Anti-HER2 / NEU Antibodies and Methods of Use

Abstract

Provided herein are anti-HER2 monoclonal antibodies or binding fragments thereof and uses thereof.

Description

[Technical field]

[0001] The present invention relates to anti-HER2 / NEU antibodies and methods of use. [Background technology]

[0002] The treatment of breast cancer has improved considerably over the last two decades as a result of, for example, earlier detection, better surgical techniques, a variety of new drugs, and novel imaging methods (e.g., to detect recurrence).

[0003] HER2 / neu is a member of the human epidermal growth factor receptor (HER / EGFR / ERBB) family. Amplification or overexpression of this oncogene has been shown to play a role in the development and progression of certain aggressive types of breast cancer. The protein is a biomarker and therapeutic target for approximately 30% of breast cancer patients.

[0004] The HER2 / neu protein is proteolytically cleaved by membrane-bound serine proteases to release the extracellular domain, which can then be detected and measured in body fluids. A HER2 / neu in vitro diagnostic (IVD) immunoassay for blood has been introduced (e.g., J. Immunol. 2003, 143:1311-1323), but the results were often confusing and the test was no longer used in clinical practice. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Carney et al., 2003, Clin.Chem., 49(10):1579-98 Summary of the Invention [Means for solving the problem]

[0006] Provided herein is an anti-HER2 monoclonal antibody or binding fragment thereof comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 95% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence at least 95% identical thereto. One embodiment provides an anti-HER2 monoclonal antibody or binding fragment thereof comprising a heavy chain and a light chain, wherein (i) the heavy chain comprises three CDR regions having the amino acid sequences of SEQ ID NOs: 5, 6, 7 or an amino acid sequence at least 95% identical thereto, and (ii) the light chain comprises three CDR regions having the amino acid sequences of SEQ ID NOs: 8, 9, 10 or an amino acid sequence at least 95% identical thereto. Another embodiment provides an anti-HER2 monoclonal antibody or binding fragment thereof described herein, wherein the antibody is conjugated to a detection agent. One embodiment provides a composition comprising an anti-HER2 antibody described herein and a carrier.

[0007] One embodiment provides a method for detecting a HER2 polypeptide or fragment thereof in a test sample, comprising: (a) contacting the test sample with an anti-HER2 monoclonal antibody or binding fragment thereof described herein under conditions that allow for the formation of a polypeptide / antibody complex; and (b) detecting the polypeptide / antibody complex of a), wherein detection of the polypeptide / antibody complex is indicative of the presence of a HER2 polypeptide in the sample. Another method provides a method for monitoring a HER2 polypeptide or fragment thereof in a sample from a subject, comprising: (a) contacting the sample with at least one of the anti-HER2 monoclonal antibodies or binding fragments thereof described herein under conditions that allow for the formation of a polypeptide / antibody complex; (b) detecting the polypeptide / antibody complex of a), wherein detection of the polypeptide / antibody complex indicates that a HER2 polypeptide or fragment thereof is present in the subject; and (c) performing steps (a) and (b) at multiple time points to monitor the HER2 polypeptide or fragment thereof in the subject over time. In one embodiment, the method further comprises contacting the sample of (a) with a second anti-HER2 antibody or fragment thereof comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence of at least 95% identity, a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence of at least 95% identity, or an anti-HER2 antibody or fragment thereof comprising a heavy chain and a light chain, wherein (i) the heavy chain comprises three CDR regions having the amino acid sequence of SEQ ID NO: 11, 12, 13 or an amino acid sequence of at least 95% identity, and (ii) the light chain comprises three CDR regions having the amino acid sequence of SEQ ID NO: 14, 15, 16 or an amino acid sequence of at least 95% identity. In one embodiment, the sample is contacted in a) with (i) a capture antibody or binding fragment thereof, and (ii) a detection antibody or binding fragment thereof.In one embodiment, the capture antibody and the detection antibody bind to HER2 or a polypeptide thereof. In one embodiment, the capture antibody is immobilized. In another embodiment, the detection antibody comprises a detection agent.

[0008] In one embodiment, the subject is being treated with a therapeutic agent. In one embodiment, the therapeutic agent is trastuzumab, trastuzumab emtansine, pembrolizumab, pertuzumab, nivolumab, atezolizumab, or a combination thereof. In another embodiment, the subject is being treated with trastuzumab, trastuzumab emtansine, pembrolizumab, pertuzumab, nivolumab, atezolizumab, or a combination thereof, and trastuzumab, trastuzumab emtansine, pembrolizumab, pertuzumab, nivolumab, atezolizumab, or a combination thereof does not interfere or only moderately interferes with the binding of the capture antibody and / or the detection antibody or their binding fragments. In one embodiment, the subject is being or can be treated for cancer, and such treatment includes small molecules, immunotherapy, surgery, chemotherapy, and / or radiation therapy. In one embodiment, the sample is a lymph node or tissue aspirate (e.g., breast), serum, whole blood, plasma, urine, saliva, tears, cerebrospinal fluid, supernatant from a normal cell lysate, supernatant from a preneoplastic cell lysate, supernatant from a neoplastic cell lysate, and / or supernatant from a cancer cell line maintained in tissue culture.

