of predictions or experimental results that indicate that a particular residue or group of residues, or the CDR as a whole, does not significantly affect antigen binding. As used herein, a CDR may refer to a CDR defined by any approach known in the art, including a combination of approaches. The methods used herein can utilize CDRs defined according to any of these approaches. For any given embodiment containing two or more CDRs, the CDRs may be defined according to any of the Kabat, Chothia, extended, AbM, contact, and / or conformational definitions. For example, the Chothia and Kabat numbering system for CDR residues is well known in the art and is further described in the art (see, e.g., Dondelinger, M., et al., Front Immunol 9:2278, 2018). In particular, mAbs herein include anti-human HER2 antibodies having the amino acid sequences of each CDR listed in Table 1. For purposes of the present disclosure, the Kabat method is used to determine the CDRs of the antibodies disclosed herein. [Table 1] [Table 2]

[0039] The antibodies may further comprise the complete heavy and / or light chains of the antibodies shown in Table 3, and / or derivatives thereof, comprising the CDRs set forth in Tables 1 and 2. In some preferred embodiments, such antibodies may have the amino acid sequence of a variable heavy chain ("VH") polypeptide comprising heavy chain framework regions (HFR1, HFR2, HFR3, HFR4 (included in the sequences listed individually in the Examples section below)) or a variable light chain ("VL") polypeptide comprising light chain framework regions (LFR1, LFR2, LFR3, LFR4 (included in the sequences listed individually in the Examples section below)) (VH or VL "chains," respectively) as set forth below for the antibodies shown in Table 3, or their equivalents. [Table 3] JPEG2025535798000007.jpg218165

[0040] In a preferred embodiment, according to Table 3, the 11D7 antibody comprises SEQ ID NOs: 41 and 42; the 2A2 antibody comprises SEQ ID NOs: 43 and 44; the 12E3 antibody comprises SEQ ID NOs: 45 and 46; the 2D7 antibody comprises SEQ ID NOs: 47 and 48; the 3G7 antibody comprises SEQ ID NOs: 49 and 50; the 8C1 antibody comprises SEQ ID NOs: 51 and 52; the 2A1 antibody comprises SEQ ID NOs: 53 and 54; the 7F9 antibody comprises SEQ ID NOs: 55 and 56; the 9E4 antibody comprises SEQ ID NOs: 57 and 58; the 11C9 antibody comprises SEQ ID NOs: 59 and 60; and the 12A6 antibody comprises SEQ ID NOs: 61 and 62.

[0041] With respect to polypeptide sequences, "conservatively modified variants" include individual substitutions, deletions, or additions to a polypeptide sequence that result in the replacement of an amino acid with a chemically equivalent amino acid. Conservative substitution tables displaying functionally equivalent amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles. In some embodiments, the term "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing that amino acid sequence. Preferably, the antibody, or antigen-binding fragment thereof, comprises one or more amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to at least one of SEQ ID NOS: 1-62 (i.e., the CDR sequences, VH sequences, and / or VL sequences shown in Tables 1-3). In some embodiments, equivalents of the 11D7, 2A2, 12E3, 2D7, 3G7, 8C1, 2A1, 7F9, 9E4, 11C9, or 12A6 antibodies include derivatives of one or more of the CDRs of the 11D7, 2A2, 12E3, 2D7, 3G7, 8C1, 2A1, 7F9, 9E4, 11C9, or 12A6 antibodies, preferably containing up to three conservative amino acid substitutions (see Table 4) of those CDRs, so long as the derivative retains the ability to bind to HER2. In some embodiments, equivalents of the 11D7, 2A2, 12E3, 2D7, 3G7, 8C1, 2A1, 7F9, 9E4, 11C9, or 12A6 antibodies include derivatives of one or more of the VH and / or VL chains of the 11D7, 2A2, 12E3, 2D7, 3G7, 8C1, 2A1, 7F9, 9E4, 11C9, or 12A6 antibodies, preferably containing up to 10 conservative amino acid substitutions (see Table 4) outside of their CDRs, so long as the derivatives retain the ability to bind to HER2.In preferred embodiments, any such substitutions allow for or do not interfere with conjugation of the antibody with one or more detectable labels, cytotoxic agents, and / or other payloads (e.g., to provide bispecific antibodies).

[0042] Using standard three letter or other abbreviations for amino acids, as will be understood by those of skill in the art, amino acid substitutions that are considered to be conservative and non-conservative are shown in Table 4 below. [Table 4]

[0043] In certain embodiments, nucleic acid molecules encoding one or more antibodies described herein can be inserted into one or more expression vectors, as described in more detail below. In such embodiments, an antibody can be encoded by nucleotides corresponding to its amino acid sequence. Specific combinations of nucleotides (codons) encoding various amino acids (AA) are well known in the art, as described in various references used by those skilled in the art (e.g., Lewin, B. Genes V, Oxford University Press, 1994). Nucleic acid variants can be identified by, for example, Table 5, and any combination of nucleotides encoding the antibody can be used. [Table 5]

[0044] In a preferred embodiment, the VH and VL amino acid sequences are encoded by the nucleotide sequences shown in Table 6. [Table 6] JPEG2025535798000011.jpg182167 JPEG2025535798000012.jpg184166 JPEG2025535798000013.jpg123167

[0045] Those skilled in the art will understand that nucleotide sequences encoding specific amino acid sequences equivalent to the 11D7, 2A2, 12E3, 2D7, 3G7, 8C1, 2A1, 7F9, 9E4, 11C9, or 12A6 antibodies can be readily derived from the amino acid sequence of any of SEQ ID NOS: 63-84 and the information set forth in Table 4. For example, from the amino acid sequence DYAMH (SEQ ID NOS: 1) and the information set forth in Table 4, it can be deduced that the amino acid sequence can be encoded by the nucleotide sequence GAT TAT GCT ATG CAT (SEQ ID NOS: 84). Those skilled in the art will understand that nucleotide sequences encoding SEQ ID NOS: 1-62 and derivatives thereof can be similarly deduced, and such nucleotide sequences are contemplated herein. The present disclosure also provides expression vectors comprising isolated nucleic acids comprising and / or consisting of such nucleotide sequences (in preferred embodiments, any of SEQ ID NOS: 63-84 or derivatives thereof), as well as host cells (e.g., cell lines) comprising such expression vectors.