[0009] In one embodiment, the detection in b) is carried out using a lateral flow assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The practice of the methods and compositions described herein may employ, unless otherwise indicated, conventional techniques of pharmaceutical chemistry, molecular biology, formulation technology, dosage regimens, immunology, and biochemistry, all of which are within the skill of one in the art.

[0011] Provided herein are recombinant rabbit monoclonal antibodies that specifically bind to HER2, such as the extracellular domain of HER2 (e.g., human HER2 receptor). In addition to their binding specificity, the antibodies described herein offer several improvements over other anti-HER2 antibodies, which exhibit little or no interference with therapeutic agents, making immunoassays based on the antibodies provided herein more beneficial in providing physicians / patients with much-needed accurate information.

[0012] Substances that alter the measurable concentration of an analyte or that alter antibody binding can potentially cause immunoassay interference. Interfering substances can lead to falsely high or low analyte concentrations in one or more assay systems, depending on the site of interference in the reaction. Interference in immunoassays can lead to misinterpretation of patient results by laboratories and incorrect treatment strategies by physicians. For example, pertuzumab is one of the most common therapies used in the treatment of breast cancer to reduce the levels of HER2 in serum; unfortunately, pertuzumab interferes with many of the diagnostic assays currently used in the clinic, greatly reducing the reliability of these assays. Interference with diagnostic assays by therapeutic antibodies can occur at any stage of the assay or at any of the components involved in the assay (e.g., capture and / or detection antibodies). Importantly, pertuzumab, trastuzumab, margetuximab, and / or HER2 small molecule inhibitors (e.g., lapatinib, neratinib) show little to no interference with the antibody / immunoassays described herein, allowing for greater accuracy when testing and / or monitoring patients receiving treatment regimens for HER2-positive breast cancer.

[0013] definition Although for clarity and conciseness of description, features may be described herein as part of the same or separate embodiments, it will be understood that the scope of the invention may include embodiments having all or a partial combination of the described features.

[0014] The terms used herein are only intended to describe certain embodiments and are not intended to limit the present invention.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.The following definitions are intended to help readers understand the present invention, but are not intended to change or otherwise limit the meaning of such terms unless specifically indicated.

[0015] As used herein, the indefinite articles "a," "an," and "the" should be understood to include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "an inhibitor" refers to one or more agents capable of inhibiting a target molecule, reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[0016] The phrase "and / or" as used herein should be understood to mean "either or both" of the elements so conjoined, e.g., elements that are conjunctively present in some cases and non-conjunctively present in other cases.

[0017] As used herein, "or" should be understood to have the same meaning as "and / or" defined above. For example, when separating a list of items, "and / or" or "or" shall be interpreted as being inclusive, e.g., including at least one of the several items, but also including two or more, optionally including further unlisted items. "Only one of," "exactly one of," or, when used in the claims, "consisting of," refers to the inclusion of exactly one element of a number or list of elements, unless the term clearly indicates otherwise. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of," etc.

[0018] As used herein, the term "about" means plus or minus 10% of the indicated value. For example, about 100 means from 90 to 110. When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the scope of the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0019] The transmembrane protein HER2 (human epidermal growth factor receptor 2) or HER2 / neu, also known as the receptor tyrosine protein kinase erbB-2, CD340 (cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent), or ERBB2 (human), is a protein encoded in humans by the ERBB2 (erythroblastic oncogene B) gene. The HER2 protein has a molecular weight of approximately 185 kilodaltons (kDa) and is composed of an intracellular tyrosine kinase domain, a transmembrane domain, and an extracellular domain.

[0020] Amplification, also known as ERBB2 gene overexpression, occurs in approximately 15-30% of breast cancers, also known as HER2-positive breast cancers. HER2-positive breast cancers are breast cancers that test positive for a protein called human epidermal growth factor receptor 2 (HER2). This protein promotes the growth of cancer cells.

[0021] About one in five women with breast cancer has an extra copy of the gene that makes the HER2 protein in their cancer cells. HER2-positive breast cancer tends to be more aggressive than other types of breast cancer. It is associated with increased disease recurrence and a poor prognosis; however, drugs that target HER2 in breast cancer have significantly improved the otherwise poor natural history of HER2-positive breast cancer. Testing for HER2 status is recommended for all invasive breast cancers, as the results can significantly influence treatment recommendations and decisions.

[0022] As used herein, "detecting" refers to the action or process of identifying the presence of what is being detected, such as HER2 / neu, in a sample. As used herein, the term "sample" is defined as blood, serum, plasma, urine, saliva, tears, cerebrospinal fluid, supernatant from normal cell lysate, supernatant from preneoplastic cell lysate, supernatant from neoplastic cell lysate, supernatant from carcinoma cell line maintained in tissue culture, and breast aspirate or biopsy. Thus, any number of biological samples can be used in the immunoassays described herein, including but not limited to blood, serum, plasma, urine, saliva, tears, cerebrospinal fluid, supernatant from cell lysate (e.g., normal cells, preneoplastic cells, neoplastic cells, carcinoma cells), or breast aspirate or biopsy.

[0023] As used herein, "monitoring" refers to the act or process of identifying the presence of something that has been detected at least twice over a period of time. The term "antibody" refers to an intact antibody or an antigen-binding portion or fragment thereof that competes with the intact antibody for antigen binding. The term "antibody" also includes any type of antibody molecule or specific binding molecule that specifically binds to HER2. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide, glycoprotein, or immunoglobulin that specifically binds to HER2 protein. Antigen-binding fragments of antibodies can be obtained from complete antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques, including the manipulation and expression of nucleic acids encoding antibody variable domains and, optionally, constant domains.

[0024] A monoclonal antibody is an antibody obtained from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies may contain minor amounts of mutants or variants. Monoclonal antibodies are highly specific and interact with a single antigen site. Each monoclonal antibody typically targets a single epitope, whereas a polyclonal antibody population typically contains a variety of antibodies that target a diverse group of epitopes. Monoclonal antibodies can be produced by many methods, including, for example, hybridoma methods (Kohler and Milstein, Nature 256:495, 1975), recombinant methods (U.S. Pat. No. 4,816,567), and isolation from phage antibody libraries (Clackson et al., Nature 352:624-628, 1991; Marks et al., J. Mol. Biol., 222:581-597, 1991).

[0025] As used herein, the terms "subject," "mammal," and "mammalian subject" refer to any animal classified as a mammal, including humans, higher non-human primates, rodents, and domestic and farm animals such as cows, horses, dogs, and cats. In some embodiments of the invention, the mammal is a human (male or female).

[0026] As used herein, the terms "including," "includes," "having," "has," "with," or variations thereof, are intended to be inclusive in the same manner as the term "comprising."

[0027] As used herein, the terms "containing", "having" and "including" include "comprising", "mainly consisting of", "essentially consisting of" and "formed of"; "mainly consisting of", "generally consisting of" and "comprising of" are broader concepts than "having", "comprising" and "containing".

[0028] The terms "comprises," "comprising," and the like can have the meaning ascribed to them in United States patent law and can mean "includes," "including," and the like. As used herein, "including" or "includes," and the like, means including without limitation.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the method and composition belongs.Methods and materials similar or equivalent to those described herein can be used in carrying out or testing the method and composition of the present invention, and suitable methods and materials are described below.In addition, the materials, methods and examples are merely illustrative and are not intended to be limiting.All publications, patent applications, patents and other references described herein are incorporated by reference in their entirety.

[0030] Rabbit Antibody / Polypeptide Rabbit monoclonal antibodies (e.g., anti-human HER2 antibodies) are useful for many applications, including immunofluorescence, immunohistochemistry, flow cytometry, Western blot, and ELISA assays. Compared to other animal models (e.g., mice and rats), rabbits provide a better system for monoclonal antibody production because the rabbit immune system responds to a broader range of antigens. Also, physically, rabbits are larger animals with larger spleens that can produce more antibodies.

[0031] Rabbit monoclonal antibodies are similar to traditional mouse monoclonal antibodies, but offer better specificity and sensitivity. Rabbits are immunized and the resulting spleen cells are fused with partner cells to create immortal cell lines expressing the antibodies. Antibodies are derived from single clones and characterized for performance in applications. One or more clones are then selected for antibody production.

[0032] Because rabbits have a more diverse natural repertoire and larger spleens than mice, their antibodies exhibit higher affinity to antigens. Thus, rabbit monoclonal antibodies tend to provide superior sensitivity in applications where clones are screened. An additional advantage of rabbit diversity is that it allows epitope recognition that may not be feasible in other systems. Other advantages include natural diversity, high affinity and specificity, novel epitope recognition, cross-reactivity to human and mouse targets, and ease of humanization. Also, as provided herein, antibodies can be provided that exhibit little or no interference with therapeutic agents such as other antibodies, peptides, or small molecules.

[0033] The light or heavy chain variable region of an antibody has four framework regions interrupted by three hypervariable regions known as complementarity determining regions (CDRs). The CDRs determine the specificity of antigen binding. The heavy and light chains each have three CDRs, called CDR1, CDR2, and CDR3 from the N-terminus, flanked by four framework regions. The amino acid sequences of the framework regions are highly conserved, allowing the CDRs to be grafted onto other antibodies. Thus, recombinant antibodies can be produced by combining the CDRs from one or more antibodies with the framework of one or more other antibodies. The antibodies of the present invention include antibodies that contain at least one, two, three, four, five, or six (or a combination thereof) of the CDRs of any of the monoclonal antibodies described herein.

[0034] Polypeptides / antibodies of the present invention include the full-length rabbit anti-HER2 / neu heavy chain variable region, the full-length rabbit light chain variable region, binding fragments or variants thereof, and combinations thereof.