[0046] In preferred embodiments, the present disclosure provides antibodies comprising: a) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 1, 2, and 3 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 4, 5, and 6 (antibody 11D7; Table 1); b) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 1, 7, and 3 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 4, 5, and 6 (antibody 2A2; Table 1); c) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 1, 9, and 3 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 4, 5, and 6 (antibody 12E3; Table 1); d) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 1, 10, and 3 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 4 a) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 1, 12, and 3 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 4, 5, and 6 (antibody 2D7; Table 1); b) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 1, 12, and 3 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 4, 5, and 6 (antibody 3G7; Table 1); c) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 1, 13, and 3 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 4, 5, and 6 (antibody 8C1; Table 1); d) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 14, 15, and 16 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 17, 18, and 19 (antibody 2A1; Table 2); h) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 20, 21, and 22 and 22 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 23, 24, and 25 (antibody 7F9; Table 2); i) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 26, 27, and 28 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 29, 30, and 31 (antibody 9E4; Table 2); j) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 32, 33, and 34 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 35, 36, and 37 (antibody 11C9; Table 2); k) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 32, 38, and 39 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 35, 36, and 40 a light chain variable region comprising the CDR sequences (antibody 12A6; Table 2); l) a heavy chain variable region comprising SEQ ID NO: 41 and a light chain variable region comprising SEQ ID NO: 42 (antibody 11D7; Table 3); m) a heavy chain variable region comprising SEQ ID NO: 43 and a light chain variable region comprising SEQ ID NO: 44 (antibody 2A2; Table 3); n) a heavy chain variable region comprising SEQ ID NO: 45 and a light chain variable region comprising SEQ ID NO: 46 (antibody 12E3; Table 3); o) a heavy chain variable region comprising SEQ ID NO: 47 and a light chain variable region comprising SEQ ID NO: 48 (antibody 2D7; Table 3); p) a heavy chain variable region comprising SEQ ID NO: 49 and a light chain variable region comprising SEQ ID NO: 50 (antibody 3G7; Table 3);q) a heavy chain variable region comprising SEQ ID NO: 51 and a light chain variable region comprising SEQ ID NO: 52 (antibody 8C1; Table 3); r) a heavy chain variable region comprising SEQ ID NO: 53 and a light chain variable region comprising SEQ ID NO: 54 (antibody 2A1; Table 3); s) a heavy chain variable region comprising SEQ ID NO: 55 and a light chain variable region comprising SEQ ID NO: 56 (antibody 7F9; Table 3); t) a heavy chain variable region comprising SEQ ID NO: 57 and a light chain variable region comprising SEQ ID NO: 58 (antibody 9E4; Table 3); u) a heavy chain variable region comprising SEQ ID NO: 59 and a light chain variable region comprising SEQ ID NO: 60 (antibody 11C9; Table 3); v) a heavy chain variable region comprising SEQ ID NO: 61 and a light chain variable region comprising SEQ ID NO: 62 (antibody 12A6; Table 3); w) a heavy chain variable region encoded by any of the polynucleotides of SEQ ID NOs: 63 to 84 (Table 6) and the corresponding light chain variable region; and / or conservative substitution derivatives thereof, optionally comprising up to three amino acid substitutions in one or more CDRs thereof and / or up to ten amino acid substitutions in the heavy and / or light chain thereof;and specifically binds to human HER2. In some preferred embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising: The present disclosure also provides, but is not limited to, combinations of at least one antibody that binds to a different epitope on human HER2, such as (preferably) any two or more of antibodies 11D7, 2A2, 12E3, 2D7, 3G7, 8C1, 2A1, 7F9, 9E4, 11C9, and 12A6, and compositions (preferably pharmaceutically acceptable compositions) thereof. In some embodiments, the combinations may include at least one antibody that competes with trastuzumab for binding to HER2 receptors on cells (e.g., antibodies 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1) and at least one antibody that does not compete with trastuzumab for binding to HER2 receptors on cells (e.g., antibodies 2A1, 7F9, 9E4, 11C9, and 12A6). In some embodiments, these combinations may include trastuzumab. In some preferred embodiments, the antibody combination can be formulated as a composition (preferably a pharmaceutically acceptable composition);

[0047] In some preferred embodiments, the antibody is derived from a human antibody, human IgG, human IgG1, human IgG2, human IgG2a, human IgG2b, human IgG3, human IgG4, human IgM, human IgA, human IgA1, human IgA2, human IgD, human IgE, dog antibody, dog IgGA, dog IgGB, dog IgGC, dog IgGD, chicken antibody, chicken IgA, chicken IgD, chicken IgE, chicken IgG, chicken IgM, chicken IgY, goat antibody, goat IgG, mouse antibody, mouse IgG, pig antibody, rat antibody, llama antibody, alpaca antibody, shark antibody, and camel antibody. ab , F ab2 , Fab' single chain antibody, F v, single chain, monospecific antibody, bispecific antibody, trimeric antibody, multispecific antibody, multivalent antibody, chimeric antibody, canine-human chimeric antibody, canine-mouse chimeric antibody, canine Fc-containing antibody, humanized antibody, human antibody, caninized antibody, CDR-grafted antibody, shark antibody, and nanobody. In some preferred embodiments, the present disclosure provides derivatives of the antibodies disclosed herein comprising an immobilized detectable label, optionally the detectable label being fluorescein, DyLight, Cy3, Cy5, FITC, HiLyte Fluor 555, HiLyte Fluor 647, 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein, 5-FAM, hydroxytryptamine, 5-hydroxytryptamine (5-HAT), 6-carboxyfluorescein (6-FAM), FITC, 6-carboxy-1,4-dichloro-2',7'-dichloro-fluorescein (TET), 6-carboxy-1,4-dichloro-2',4',5',7'-tetra-chlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxy-fluorescein (6-JOE), Alexa fluor, Alexa fluor fluor 350, Alexa fluor 405, Alexa fluor 430, Alexa fluor 488, Alexa fluor 500, Alexa fluor 514, Alexa fluor 532, Alexa fluor 546, Alexa fluor 555, Alexa fluor 568, Alexa fluor 594, Alexa fluor 610, Alexa fluor 633, Alexa fluor 635, Alexa fluor 647, Alexa fluor 660, Alexa fluor 680, Alexa fluor 700, Alexa fluor 750, BODIPY fluorophore, BODIPY 492 / 515, BODIPY 493 / 503, BODIPY 500 / 510, BODIPY 505 / 515, BODIPY 530 / 550, BODIPY 542 / 563, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY576 / 589, BODIPY 581 / 591, BODIPY 630 / 650-X, BODIPY 650 / 665-X, BODIPY 665 / 676, FL, FL ATP, FI-ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X SE, TR, TR ATP, TR-X SE, rhodamine, rhodamine 110, rhodamine 123, rhodamine B, rhodamine B 200, rhodamine BB, rhodamine BG, rhodamine B Extra, 5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-carboxyrhodamine 6G, Lissamine, Lissamine rhodamine B, Phallicidine, Phalloidin, Rhodamine Red, Rhodo-2, 6-carboxy-X-rhodamine (ROX), carboxy-X-rhodamine (5-ROX), sulforhodamine B canC, sulforhodamine G extra, 6-carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TRITC), rhodamine WT, Texas Red, and Texas Red-X. In some preferred embodiments, the present disclosure provides derivatives of the antibodies disclosed herein comprising an attached effector moiety, optionally the effector moiety being selected from the group consisting of a cytotoxic drug, a toxin, diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, and a radiochemical, and optionally a cleavable linker disposed between the antibody and the effector moiety, wherein the cleavable linker releases the effector moiety into or within a cell. In some preferred embodiments, the present disclosure provides isolated polynucleotides encoding the antibodies of the present disclosure, optionally having the nucleic acid sequence of at least one of SEQ ID NOs: 63-84, as well as expression vectors and host cells comprising them. In some preferred embodiments, the present disclosure provides compositions comprising at least one antibody or derivative disclosed herein; at least one isolated polynucleotide encoding such an antibody or derivative; or at least one expression vector comprising such a polynucleotide; and / or at least one host cell comprising such a polynucleotide and / or expression vector; or combinations thereof, together with a pharmaceutically acceptable carrier.

[0048] In some preferred embodiments, the present disclosure provides methods for detecting HER2 on cells, the methods comprising contacting a test biological sample with an antibody or derivative, combination, or composition (preferably a pharmaceutical composition) comprising any of the disclosed antibodies and detecting antibody binding to the biological sample or a component thereof. In some embodiments, these methods can comprise comparing the amount of binding to the test biological sample or a component thereof with the amount of binding to a control biological sample or a component thereof, where increased binding to the test biological sample or a component thereof relative to the control biological sample or a component thereof indicates the presence of HER2-expressing cells in the test biological sample (e.g., the test biological sample is a mammalian cell, tissue, or blood). The methods can be in vivo or in vitro methods.

[0049] In some preferred embodiments, the present disclosure provides methods for treating, preventing, and / or ameliorating cancer in a mammal by administering to the mammal at least one effective dose of a pharmaceutical composition comprising an antibody, derivative, and / or combination of the present disclosure. In some embodiments, the present disclosure provides such antibodies comprising an attached cytotoxic effector moiety, optionally selected from the group consisting of a cytotoxic drug, a toxin, diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, and a radiochemical. In some embodiments, such antibodies can comprise a cleavable linker disposed between the antibody and the effector moiety, the cleavable linker releasing the effector moiety into or within a cell. In some such embodiments, the antibody is administered as an antibody-drug conjugate. In some embodiments, multiple doses are administered to the animal; and / or the antibody is administered at a dosage of about 1-50 mg / kg. In some embodiments, the present disclosure provides a kit for detecting HER2 expression in or on a cell, the kit comprising an antibody or derivative of the present disclosure and instructions for use. In some embodiments, the antibody or derivative may be in lyophilized form. As will be appreciated by those skilled in the art, other embodiments are also provided. All references cited within this disclosure are incorporated herein by reference in their entirety. Certain embodiments are further described in the following examples. These embodiments are presented by way of example only and are not intended to limit the scope of the claims in any way. [Example]

[0050] The following are examples of antibodies described herein: It should be understood by those of skill in the art that the methods described herein are exemplary only, as many other methods for doing so are available to those of skill in the art.

[0051] Example 1 : Development of a fully human anti-HER2 monoclonal antibody Described herein are methods used to produce fully human anti-HER2 monoclonal antibodies. The first group of fully human anti-HER2 antibodies internalizes and competes with trastuzumab for binding to HER2 (6 antibodies); these antibodies can be used in in vitro or in vivo animal models, e.g., for their internalization properties as well as neutralization properties. The second group of fully human anti-HER2 antibodies internalizes and does not compete with trastuzumab for binding to the HER2 receptor (5 antibodies); these antibodies can be used in in vitro or in vivo animal models for their internalization properties. To generate the fully human antibodies described herein, three humanized mice (TC-mAb™) were immunized with human recombinant His-tagged HER2 extracellular domain. The characteristics of these mice have been reported (Moriwaki, T., Abe, S., Oshimura, M. & Kazuki, Y. 2020, Transchromosomic technology for genomically humanized animals. Exp. Cell Res. 390, 111914.).

[0052] Human Ab-producing TC mice (TC-mAb mice) stably maintain a mouse-derived artificial chromosome containing the entire human Ig heavy and kappa chain loci in a mouse Ig knockout background. Transchromosomal (TC) mice carrying a minichromosome containing human immunoglobulin (Ig) loci can contribute to the development of fully human therapeutic monoclonal antibodies (Abs) by immunization with antigens of interest. In this case, TC-mAb mice were immunized with a human recombinant His-tagged HER2 extracellular domain according to a previously validated schedule. Thirty days later, serum titers from immunized mice were confirmed by enzyme immunoassay (ELISA) using human His-tagged HER2 ECD immobilized on nickel plates, followed by incubation with an HRP-conjugated goat anti-human Fc secondary antibody. From the mouse with the highest anti-HER2 titer, spleen and lymph node B cells were collected and fused with mouse myeloma HL-1 cells by electroporation. The fused hybridomas were seeded as single cells in semi-solid hybridoma culture Clonal Cell™ HY medium D (StemCell Technology) in 10 cm tissue culture plates. After 11 days, 1,152 single hybridoma clones were harvested from the semi-solid medium plates and transferred to 96-well dishes in Hybridoma Culture Medium E (StemCell Technology) (one clone per well). After 4 days, the culture medium from the hybridoma clones was assayed by HER2-ECD EIA as described above. From the initial screening, the top 313 clones with OD > 2.0 were selected. These clones were transferred to 48-well plates for confirmatory screening by HER2-ECD EIA as described above. 207 clones were confirmed as strongly positive. These clones were transferred in duplicate to 6-well plates in Medium E. Cells were cryopreserved under appropriate culture conditions for long-term storage in liquid nitrogen, and the culture medium containing the secreted antibodies was collected and stored for future evaluation and selection of the desired hybridomas.The antibodies produced by the hybridoma clones were tested for the following: 1) competition with trastuzumab for binding to the HER2-ECD by competitive enzyme immunoassay (EIA), 2) internalization in HER2-overexpressing cells, such as SKBR3 and AU565 cells, 3) inhibition of cell signaling in HER2-overexpressing breast cancer cells, SKBR3, 4) inhibition of migration of HER2-overexpressing breast cancer cells, and 5) inhibition of proliferation of HER2-overexpressing SKBR3 cells by incubation with anti-HER2 antibodies in the presence of Fab-αHFc-NC-DM1. Fab-αHFc-NC-DM1 is a Fab fragment of an anti-human IgG Fc-specific antibody conjugated to the maytansinoid DM1 via a non-cleavable linker. The antibody portion is a polyclonal antibody specific for the Fc region of human IgG. DM1 is a cytotoxic small molecule that inhibits cell division by blocking tubulin polymerization. The non-cleavable linker connecting DM1 to the antibody is stable in extracellular fluids but can be cleaved by an unspecified mechanism upon entry into cells. Alternatively, killing assays were also performed using anti-HER2 antibodies directly linked to DM1 via the linker. Hybridoma clones selected for their activity were further characterized by nucleotide and amino acid sequencing and CDR determination, as shown in Example 2 below.

[0053] 1. Competition with trastuzumab for binding to HER2-ECD by EIA Rationale: Trastuzumab is an FDA-approved anti-Her2 therapeutic antibody currently used in standard of care as a naked antibody or antibody-drug conjugate. Therefore, one of the screening assays and selection criteria was to examine the ability of fully human HER2 antibodies to compete with trastuzumab for binding to the HER2 protein. Methods: A 96-well high-binding EIA plate was coated with 10 ng of HER2 protein overnight at 4°C. After washing and blocking with 4% milk protein for 1 hour at 37°C, the plate was washed three times and then incubated for 1 hour at 37°C with either hybridoma supernatants or fully human purified antibodies from 202 clones selected in the confirmation EIA. Trastuzumab was used as a positive control, and human IgG was used as a negative control. After 1 hour of incubation at 37°C, the plate was washed six times and then incubated with a biotin-labeled anti-HER2 antibody (anti-HER2 TRAST-Biotin) for 1 hour at 37°C. Following washing, horseradish peroxidase (HRP)-conjugated streptavidin was added. After washing, the colorimetric substrate 3,3',5,5'-tetramethylbenzidine (TMB) was added, and absorbance was measured at 620 nm after 10 minutes. The results led to the investigation of the ability of anti-fully human antibodies to inhibit the binding of anti-HER2 trastuzumab conjugated with biotin (anti-HER2TRAST-biotin) to coated HER2. Results: Of 207 hybridoma clones tested, 27 hybridomas produced antibodies that could compete with anti-HER2 TRAST-biotin.

[0054] 2.Comparison of internalization with trastuzumab using Hab probe Rationale: Trastuzumab is an internalizing antibody. Upon binding to HER2 on the surface of HER2-expressing cells, the antibody-HER2 receptor complex translocates into the cell in a temperature-dependent manner. Based on this characteristic, antibody-drug conjugates in which trastuzumab is linked to a cytotoxic payload via a specific linker can deliver the cytotoxic drug to target cells, resulting in their killing. Two trastuzumab-based ADCs have been developed: T-DM1 (Kadcyla) and a conjugate of trastuzumab and deruxtecan (Enhertz), both of which are approved by the U.S. Food and Drug Administration (FDA). Therefore, we were interested in examining whether any internalizing, fully human anti-HER2 antibodies were present among the antibodies produced by the selected 207 hybridomas. Methods: Goat anti-human IgG (H+L) secondary antibody was conjugated to a pH probe (Promega) according to the manufacturer's instructions. 5 μg / ml of anti-HER2 antibody was premixed with 10 μg / ml of pHab-labeled goat anti-human antibody at 22-25°C for 30 minutes (antibody combination). SKBR3 or AU565 cells were detached with 5 mM EDTA and 2-4 × 10 5 Cells were incubated with the antibody combination and placed in a 5% CO2 incubator for 20 hours. At the end of the incubation, cells were washed twice with cold PBS, resuspended in PBS, and fluorescence was measured using a Flow IntelliCyte in the Mean FL2-H channel. If the antibody is internalized, the measured fluorescence units will increase. Results: Using a cutoff value of 10,000 RFU (relative fluorescence units), 96 hybridoma clones producing internalizing antibodies were identified from 207 clones tested. All 27 clones that produced antibodies competitive with trastuzumab were internalizing antibodies. The 27 internalizing antibodies were then examined for their biological activities, including inhibition of HER2 signaling compared with trastuzumab, and further screened by inhibition of migration assays. Selected antibodies were then tested in killing assays.