[0035] 1C5 (capture antibody) sequence: Heavy chain (SEQ ID NO:1 (below); CDR1, 2, and 3 are SEQ ID NOs:5, 6, and 7, respectively, as provided in Table A):

[0036] [ka]

[0037] Light chain (SEQ ID NO:2 (below); CDR1, 2, and 3 are SEQ ID NOs:8, 9, and 10, respectively, as provided in Table B):

[0038] [ka]

[0039] [Table 1]

[0040] [Table 2]

[0041] 1B7 (labeled antibody) sequence: Heavy chain (SEQ ID NO: 3 (below); CDR1, 2, and 3 are SEQ ID NOs: 11, 12, 13, respectively, as provided in Table C):

[0042] [ka]

[0043] Light chain (SEQ ID NO: 4 (below); CDR1, 2 and 3 are SEQ ID NOs: 14, 15, 16, respectively, as provided in Table D):

[0044] [ka]

[0045] [Table 3]

[0046] [Table 4]

[0047] CDR identification methods are described in E. Kabat, T. Wu, H. Perry, Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, National Institutes of Health, Bethesda MD, 1992.

[0048] A polypeptide variant, antibody variant or variant CDR differs from a polypeptide or fragment thereof shown in SEQ ID NO: 1-16 by, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60 or more amino acid residues (e.g., addition, substitution or deletion of amino acids). If this comparison requires alignment, the sequences are aligned for maximum homology. The mutation site may be located anywhere in the polypeptide. In one embodiment of the present invention, the variant polypeptide has substantially similar activity to the polypeptide shown in SEQ ID NO: 1-16. Substantially similar activity means that when the polypeptide is used to construct an antibody, the antibody has the same or substantially the same activity / binding as the wild-type antibody.

[0049] As used herein, the percent identity of two amino acid sequences (or two nucleic acid sequences) is determined using the algorithm of Karlin and Altschul (PNAS USA 87:2264-2268, 1990), modified in Karlin and Altschul (PNAS USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol., 215:403-410, 1990). BLAST nucleotide searches are performed using the NBLAST program, score=100, word length=12. BLAST protein searches are performed using the XBLAST program, score=50, word length=3. To obtain gapped alignments for comparison purposes, GappedBLAST is utilized as described in Altschul et al. (Nucleic Acids Res., vol. 25:3389-3402, 1997). When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention.

[0050] Identity or identical refers to amino acid sequence (or nucleic acid sequence) similarity and has the art-recognized meaning. Sequences with identity share identical or similar amino acids (or nucleic acids). Sequence identity refers to the percentage of amino acids that are the same as the original amino acid sequence of the antibody, and is determined after aligning the sequences and appropriately introducing gaps, if necessary, to maximize sequence identity. Thus, a candidate sequence that shares 85% amino acid sequence identity with a reference sequence requires that after alignment of the candidate sequence with the reference sequence, 85% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence and / or contain conservative amino acid changes.

[0051] The present invention also includes polypeptide variants or CDR variants of SEQ ID NOs: 1-16. Polypeptide variants or CDR variants of SEQ ID NOs: 1-16 can include one or more amino acid substitutions, additions or deletions. In one embodiment, the variant polypeptide or variant CDR comprises an amino acid sequence at least about 75% identical to the sequence shown as SEQ ID NOs: 1-16. In one embodiment, the variant tripeptide or CDR is at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or more identical to SEQ ID NOs: 1-16. The variant polypeptide or variant CDR encodes a variant antibody, which is an antibody comprising the amino acid sequence of SEQ ID NOs: 1-16 in which one or more amino acid residues have been added, substituted or deleted. For example, the variable region of the antibody can be modified to improve its biological properties (e.g., antigen binding). Such modifications can be achieved, for example, by site-directed mutagenesis, PCR-based mutagenesis, cassette mutagenesis. The variant antibody comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or more identical to the amino acid sequence of the heavy or light chain variable region of SEQ ID NOs: 1-16.

[0052] Methods for introducing mutations into amino acid sequences are well known to those skilled in the art. See, for example, Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994); Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, NY (1989). Mutations can also be introduced using commercially available kits such as the "QuikChange™ Site-Directed Mutagenesis Kit" (Stratagene). The generation of functionally active variant polypeptides by substituting amino acids that do not affect the function of the polypeptide can be accomplished by those skilled in the art.

[0053] The variant polypeptide may have conservative amino acid substitutions at one or more predicted non-essential amino acid residues. Conservative substitutions are those in which an amino acid is replaced with another amino acid having similar properties, such that one skilled in the art of peptide chemistry would predict that the secondary structure and hydropathic nature of the polypeptide will not be substantially altered. In general, the following amino acid groups represent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

[0054] The polypeptide or antibody of the present invention can be covalently or non-covalently linked to an amino acid sequence that the polypeptide or antibody is not normally bound to in nature. In addition, the polypeptide or antibody of the present invention can be covalently or non-covalently linked to compounds or molecules other than amino acids. For example, the polypeptide or antibody can be linked to an indicator reagent (indicator reagents can include colorimetric agents, catalysts (e.g., enzyme conjugates), fluorescent compounds (e.g., fluorescein and rhodamine), chemiluminescent compounds (e.g., dioxetanes, acridinium, phenanthridinium, ruthenium, and luminol), radioactive elements, direct visual labels, as well as cofactors, inhibitors, magnetic particles, and the like; examples of enzyme conjugates include alkaline phosphatase, horseradish peroxidase, β-galactosidase, and the like), amino acid spacers, amino acid linkers, signal sequences, stop transport sequences, transmembrane domains, protein purification ligands (e.g., glutathione-S-transferase, histidine tags, and Staphylococcus aureus protein A), or combinations thereof. In one embodiment of the invention, the protein purification ligand can be, for example, one or more C amino acid residues at the amino or carboxy terminus of a polypeptide of the invention. The amino acid spacer is a sequence of amino acids not normally associated with the polypeptide or antibody of the invention in nature. The amino acid spacer can comprise about 1, 5, 10, 20, 100, or 1,000 amino acids.

[0055] The polypeptide of the present invention can be isolated from cells or tissue sources using standard protein purification techniques.The polypeptide of the present invention can also be chemically synthesized or produced by recombinant DNA technology.For example, the polypeptide of the present invention can be synthesized using a conventional peptide synthesizer.

[0056] The polypeptide of the present invention can be produced recombinantly. The polynucleotide encoding the polypeptide of the present invention can be introduced into a recombinant expression vector, which can be expressed in a suitable expression host cell system using techniques well known in the art. A variety of bacterial, yeast, plant, mammalian, and insect expression systems are available in the art, and any such expression system can be used. If necessary, the polynucleotide encoding the polypeptide can be translated in a cell-free translation system.

[0057] join The antibody / its binding portion (antigen-binding fragment) of the present invention specifically binds to HER2 (e.g., human HER2). "Specifically binds" means that the antibody recognizes and binds to HER2 with higher affinity than other non-specific molecules that are not HER2. For example, an antibody raised against an antigen (polypeptide) that binds more efficiently than a non-specific antigen (e.g., a protein that is not related to or homologous to HER2) can be described as specifically binding to that antigen. Binding specificity can be tested, for example, using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or Western blot assay using methodologies well known in the art.

[0058] How to Generate Antibodies The antibodies of the present invention can be produced using methods known to those skilled in the art. For example, HER2 antigen or a fragment thereof can be used to immunize animals, including rabbits. HER2 or a fragment thereof can be conjugated to a carrier protein and / or administered to animals with an adjuvant. HER2 antigen can contain one or more epitopes (i.e., antigenic determinants). Epitopes can be linear, continuous or conformational epitopes. Epitopes within the polypeptides of the present invention can be identified by several methods. See, for example, U.S. Pat. No. 4,554,101; Jameson and Wolf, CABIOS 4:181-186 (1988). For example, HER2 can be isolated and screened. A series of short peptides that together make up the entire HER2 polypeptide sequence can be prepared by proteolytic cleavage. For example, starting with a 100-mer polypeptide fragment, each fragment is tested for the presence of a recognized epitope in an ELISA. For example, in an ELISA assay, a HER2 antigen (e.g., a 100-mer polypeptide fragment) is attached to a solid support (e.g., the wells of a plastic multi-well plate). A population of antibodies is labeled, added to the solid support, and allowed to bind to the unlabeled antigen under conditions that block nonspecific absorption, and any unbound antibodies and other proteins are washed away. Antibody binding is detected, for example, by a reaction that converts a colorless substrate into a colored reaction product. From the identified 100-mer, smaller, overlapping fragments are then tested in sequence to map the location of the epitope of interest.

[0059] Methods for preparing monoclonal antibodies from hybridomas are well known to those skilled in the art and include, for example, standard cell culture and ascites production methods. Recombinant antibodies or fragments thereof produced by genetic engineering can be made using the polynucleotide sequences of the present invention. Genes encoding antibodies or fragments thereof can be isolated from the hybridomas of the present invention or other hybridomas. The genes can be inserted into appropriate vectors and introduced into host cells. See, for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, Macmillan Publ.Ltd, 1990.

[0060] In one embodiment, highly specific monoclonal antibodies were developed by immunizing rabbits, selecting spleen cells, and constructing monoclonal antibodies in commercial quantities suitable for clinical use. The use of monoclonal rabbit antibodies in the clinical field is specific, since most recombinant rabbit monoclonal antibodies are only used in research. In addition, the antibodies described herein are superior for several reasons. For example, recombinant rabbit mAbs exhibit higher binding affinity to their ligands compared to recombinant mouse mAbs, thereby providing more reproducible results. Furthermore, the rabbit monoclonal antibodies provided herein exhibit limited / moderate or no therapeutic drug interference in immunoassays.

[0061] Rabbit monoclonal antibodies (mAbs) are recognized for their advantages as research and diagnostic reagents: they have 10-100 times higher affinity than mouse mAbs; superior specificity that can distinguish even single amino acid differences and reduce cross-reactivity; broad epitope recognition that increases mAb diversity; great stability for consistent performance; and a longer shelf life due to excess disulfide bonds in rabbit IgG (Feng et al., Am J Transl Res., 2011;3(3):269-74; Rossi S. et al., American Journal of Clinical Pathology., 2005;124(2):295-302; Vilches-Moure JG et al., J Vet Diagn Invest., 2005;17(4):346-50).