[0055] 3. Inhibition of p-AKT phosphorylation (inhibition of cell signaling in HER2-overexpressing breast cancer cells SKBR3) Rationale: In HER2-overexpressing cells, such as breast cancer cells SKBR3, HR2 is phosphorylated, leading to activation of the AKT signaling pathway without affecting the ERK1 / 2 pathway. Therefore, antibodies that inhibit HER2 function inhibit AKT phosphorylation (P-AKT). Trastuzumab (an anti-HER2 antibody) is known to inhibit such phosphorylation. Therefore, examining the P-AKT inhibitory ability of selected antibodies is a rational approach for further antibody selection. Methods: These studies used HER2-overexpressing SKBR3 cells. 8 × 10 4Cells were seeded in 35-μm dishes in 2 ml of McCoy's-5A medium supplemented with 10% fetal bovine serum. After 72 hours, cells were washed twice with serum-free medium and serum-starved for 2 hours. Test antibodies were added at 10 μg / ml, and cells were harvested in RIPA buffer in the presence of protease and phosphatase inhibitors. Cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, transferred to a pdf membrane, and Western blot analysis was performed for phospho-AKT and phospho-ERK1 / 2. Test antibodies were added along with human IgG as a negative control and the anti-Her2 antibody trastuzumab as a positive control. Results: Antibodies capable of inhibiting P-HER2, P-ERK1 / 2 and / or P-AKT expression were further selected.

[0056] 4. Inhibition of migration by HER2-overexpressing breast cancer cells SKBR3 Rationale: HER2-overexpressing cells have a greater ability to migrate to distant sites. Therefore, examining the ability of anti-HER2 antibodies to inhibit cell migration is a rational approach to investigating and selecting effective anti-HER2 antibodies. Methods: Migration was measured using the transwell method. Here, cells were seeded onto collagen-coated 8 μm filters, as described by Guha et al., 2020, and allowed to migrate through the filters. Antibodies were added at increasing concentrations from 0 to 50 μg / ml. Human IgG was used as a negative control, and the anti-HER2 antibody trastuzumab was used as a positive control. Results: The antibody inhibited migration in a dose-dependent manner, with 50% inhibition at 50 μg / ml.

[0057] 5. Stimulation of Cytotoxicity or Inhibition of Cell Proliferation in the Presence of Fab Anti-Human Fc Conjugated to DM1 Cytotoxic Payload Rationale: Internalizing antibodies can deliver a cytotoxic payload to the target-bearing cells for which the internalizing antibody was developed. Measuring the killing efficacy of selected antibodies is a way to determine their internalization capacity and ability to deliver a cytotoxic payload. Methods: The ability of selected antibodies to deliver the cytotoxic payload DM-1 conjugated to goat anti-human Fc to SKBR3 cells was assessed using the method described in (Marquez, J., J. Dong, C. Dong, C. Tian and G. Serrero (2021). “Identification of Prostaglandin F2 Receptor Negative Regulator (PTGFRN) as an Internalizable Target in Cancer Cells for Antibody-Drug Conjugate Development.” PLoS One 16(1): e0246197. Briefly, 8000 SKBR3 cells were seeded in a 96-well plate in culture medium supplemented with an anti-HER2 antibody and various concentrations of Fab-αHFc-NC-DM1. Human IgG containing Fab-αHFc-NC-DM1 was used as a negative control. After 72 hours, cell proliferation and cell killing were measured using the Cell-Glo assay (Promega) according to the manufacturer's instructions.

[0058] Results: The results for antibodies shown to stimulate cell killing are shown below. Overall results and antibody selection: Based on the above five assays, six hybridomas were selected that produced antibodies that showed the highest activity in internalization and in competing with trastuzumab for binding to HER2 protein. These six selected antibodies were 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1. Figure 1 shows the results of a competition assay for trastuzumab binding to HER2 protein using the above-described method, with increasing doses of selected fully human anti-HER2 antibodies 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1. Trastuzumab and human IgG were used as positive and negative controls. Figure 1 also shows the results of a competition assay for trastuzumab binding to HER2 protein using six selected fully human antibodies at concentrations ranging from 0.01 μg / ml to 2.5 μg / ml. Five of the six antibodies achieved an ED50. Five of the six antibodies strongly inhibited the binding of trastuzumab-biotin to HER2, with ED50 values ​​of 0.04ug / ml for 12E3, 0.06ug / ml for 11D7 and 3G7, 0.09ug / ml and 0.1ug / ml for 2A2 and 8C1, respectively, with 2D7 showing an ED50 similar to that of trastuzumab. Antibodies 11D7, 12E3, 2D7, and 3G7 were determined to be of the isotype IgG1. Antibodies 2A2 and 8C1 were of the isotype IgG2a. The affinity of 11D7, 2A2, 12E3, 2D7, 3G7, 8C1 and trastuzumab for binding to HER2 was investigated by biolayer interference using Octet Red 96, as shown in Table 6. Antibodies 11D7, 2A2, 12E3, 2D7, 3G7 and 8C1 each had a 3.6×10 -9 It was found to have an excellent Kd ranging from 3.1 × 10 to 10 M, which was found to be 3.1 × 10 in these experiments. -10 Commercially available trastuzumab with a Kd of 5 x 10 -9 It has been reported to have a Kd of M, which is within the range of the Kd measured for reported and manufactured trastuzumab. [Table 7]

[0059] The migration assay, which measures the ability of cells to migrate through collagen-coated transwells, is a well-recognized assay for assessing one of the hallmarks of tumorigenicity and metastasis in cancer cells. Determining whether an antibody can inhibit migration provides an indication of future therapeutic potential and aids in the selection of antibodies for further preclinical testing. The method used has been previously described, except that the cells used were HER2-overexpressing breast cancer SKBR3 cells and the migration time was 40–48 hours at 75,000 cells in medium supplemented with 2% fetal bovine serum (Guha, R., B. Yue, J. Dong, A. Banerjee and G. Serrero (2021). "Anti-progranulin / GP88 antibody AG01 inhibits triple-negative breast cancer cell proliferation and migration." Breast Cancer Res Treat 186(3): 637-653). Six selected antibodies were tested for their ability to inhibit migration, a hallmark of tumorigenesis and metastasis. Table 7 summarizes the data from all assays performed with the 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1 antibodies, demonstrating inhibition of migration of SKBR3 cells.

[0060] The selected 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1 fully human anti-HER2 antibodies have the ability to induce cell killing of HER2-overexpressing SKBR3 cells compared to trastuzumab, as shown in Table 7A. These data confirm that the 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1 fully human anti-HER2 antibodies are capable of internalizing and delivering a cytotoxic payload, and are therefore suitable for the development of antibody-drug conjugates (ADCs). [Table 8] 12E3 was directly conjugated to DM1 using Mal-VC-PAB-DM1 linker payload moiety (MEDCHEM express) according to the manufacturer's instructions. Table 7B shows that direct conjugation of 12E3 can inhibit cell proliferation at concentrations as low as 0.01 nM. [Table 9]

[0061] In another set of experiments, six mAb clones from group A (which compete with trastuzumab for binding to HER2) and five clones from group B (which do not compete with trastuzumab for binding to HER2) were selected. Among these, 12E3 from group A and 12A6 from group B were examined for their biochemical and biological properties. 12E3 and 12A6 were separately conjugated with DM1 to form the ADCs 12E3-DM1 and 12A6-DM1, respectively. These ADCs were then tested for their ability to inhibit the proliferation of HER2-overexpressing breast cancer cells alone or in combination with the HER2-overexpressing breast cancer cell lines AU565 and SKBR3 in vitro. As shown in Figure 2, both conjugates, alone or in combination, dose-dependently reduced the proliferation of AU565 cells. As shown in Figure 3, both conjugates, alone or in combination, dose-dependently reduced the proliferation of SKBR3 cells. Therefore, this data demonstrates that the antibodies in Group A (e.g., 12E3-DM1) and Group B (e.g., 12A6-DM1) anti-HER2 ADCs inhibit HER2 + The results show that the combination of ADCs in each group inhibited cell proliferation in a dose-dependent manner. The combination of ADCs in each group showed an enhanced effect in inhibiting cell proliferation, demonstrating the advantage of combining anti-HER2 MAbs targeting different HER2 epitopes.

[0062] Example 4 Sequence analysis of fully human antibodies 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1 The hybridoma cell lines were thawed and cultured. Cells were counted, and the RNA was converted to cDNA by reverse transcription. Several sets of mouse heavy and light chain-specific primers were used to perform PCR amplification of heavy and light chain variable region sequences. A small number of PCR products were loaded onto an agarose gel and electrophoresed to confirm PCR bands. The PCR products were mixed and sequenced using a Miseq sequencer. NGS sequencing data was processed using BCR analysis software. DNA sequence data from all constructs was analyzed to determine consensus sequences for the heavy and light chains. The consensus sequences were compared with known variable region sequences to rule out artifacts and / or process contamination. The consensus sequences were then analyzed using an online tool to confirm whether they could encode the resulting immunoglobulin (e.g., one containing both heavy and light chains). After determining the amino acid sequences of these antibodies, the amino acid sequences of the antibody CDRs were determined according to the Kabat method. The amino acid and polynucleotide sequences of the antibodies are shown below.

[0063] 1. Fully human 11D7 antibody AGRD017-11D7-IgG (variable heavy chain (VH)) Amino acid sequence (CDRs underlined): EVQLVESGGGLVQPGRSLRLSCAASGFTFD DYAMH WVRQAPGKGLEWVS GIAWNGGILGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCAK DTGSYSHWYFDL WGRGTLVTVSS (SEQ ID NO: 41) DNA sequence: GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTC TCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCT CCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTGCTTGGAATGGTGGTATCTTAGGCTAT GCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTAT CTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATACG GGGAGTTATTCTCACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCC TCAG (SEQ ID NO: 63) [Table 10]

[0064] AGRD017-11D7-IgK (variable light chain (VL)) Amino acid sequence (CDRs underlined): EIVMTQSPATLSVSPGERATLSC RASQSVRSNLA WYQQKPGQAPRLLIY GASTRAT GIPA RFSGSGSGTEFTLTISSLQSEDFAVYCC QQYNNWPT FGQGTKVEIK (SEQ ID NO: 42) DNA sequence: GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACC CTCTCCTGCAGGGCCAGTCAGAGTGTTAGGAGCAACTTAGCCTGGTATCAGCAGAAACCT GGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCT GAAGATTTTGCAGTTTATTGCTGTCAGCAGTATAATAACTGGCCTACGTTCGGCCAAGGG ACCAAGGTGGAAATCAAAC (SEQ ID NO: 64) [Table 11]

[0065] 2. Fully human 2A2 antibody AGRD017-2A2-IgG (variable heavy chain (VH)) Amino acid sequence (CDRs underlined): EVQLVESGGGLVQPGRSLRLSCAASGFTFD DYAMH WVRQAPGKGLEWVS GISWNGGTLGY ADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCAK DTGSYSHWYFDL WGRGTLVTVS S (SEQ ID NO: 43) DNA sequence: GAAGTGCAGTTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTC TCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCT CCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATGGTGGTACCTTAGGCTAT GCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTAT CTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATACG GGGAGTTATTCTCACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCC TCAG (SEQ ID NO: 65) [Table 12]

[0066] AGRD017-2A2-IgK (variable light chain (VL)) Amino acid sequence (CDRs underlined): EIVMTQSPATLSVSPGERATLSC RASQSVRSNLA WYQQKPGQAPRLLIY GASTRAT GIPA RFSGSGSGTEFTLTISSLHSEDFAVYYC QQYNNWPT FGQGTKVEIK (SEQ ID NO: 44) DNA sequence: GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACC CTCTCCTGCAGGGCCAGTCAGAGTGTTAGGAGCAACTTAGCCTGGTATCAGCAGAAACCT GGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATTAGCAGCCTGCACTCT GAAGATTTTGCAGTTTATTACTGTCAGCAGTATAACAACTGGCCTACGTTCGGCCAAGGG ACCAAGGTGGAAATCAAAC (SEQ ID NO: 66) [Table 13]

[0067] 2. Fully human 12E3 antibody AGRD017-12E3-IgG (variable heavy chain (VH)) Amino acid sequence (CDRs underlined): EVQLVGSGGGLVQPGRSLRLSCAASGFTFD DYAMH WVRQAPGKGLEWVS GISWNGGTLGY AGSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCAK DTGSYSHWYFDL WGRGTLVTVS S (SEQ ID NO: 45) DNA sequence: GAAGTGCAGCTGGTGGGGTCTGGGGGAGGTTTGGTACAGCCTGGCAGGTCCCTGAGACTC TCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCT CCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATGGTGGTACGTTGGGCTAT GCGGGCTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTAT CTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATACG GGGAGTTATTCTCACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCC TCAG (SEQ ID NO: 67) [Table 14]

[0068] AGRD017-12E3-IgK (variable light chain (VL)) Amino acid sequence (CDRs underlined): EIVMTQSPATLSVSPGERATLSC RASQSVRSNLA WYQQKPGQAPRLLIY GASTRAT GIPA RCSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPT FGQGTKVEIK (SEQ ID NO: 46) DNA sequence: GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACC CTCTCCTGCAGGGCCAGTCAGAGTGTTAGAAGCAACTTAGCCTGGTACCAGCAGAAACCT GGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCC AGGTGCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCT GAGGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCTACGTTCGGCCAAGGG ACCAAGGTGGAAATCAAAC (SEQ ID NO: 68) [Table 15]

[0069] 3. Fully human 2D7 antibody AGRD017-2D7-IgG (variable heavy chain (VH)) Amino acid sequence (CDRs underlined): EVQLVESGGGLVQPGRSLRLSCAASGFTFD DYAMH WVRQAPGKGLEWVS GISWNSGTMGY AGSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCAK DTGSYSHWYFDL WGRGTLVTVS S (SEQ ID NO: 47) DNA sequence: GAAGTGCAGCTGGTGGAGTCTGGGGGAGGTTTGGTACAGCCTGGCAGGTCCCTGAGACTC TCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCT CCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTACCATGGGCTAT GCGGGCTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTAT CTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATACG GGGAGTTATTCTCACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCC TCAG (SEQ ID NO: 69) [Table 16]

[0070] AGRD017-2D7-IgK (variable light chain (VL)) Amino acid sequence (CDRs underlined): EIVMTQSPATLSVSPGERATLSC RASQSVRTNLA WYQQKPGQAPRLLIY GASTRAT GIPA RFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPT FGQGTKVEIK (SEQ ID NO: 48) DNA sequence: GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACC CTCTCCTGCAGGGCCAGTCAGAGTGTTAGACCAACTTAGCCTGGTACCAGCAGAAACCT GGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGCATCCCAGCC AGGTTCAGTGGTAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCT GAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCTACGTTCGGCCAAGGG ACCAAGGTGGAAATCAAAC (SEQ ID NO: 70) [Table 17]

[0071] 4. Fully human 3G7 antibody AGRD017-3G7-IgG (variable heavy chain (VH)) Amino acid sequence (CDRs underlined): EVQLVESGGGLVQPGRSLRLSCAASGFTFD DYAMH WVRQAPGKGLEWVS GVSWNGGTMGY AGSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCAK DTGSYSHWYFDL WGRGTLVTVS S (SEQ ID NO: 49) DNA sequence: GAAGTGCAGCTGGTGGAGTCTGGGGGAGGTTTGGTACAGCCTGGCAGGTCCCTGAGACTC TCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCT CCAGGGAAGGGCCTGGAGTGGGTCTCAGGTGTTAGTTGGAATGGTGGTACCATGGGCTAT GCGGGCTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTAT CTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATACG GGGAGTTATTCTCACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCC TCAG (SEQ ID NO: 71) [Table 18]