[0062] To effectively discover specific mAbs, methods available in the art can be used, such as the single B cell-based SMab™ platform for efficient high-throughput screening for specific rabbit mAbs of interest. Briefly, protein-recognizing B cells are first enriched and sorted from spleen cells using fluorescence-activated cell sorting (FACS); sorted cells are cultured and stimulated at 1 cell / well; positive clones are identified from single B cells using enzyme-linked immunosorbent assay (ELISA) and other desired assays for the protein of interest; supernatants and RNA are collected for further analysis; naturally paired IgG light and heavy chain genes are then cloned from the positive clones; selected mAb clones are then expressed and validated. The SMab™ platform routinely generates 300-500 testable clones of mAbs in 3-4.5 months, approximately 30%-50% faster than traditional hybridoma and display platforms. The use of large amounts of spleen cells and a scalable high-throughput design improves the diversity of the initial mAb pool that recognizes the protein of interest. The SMab™ platform provides the earliest functional characterization of protein-specific mAbs using culture supernatants from intermediate steps to shorten antibody development time by removing unnecessary workload.

[0063] Conjugates The antibody of the present invention may be covalently linked to other molecules, provided that the covalent linkage does not affect the ability of the antibody to bind to HER2. For example, the antibody may be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups (e.g., methyl groups, ethyl groups, carbohydrate groups), proteolytic cleavage, linkage to cellular ligands or other proteins, etc.

[0064] Conjugated antibodies can be coupled to a variety of molecules including, for example, polymers, hyaluronic acid, fluorescent agents, luminescent agents, haptens, enzymes, metal chelates, cytotoxic agents, radionuclides, and drugs.

[0065] Detection Method One embodiment of the present invention provides a method of detecting a HER2 polypeptide in a sample. The method includes contacting a sample suspected of containing a HER2 polypeptide with an antibody, or antigen-binding portion thereof, of the invention to form a HER2 / antibody complex. The presence of the HER2 / antibody complex is detected, thereby detecting the presence of the HER2 polypeptide. In some embodiments, two different antibodies, or antigen-binding portions thereof, of the invention are used in detecting HER2 (e.g., contacting an antibody comprising SEQ ID NOs: 5-10 and an antibody comprising SEQ ID NOs: 11-16; including a capture antibody and a labeled antibody, 1C5 and 1B7).

[0066] The test sample may be, for example, lymph node or tissue aspirate, serum, whole blood, plasma, circulating tumor cells, tumor cells or tissue (e.g., tissue biopsy) or ascites. The polypeptide / antibody complex may be detected by any method known in the art, including, but not limited to, enzyme-linked immunosorbent assay (ELISA), multiplex fluorescent immunoassay (MFI or MFIA), radioimmunoassay (RIA), sandwich assay, Western blotting, immunoblotting analysis, immunohistochemistry, immunofluorescence assay, fluorescence-activated cell sorting (FACS) or a combination thereof.

[0067] The immunoassay for HER2 can utilize one antibody or several different antibodies.The immunoassay protocol can be based on, for example, competitive, direct reaction, or sandwich-type assay, for example, using labeled antibody.The antibody of the present invention can be labeled with any type of label known in the art, including, for example, fluorescent label, chemiluminescent label, radioactive label, enzyme label, colloidal metal label, radioisotope label and bioluminescent label.

[0068] The antibodies or antigen-binding portions thereof of the invention may be bound to supports and used to detect the presence of HER2, including, for example, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified celluloses, polyacrylamide, agarose, and magletite.

[0069] The antibodies of the present invention can be used in a method of diagnosis of a hyperproliferative disorder, for example, by obtaining a test sample from a human or animal suspected of having a hyperproliferative disorder. The test sample is contacted with the antibody or antigen-binding portion thereof of the present invention under conditions that allow for the formation of an antibody-antigen complex (i.e., immune complex). Those skilled in the art will recognize conditions that allow and are suitable for the formation of an antigen / antibody complex. The amount of antibody-antigen complex (e.g., including a complex of an antibody or antigen-binding portion thereof with HER2) can be determined by methodologies known in the art. A higher level than that formed in a control sample indicates the presence of a hyperproliferative disorder. The amount of antibody / antigen complex can be determined by methods known in the art.

[0070] The HER2-positive hyperproliferative disorder may be a neoplastic disorder including breast cancer, ovarian cancer, pancreatic cancer, bladder cancer, adenocarcinoma of the lung, uterine cancer (such as uterine serous endometrial cancer), gastric cancer, esophageal cancer, colon cancer, and head and neck and / or salivary duct cancer.

[0071] The antibody / assay described herein can be used to identify and monitor patients with tumors that overexpress HER2, and thus are candidates for targeted drug therapy.The antibody / assay described herein shows limited or no interference with therapeutic agents.Therefore, the immunoassay described herein fulfills an unmet need in the field of breast cancer care.

[0072] The immunoassays described herein can be used to test for HER2-positive breast cancer, to monitor serum levels of HER2 in patients undergoing drug therapy, to detect recurrence, or to detect HER2 disease in women who have been tested as tissue HER2 negative.The immunoassays described herein can also be used to detect elevated or rising levels of serum HER2 in women, which can indicate the emergence of HER2 disease in women who were previously considered HER2 negative (e.g., by tissue testing).The immunoassays described herein can also be used in conjunction with measuring circulating tumor cells (CTCs) or as an adjunct to identify or help identify patients who may need or benefit from positron emission tomography (PET) scans.