[0072] AGRD017-3G7-IgK (variable light chain (VL)) Amino acid sequence (CDRs underlined): EIVMTQSPATLSVSPGERATLSC RASQSVRSNLA WYQQKPGQAPRLLIY GASTRATGIPG RFSGSGSGTEFILTISSLQSEDFAVYYC QQYNNWPT FGQGTKVEIK (SEQ ID NO: 50) DNA sequence: GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACC CTCTCCTGCAGGGCCAGTCAGAGTGTTAGAGCAACTTAGCCTGGTACCAGCAGAAACCT GGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGGC AGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCATTCTCACCATCAGCAGCCTGCAGTCT GAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCTACGTTCGGCCAAGGG ACCAAGGTGGAAATCAAAC (SEQ ID NO: 72) [Table 19]

[0073] 5. Fully human 8C1 antibody AGRD017-8C1-IgG (variable heavy chain (VH)): Amino acid sequence (CDRs underlined): EVQLVESGGGLVQPGRSLRLSCTASGFTFD DYAMH WVRQAPGKGLEWVS GISWNGGTLGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCAK DTGSYSHWYFDL WGRGTLVTVSS (SEQ ID NO: 51) DNA sequence: GAAGTGCAGTTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTACAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATGGTGGTACCT tagCTATGCG GACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATACGGGGAGTTATTCTCACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAG (Sequence number 73) [Table 20]

[0074] AGRD017-8C1-IgK (variable light chain (VL)): Amino acid sequence: EIVMTQSPATLSVSPGERATLSC RASQSVRSNLA WYQQKPGQAPRLLIY GASTRAT GIPARFSGSGSGTEFTLTISSLHSEDFAVYYC QQYNNWPT FGQGTKVEIK (SEQ ID NO: 52) DNA sequence: GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACC CTCTCCTGTAGGGCCAGTCAGAGTGTTAGGAGCAACTTAGCCTGGTATCAGCAGAAACCT GGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATTAGCAGCCTGCACTCT GAAGATTTTGCAGTTTATTACTGTCAGCAGTATAACAACTGGCCTACGTTCGGCCAAGGG ACCAAGGTGGAAATCAAAC (SEQ ID NO: 74) [Table 21]

[0075] To align the amino acid sequences of 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1 with trastuzumab and pertuzumab, the sequences of both antibodies were obtained from published reports and are shown below. Trastuzumab AA sequence Anti-HER2 light chain (1 and 2) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 133) Anti-HER2 heavy chain (1 and 2) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 134) Pertuzumab AA sequence Pertuzumab light chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 135) Pertuzumab heavy chain amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 136) The amino acid sequences of the variable regions of the 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1 fully human antibodies were aligned with those of trastuzumab and pertuzumab, whose CDRs were determined according to Kabat, and the respective CDR1, CDR2, and CDR3 are underlined.

[0076] Heavy Chain Alignment [Table 22] These alignments reveal that the CDR3 regions (CDRs underlined) of the variable heavy chains (VH) of 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1 differ from those of trastuzumab ("Trastuzu") and pertuzumab ("Pertuzu").

[0077] Light Chain Alignment [Table 23] From these alignments, it is clear that the CDR3 regions of the variable light chains (VH) of 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1 are distinct from those of trastuzumab and pertuzumab. AGRD017 6 clone alignment, H chain [Table 24] AGRD017 6 clone alignment, light chain [Table 25] From these studies, it can be concluded that the 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1 AGRD017 clones have similar sequences but are distinct antibodies from trastuzumab and pertuzumab.

[0078] Example 5 : A fully human anti-HER-2 antibody that does not compete with trastuzumab for binding to the HER-2 receptor We also focused our efforts on characterizing fully human anti-HER2 antibodies that bind to the HER-2 receptor and internalize but do not compete with trastuzumab for binding to HER-2. The rationale was to select fully human antibodies against alternative HER2 epitopes from those targeted by currently available antibodies, such as pertuzumab and trastuzumab, for therapeutic applications as internalizing antibodies that can be used in antibody-drug conjugates. These antibodies were developed using the same antibody development procedures described above, using human HER-ECD as the immunogen. Antibody selection was based on 1) ELISA binding to HER-ECD, 2) flow binding to SKBR3 cells, 3) lack of competition with trastuzumab for binding to HER2, 4) internalization, 6) antibody Kd determination and epitope binning, 7) cytotoxicity (killing) assays with the anti-HER2-selected antibodies in the presence of Fab-αHFc-NC-DM1 as described above, and 8) IgG sequencing. Among the antibodies examined, five were selected based on these characteristics, particularly their lack of competitive binding with trastuzumab for internalization and HER-2 binding (2A1, 7F9, 9E4, 11C9, and 12A6; all of isotype IgG1-κ (IgG1κ)). Epitope binning studies also revealed that 2A1, 7F9, 9E4, 11C9, and 12A6 bind to epitopes on HER2 distinct from trastuzumab. Table 8 summarizes the functional properties of these antibodies ("clones"). [Table 26] All experiments were performed using SKBR3 cells.

[0079] The amino acid and nucleic acid sequences of the variable heavy (VH) and variable light (VL) polypeptide chains of the fully human 2A1, 7F9, 9E4, 11C9, and 12A6 antibodies were determined as described above. The CDRs were determined according to the Kabat method and are underlined in the amino acid sequences shown below.

[0080] 1.2A1 antibody AGRD017-2A1-IgG(VH) Amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYWMS WVRQAPGKGLEWVA NIKQDGSEKYY VDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR EVGPGDY WGQGTLVTVSS (SEQ ID NO: 53) DNA sequence: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTC TCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGAGCTGGGTCCGCCAGGCT CCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATACTAT GTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTAT CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGTG GGGCCCGGGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO: 75) [Table 27]

[0081] AGRD017-2A1-IgK(VL) Amino acid sequence: DIQMTQSPSTLSASVGDRVTITC RASQSISNWVA WYQQKPGKAPKLLIH TASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYYSYSPT FGQGTKVEIK (SEQ ID NO: 54) DNA sequence: GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCCGGGCCAGTCAGAGTATTAGTAACTGGGTGGCCTGGTATCAGCAGAAACCA GGGAAAGCCCCTAAGCTCCTGATCCATACGGCATCTAGCTTAGAAAGTGGGGTCCCATCA AGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCT GATGATTTTGCAACTTATTACTGCCAACAGTATTATAGTTATTCTCCGACGTTCGGCCAA GGGACCAAGGTGGAAATCAAAC (SEQ ID NO: 76)

Table 28

[0082] 2.7F9 antibody AGRD017-7F9-IgG(VH) Amino acid sequence: QMQLVQSGPEVKKPGTSVKVSCKASGFTFT SSAVQ WVRQARGQRLEWIG WIVVGSGNTNY AQKFQE RVTITRDMSTSTAYMELSSLRSEDTAVYYCAA GYYGSGSPYYYYYGMDV WGQGT TVTVSS (SEQ ID NO: 55) DNA sequence: CAAATGCAGCTGGTGCAGTCTGGGCCTGAGGTGAAGAAGCCTGGGACCTCAGTGAAGGTC TCCTGCAAGGCTTCTGGATTCACCTTTACTAGCTCTGCTGTGCAGTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGATGGATCGTCGTTGGCAGTGGTAACACAAACTAC GCACAGAAGTTCCAGGAAAGAGTCACCATTACCAGGGACATGTCCACAAGCACAGCCTAC ATGGAGCTGAGCAGCCTGAGATCCGAGGACACGGCCGTGTATTACTGTGCGGCGGGGTAC TATGGTTCGGGGAGTCCTTACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACC ACGGTCACCGTCTCCTCAG (SEQ ID NO: 77) [Table 29]