[0073] Lateral Flow Assay (LFA) Lateral flow assays (LFAs) are based on the migration of a liquid sample through a polymer strip with attached molecules that interact with the analyte, providing a signal that can be visually detected. LFAs are generally paper-based platforms for the detection and / or quantification of analytes (e.g., proteins, haptens, nucleic acids and amplicons), which are often complex mixtures, where the sample is placed on the test device and results are displayed within about 5-30 minutes (e.g., 5-10 minutes). The low development costs and ease of manufacture of LFAs have led to the expansion of their application to multiple fields where rapid testing is required, such as biomedicine, agriculture, food and environmental science. LFA-based tests are widely used in hospitals, clinics and clinical laboratories for the qualitative and quantitative detection of specific antigens and antibodies, as well as products of gene amplification, in contexts such as veterinary medicine, quality control, product safety in food production, and environmental health and safety, including screening for animal and human diseases, pathogens, chemicals, toxins and water contaminants, among others.

[0074] In LFAs, liquid samples (such as urine, saliva, sweat, serum, plasma, whole blood, and other fluids) containing the analyte of interest migrate, without the assistance of external forces (capillary action), through various zones of a polymer strip to which molecules capable of interacting with the analyte are attached. A typical lateral flow test strip may be composed of overlapping membranes mounted on a backing card for better stability. The sample is applied to one end of the strip on an adsorbent sample pad, which can be loaded with buffer salts and surfactants that make the sample suitable for interaction with the detection system. The sample migrates through a conjugate release pad that contains antibodies that are specific for the target analyte and conjugated to colored or fluorescent particles (e.g., gold colloids and latex microspheres) (depending on the recognition element used, LFAs can be classified into different types (e.g., "lateral flow immunoassays" (LFIAs), where antibodies are used as recognition elements, and nucleic acid LFAs (NALFAs), where the detection of amplicons that may be formed during the polymerase chain reaction (PCR) is used). The sample, together with the conjugated antibodies bound to the target analytes, migrates along the strip into the detection zone. This is generally a porous membrane (usually composed of nitrocellulose) with specific biological components (mainly antibodies or antigens) immobilized in lines. Their role is to react with the analytes bound to the conjugated antibodies. The recognition of the sample analyte produces an appropriate response on the test line, while the response on the control line indicates an appropriate liquid flow through the strip. The read-out, represented by lines appearing at different intensities, can be evaluated visually or using a dedicated reader (device).

[0075] Provided herein is a point-of-care multiplex diagnostic assay using multiple test lines that allows for rapid and simultaneous detection of multiple analytes present in a sample, including HER2-positive hyperproliferative diseases such as breast cancer, ovarian cancer, gastric cancer, adenocarcinoma of the lung, uterine cancer (e.g., serous endometrial intraepithelial carcinoma), gastric cancer, and / or salivary gland cancer, providing a powerful tool for the detection and progression of cancer, for example, before, after, and / or during treatment. To simultaneously test multiple analytes under the same conditions, additional test lines of antibodies specific for different analytes can be immobilized in an array format. Meanwhile, multiple test lines loaded with the same antibody can be used for semi-quantitative assays. The principle of this "ladder bars" assay is based on the stepwise capture of colorimetric conjugated antigen complexes by immobilized antibodies on each successive line, and the number of lines appearing on the strip is directly proportional to the concentration of the analyte. The liquid flows across the device due to the capillary forces of the strip material, and to maintain this movement, absorbent pads can be attached to the ends of the strip. The role of the absorbent pad is to soak up excess reagent and prevent backflow of liquid. A current example of an LFA is the pregnancy test stick.

[0076] Two forms of LFIA can be distinguished: direct and competitive. Direct tests are used for larger analytes, such as the p24 antigen used in human immunodeficiency virus (HIV) tests, as well as for analytes with multiple antigenic sites, such as human chorionic gonadotropin (hCG) used in pregnancy tests. The hCG test is an example of a sandwich-based assay in which the target is immobilized between two complementary antibodies. In a direct test, the presence of a test line indicates a positive result, while the control line usually contains a species-specific anti-immunoglobulin antibody specific for the antibody in the particular conjugate. For small molecules with a single antigenic determinant that cannot bind simultaneously to two antibodies, a competitive test is used. In this type of test, the analyte blocks the binding sites on the antibodies on the test line, preventing their interaction with the colored conjugate. A positive result is therefore indicated by the absence of a signal at the test line, while the control line should be visible independent of the test result.

[0077] Concerning labels, gold colloids are a widely used label in commercial LFIAs. Another common label is latex, which can be tagged with various detection reagents (e.g., colored or fluorescent dyes, and magnetic or paramagnetic components). Latex can be produced in multiple colors, so it is applied to multiplex assays that require discrimination between many lines. Carbon and fluorescent labels, or enzymatic modification of labels, are also used. Carbon nanotubes, fluorescent labels, quantum dots, upconverting phosphors can all be used as labels. Another detection system that can be used is FACTT, an acronym for Highly Sensitive Protein Detection System, where amplification of the detection mAb occurs when bound to T7 polymerase. Rather than measuring the mAb directly, the reader detects the RNA molecules generated by the polymerase, thus greatly amplifying the result. This test can result in a qualitative color change, but can also benefit from a reader (device). It can take 20-30 minutes.