[0083] AGRD017-7F9-IgK(VL) Amino acid sequence: DIQMTQSPSSVSASVGDRVTITC RASQGISSWLA WYQQKSGKAPKLLIY AASSLQS GVPS RFSGSGSGTDFTLTINSLQPEDFATYYC QQVDSFPFT FGGGTKVESK (SEQ ID NO:56) DNA sequence: GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAATCA GGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCTAGTTTGCAAAGTGGGGTCCCATCA AGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCT GAAGATTTTGCAACTTACTATTGTCAACAGGTTGACAGTTTCCCGTTCACTTTCGGCGGA GGGACCAAGGTGGAGAGCAAAC (SEQ ID NO: 78) [Table 30]

[0084] 3.9E4 antibody AGRD017-9E4-IgG(VH) Amino acid sequence: QVQLVESGGGLVKPGGSLRLSCAASGFTFS DYYMS WLRQAPGRGLEWVS YISSSGITIYY ADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DGQQLADYYYYGMDV WGQGTTV TVSS (SEQ ID NO: 57) DNA sequence: CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTC TCCTGTGCAGCCTCTGGATTCACCTTCATGTGACTACTACATGAGCTGGCTCCGCCAGGCT CCAGGGAGGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTATTACCATATACTAC GCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTAT CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGA CAGCAGCTGGCCGACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTC ACCGTCTCCTCAG (SEQ ID NO: 79) [Table 31]

[0085] AGRD017-9E4-IgK(VL) Amino acid sequence: EIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLT WYQQKPGQAPRLLIY GASSRAT GIP DRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSLT FGGGTKVEIK (SEQ ID NO: 58) DNA sequence: GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACC CTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAACCTGGTACCAGCAGAAA CCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAG CCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACTCACTTTCGGCGGA GGGACCAAGGTGGAGATCAAAC (SEQ ID NO: 80) [Table 32]

[0086] 4.11C9 antibody AGRD017-11C9-IgG(VH) Amino acid sequence: QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA VIWYDGGNKDY ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR VGQLAHYYYGMDV WGQGTTVTV SS (SEQ ID NO: 59) DNA sequence: CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTC TCCTGTGCAGCGTCTGGATTCACGTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCT CCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAGGTAATAAAGACTAT GCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGTAGGG CAGCTGGCACACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCCTCAG (SEQ ID NO: 81) [Table 33]

[0087] AGRD017-11C9-IgK(VL) Amino acid sequence: DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLD WYLQKPGQSPQLLIY LGSNRA S GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQSPPT FGQGTKVEIK (SEQ ID NO: 60) DNA sequence: GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCC ATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGG TACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCC TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATC AGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAAGTCCT CCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC (SEQ ID NO: 82) [Table 34]

[0088] 5.12A6 antibody AGRD017-12A6-IgG(VH) Amino acid sequence: QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA VIWYDGSDDYY ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR ELVRGASFDY WGQGTLVTVSS (SEQ ID NO: 61) DNA sequence: CAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTC TCCTGTGCAGCGTCTGGCTTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCT CCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTGATGACTACTAT GCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAGCTG GTTCGGGGAGCCTCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO: 83) [Table 35]

[0089] AGRD017-12A6-IgK(VL) Amino acid sequence: EILMTQSPLSLPVTPGEPATISC RSSQSLLHSNGYNYLD WYLQKPGQSPQLLIY LGSNRAS GVPDRFSGSGSGTDFTLRISRVEAEDVGVYYC MQALQTPLT FGGGTKVEIK (SEQ ID NO: 62) DNA sequence: GAAATTTTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCACC ATCTCCTGCAGGTCTAGTCAGAGCCTCCTCCATAGTAATGGATACAACTATTTGGATTGG TACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCC TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAGAATC AGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCG CTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC (SEQ ID NO: 84) [Table 36]

[0090] A comparison of the variable heavy (VH) and variable light (VL) polypeptide chains by the alignment below shows that these antibodies differ from each other (CDRs as determined by Kabat are underlined). [Table 37-1] [Table 37-2]

[0091] The alignment shown below also shows that the VH and VL CDR sequences of these antibodies differ from Trastuzumab and Pertuzumab. [Table 38-1] [Table 38-2]

[0092] Other embodiments will be apparent to one skilled in the art from consideration of the specification and instruction provided herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the present invention being indicated by the following claims.

Claims

1. a) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 1, 2, and 3 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 4, 5, and 6 (antibody 11D7); b) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 1, 7, and 3 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 4, 5, and 6 (antibody 2A2); c) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 1, 9, and 3 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 4, 5, and 6 (antibody 12E3); d) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 1, 10, and 3 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 4, 5, and 6 (antibody 2D7); e) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 1, 12, and 3 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 4, 5, and 6 (antibody 3G7); f) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 1, 13, and 3 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 4, 5, and 6 (antibody 8C1); g) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 14, 15, and 16 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 17, 18, and 19 (antibody 2A1); h) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 20, 21, and 22 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 23, 24, and 25 (antibody 7F9); i) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 26, 27, and 28 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 29, 30, and 31 (antibody 9E4); j) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 32, 33, and 34 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 35, 36, and 37 (antibody 11C9); k) a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 32, 38, and 39 and a light chain variable region comprising the CDR sequences of SEQ ID NOs: 35, 36, and 40 (antibody 12A6); l) a heavy chain variable region comprising SEQ ID NO: 41 and a light chain variable region comprising SEQ ID NO: 42 (antibody 11D7); m) a heavy chain variable region comprising SEQ ID NO: 43 and a light chain variable region comprising SEQ ID NO: 44 (antibody 2A2); n) a heavy chain variable region comprising SEQ ID NO: 45 and a light chain variable region comprising SEQ ID NO: 46 (antibody 12E3); o) a heavy chain variable region comprising SEQ ID NO: 47 and a light chain variable region comprising SEQ ID NO: 48 (antibody 2D7); p) a heavy chain variable region comprising SEQ ID NO: 49 and a light chain variable region comprising SEQ ID NO: 50 (antibody 3G7); q) a heavy chain variable region comprising SEQ ID NO: 51 and a light chain variable region comprising SEQ ID NO: 52 (antibody 8C1); r) a heavy chain variable region comprising SEQ ID NO: 53 and a light chain variable region comprising SEQ ID NO: 54 (antibody 2A1); s) a heavy chain variable region comprising SEQ ID NO: 55 and a light chain variable region comprising SEQ ID NO: 56 (antibody 7F9); t) a heavy chain variable region comprising SEQ ID NO: 57 and a light chain variable region comprising SEQ ID NO: 58 (antibody 9E4); u) a heavy chain variable region comprising SEQ ID NO: 59 and a light chain variable region comprising SEQ ID NO: 60 (antibody 11C9); v) a heavy chain variable region comprising SEQ ID NO: 61 and a light chain variable region comprising SEQ ID NO: 62 (antibody 12A6); w) a heavy chain variable region encoded by any of the polynucleotides of SEQ ID NOs: 63-84 and a corresponding light chain variable region; and / or optionally comprising a conservatively substituted derivative thereof, comprising up to three amino acid substitutions in one or more CDRs thereof and / or up to ten amino acid substitutions in the heavy and / or light chains thereof; An isolated antibody or antigen-binding fragment thereof that specifically binds to human HER2.

2. The antibody of claim 1, which is internalized into cells that express HER2 in vitro and / or in vivo.

3. The antibody of any one of claims 1 to 2, which competes with trastuzumab for binding to the HER2 receptor on cells.

4. 4. The antibody of claim 3, selected from the group consisting of antibodies 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1.

5. The antibody of any one of claims 1 to 4, which does not compete with trastuzumab for binding to the HER2 receptor on cells.

6. 6. The antibody of claim 5, selected from the group consisting of antibodies 2A1, 7F9, 9E4, 11C9, and 12A6.

7. The antibody of any one of claims 1 to 6, which is an isolated monoclonal antibody.

8. The antibody of claim 8, wherein the monoclonal antibody is a human monoclonal antibody.

9. 9. The antibody of any one of claims 1 to 8, wherein the antibody is derived from a human antibody, human IgG, human IgG1, human IgG2, human IgG2a, human IgG2b, human IgG3, human IgG4, human IgM, human IgA, human IgA1, human IgA2, human IgD, human IgE, dog antibody, dog IgGA, dog IgGB, dog IgGC, dog IgGD, chicken antibody, chicken IgA, chicken IgD, chicken IgE, chicken IgG, chicken IgM, chicken IgY, goat antibody, goat IgG, mouse antibody, mouse IgG, pig antibody, rat antibody, llama antibody, alpaca antibody, shark antibody, and camel antibody.

10. Optionally, F ab , F ab2 , Fab' single chain antibody, F v 9. A derivative of the antibody of any one of claims 1 to 8, selected from the group consisting of: a single chain, a monospecific antibody, a bispecific antibody, a trimeric antibody, a multispecific antibody, a multivalent antibody, a chimeric antibody, a canine-human chimeric antibody, a canine-mouse chimeric antibody, an antibody comprising a canine Fc, a humanized antibody, a human antibody, a caninized antibody, a CDR-grafted antibody, a shark antibody, and a nanobody.

11. and an immobilized detectable label, optionally the detectable label being selected from the group consisting of fluorescein, DyLight, Cy3, Cy5, FITC, HiLyte Fluor 555, HiLyte Fluor 647, 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein, 5-FAM, hydroxytryptamine, 5-hydroxytryptamine (5-HAT), 6-carboxyfluorescein (6-FAM), FITC, 6-carboxy-1,4-dichloro-2',7'-dichlorofluorescein (TET), 6-carboxy-1,4-dichloro-2',4',5',7'-tetra-chlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (6-JOE), Alexa fluor, Alexa fluor 350, Alexa fluor 405, Alexa fluor 430, Alexa fluor 488, Alexa fluor 500, Alexa fluor 514, Alexa fluor 532, Alexa fluor 546, Alexa fluor 555, Alexa fluor 568, Alexa fluor 594, Alexa fluor 610, Alexa fluor 633, Alexa fluor 635, Alexa fluor 647, Alexa fluor 660, Alexa fluor 680, Alexa fluor 700, Alexa fluor 750, BODIPY fluorophore, BODIPY 492 / 515, BODIPY 493 / 503, BODIPY 500 / 510, BODIPY 505 / 515, BODIPY 530 / 550, BODIPY 542 / 563, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650-X, BODIPY 650 / 665-X, BODIPY 665 / 676, FL, FL ATP, FI-ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X SE, TR, TR ATP, TR-X SE, rhodamine, rhodamine 110, rhodamine 123, rhodamine B, rhodamine B 200, rhodamine BB, rhodamine BG, rhodamine B extra, 5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-carboxyrhodamine 6G, lissamine, lissamine rhodamine B, phallicidin, phalloidin, rhodamine red, rhod-2, 6-carboxy-X-rhodamine (ROX), carboxy-X-rhodamine (5-ROX), sulforhodamine B can The antibody derivative of any one of claims 1 to 10, wherein the antibody derivative is selected from the group consisting of Rhodamine C, sulforhodamine G extra, 6-carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TRITC), rhodamine WT, Texas Red, and Texas Red-X.

12. 12. The antibody of any one of claims 1 to 11, comprising an attached effector moiety, optionally said effector moiety selected from the group consisting of a cytotoxic drug, mertansine (DM1), a toxin, diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, and a radiochemical.

13. 13. The antibody of claim 12, further comprising a cleavable linker disposed between the antibody and the effector moiety, the cleavable linker releasing the effector moiety into or within a cell.

14. An isolated polynucleotide encoding the antibody of any one of claims 1 to 13, optionally having at least one nucleic acid sequence of SEQ ID NOs: 63 to 84.

15. 15. An expression vector comprising one or more polynucleotides according to claim 14.

16. 16. A host cell comprising the isolated polynucleotide of claim 14 and / or the expression vector of claim 15.

17. A composition comprising at least one antibody or derivative according to any one of claims 1 to 13; at least one isolated polynucleotide according to claim 14; at least one expression vector according to claim 15; and / or at least one host cell according to claim 16; or a combination thereof; and a pharmaceutically acceptable carrier.

18. The composition of claim 17, comprising at least one first antibody or derivative of any one of claims 1 to 14 and trastuzumab.

19. The composition according to claim 17, comprising at least two or more antibodies and / or derivatives according to any one of claims 1 to 14.

20. 19. The composition of claim 18, wherein the at least one first antibody is internalized by cells that express HER2 in vitro and / or in vivo and / or does not compete with trastuzumab for binding to HER2.

21. 21. The composition of claim 19 or 20, further comprising trastuzumab.

22. 22. The composition of any one of claims 17 to 21, comprising at least one antibody selected from the group consisting of 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1 and at least one antibody selected from the group consisting of 2A1, 7F9, 9E4, 11C9, and 12A6.

23. 14. A method for detecting HER2 on a cell, comprising contacting a test biological sample with an antibody or derivative according to any one of claims 1 to 13, and detecting antibody bound to the biological sample or a component thereof.

24. 24. The method of claim 23, further comprising comparing the amount of binding to the test biological sample or its component with the amount of binding to a control biological sample or its component, wherein increased binding to the test biological sample or its component compared to the control biological sample or its component indicates the presence of HER2-expressing cells in the test biological sample.

25. 25. The method of claim 23 or 24, wherein the test biological sample is mammalian cells, tissue, or blood.

26. The method according to any one of claims 23 to 25, which is an in vivo method or an in vitro method.

27. 14. A method for treating, preventing and / or ameliorating cancer in a mammal, comprising administering to said mammal at least one effective dose of a pharmaceutical composition comprising at least one antibody or derivative according to any one of claims 1 to 13.

28. 29. The method of claim 28, wherein the antibody comprises a cytotoxic effector moiety attached thereto, optionally the effector moiety is selected from the group consisting of a cytotoxic drug, mertansine (DM1), a toxin, diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, and a radiochemical.

29. 30. The method of claim 29, further comprising a cleavable linker disposed between the antibody and the effector moiety, wherein the cleavable linker releases the effector moiety into or within the cell.

30. The method of any one of claims 27 to 29, wherein the antibody is administered as an antibody-drug conjugate.

31. 28. The method of claim 27, comprising administering at least one first antibody or derivative according to any one of claims 1 to 13 and trastuzumab.

32. The method according to claim 27, comprising administering at least two or more antibodies and / or derivatives according to any one of claims 1 to 13.

33. 28. The method of claim 27, comprising administering at least two antibodies, wherein the at least two antibodies are internalized by cells that express HER2 in vitro and / or in vivo and / or do not compete with trastuzumab for binding to HER2.

34. 33. The method of claim 32, wherein the at least one antibody is internalized by cells that express HER2 in vitro and / or in vivo and / or the at least one antibody competes with trastuzumab for binding to the HER2 receptor on cells.

35. 35. The method of any one of claims 32-34, comprising administering at least one antibody selected from the group consisting of 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1 and at least one antibody selected from the group consisting of 2A1, 7F9, 9E4, 11C9, and 12A6, and optionally further comprising administering trastuzumab.

36. 36. The method of any one of claims 27-35, wherein multiple doses are administered to the animal; and / or the antibody is administered at a dose of about 1-50 mg / kg.

37. A kit for detecting HER2 expression in or on cells, comprising at least one antibody or derivative according to any one of claims 1 to 36 and instructions for use.

38. 38. The kit of claim 37, wherein the antibody or derivative is in lyophilized form.

39. 39. The kit of claim 37 or 38, wherein the at least one antibody is selected from the group consisting of 11D7, 2A2, 12E3, 2D7, 3G7, 8C1, 2A1, 7F9, 9E4, 11C9, and 12A6.

40. 39. The kit of claim 37 or 38, wherein the at least one antibody is selected from the group consisting of 11D7, 2A2, 12E3, 2D7, 3G7, and 8C1; and further comprising at least one antibody selected from the group consisting of 2A1, 7F9, 9E4, 11C9, and 12A6.

Images