[0078] There are many advantages to using such assays, including, for example, point-of-care, offering cheap, fast and easy testing that is desirable in many industries / countries, and due to their long shelf life and the fact that they often do not require refrigeration for storage, these tests are well suited for use in developing countries, small outpatient care settings, remote areas and on the battlefield. Furthermore, as the visual result is usually clear, no additional equipment is required, but any device can be used for readout.

[0079] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention which is described in broad terms above. EXAMPLES

[0080] Example I A. Linearity A linearity assay was utilized to assess the reportable range. Two native samples with appropriate HER2 levels, 35SC and 40SC, were diluted 1:25, 1:100, and 1:200 in addition to the 1:50 dilution specified in the assay's standard operating procedure (SOP). After measurement, linearity was demonstrated by quantifying these values ​​as a percentage of the expected HER2 concentration for each dilution tested. A range of 80%-120% of the expected values ​​indicates linearity that is acceptable.

[0081] Linearity was well within acceptable tolerance limits for both natural samples tested. For 1:25, 1:100 and 1:200 dilutions, serum 35SC gave corrected values ​​of 98%, 101% and 105% of the expected concentration, respectively; and serum 40SC gave corrected values ​​of 101%, 99% and 100%, respectively. See Table 1.

[0082] [Table 5]

[0083] B. Cross-reactivity For cross-reactivity, the following four recombinant human (rh) proteins (three family members related to HER2 and one unrelated protein) were individually tested at concentrations as high as 200 ng / mL in assays alongside standard curves: rhEGFR, rhHER3, rhHER4, and rhPD-L1 (each provided by Sino Biological). Cross-reactivity was assessed by calculating the percentage of the measured recombinant protein concentration relative to the loaded initial concentration of 200 ng / mL. Any recombinant protein found to yield values ​​greater than 5.0% of the value predicted for HER2 was considered cross-reactive.

[0084] None of the four recombinant proteins tested cross-reacted in the HER2 ELISA at 200 ng / mL, meeting the acceptability requirements. See Table 2.

[0085] [Table 6]

[0086] C. Protein interference To determine interference with related family proteins, the same high concentration of 200ng / mL of EGFR, HER3 and HER4 were individually added to a midpoint rhHER2 concentration of approximately 7ng / mL (considered the reference sample). After performing the assay, the measured HER2 concentration was compared to the expected concentration and the percent recovery was calculated. Any protein, if present, that produced a measured concentration less than 80% or more than 120% of the concentration predicted for HER2 was considered to be interfering.

[0087] The percent recovery was 96.7% for rhEGFR, 99.7% for rhHER3, and 109.6% for rhHER4. Thus, these related recombinant proteins showed no interference with the HER2 ELISA at 200 ng / mL, and the results met the acceptance requirements. See Table 3.

[0088] [Table 7]

[0089] D. Drug Interference To determine interference with related drugs, three therapeutic antibodies were run for interference in serum samples: trastuzumab (Herceptin; Roche), pertuzumab (Perjeta; Roche), and pembrolizumab (Keytruda; Merck). Each drug was spiked into an endogenous sample with known assay measurements (natural reference point) at a physiologically relevant concentration of 100 μg / mL. Any change from expected concentration was determined and expressed as percent interference. Therapeutic antibodies that, if present, caused a change in measured concentration of more than 10% of that expected for HER2 were considered to be interfering.

[0090] Using two samples (B54 and 20SC), the average percentage of interference was 6.13% for Herceptin® and 3.72% for Perjeta®. Using eight sera and two experimental days to confirm the results, the average percentage of interference was 4.5% for Perjeta®. Thus, all three antibody drugs tested, Herceptin®, Keytruda®, and Perjeta®, showed no interference with the HER2 ELISA at 100 μg / mL. See Table 4.

[0091] [Table 8]

[0092] All publications, nucleotide and amino acid sequences identified by their accession numbers, patents and patent applications are incorporated herein by reference. In the above specification, the invention has been described in connection with certain preferred embodiments thereof, and numerous details have been set forth for purposes of illustration, but it will be apparent to those skilled in the art that the invention is susceptible to further embodiments, and that the specific details described herein may be modified considerably without departing from the basic principles of the invention.

[0093] The specific methods and compositions described herein are representative of preferred embodiments and are illustrative and are not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification and are encompassed within the spirit of the invention as defined by the claims. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention as illustratively described herein may be practiced in the absence of any element(s) or limitation(s) not specifically disclosed herein as essential. The methods and processes as illustratively described herein may have steps performed in different orders, and the methods and processes are not necessarily limited to the order of steps set forth herein or in the claims. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to an "anucleic acid" or "apolypeptide" includes a plurality of such nucleic acids or polypeptides (e.g., a solution of a nucleic acid or polypeptide or a series of nucleic acid or polypeptide preparations), etc. As used herein, the term "or" is used to refer to non-exclusive, and "A or B" includes "A but not B," "B but not A," and "A and B," unless otherwise indicated.

[0094] Under no circumstances should this patent be construed as limited to the particular examples or embodiments or methods specifically disclosed herein. Under no circumstances should this patent be construed as limited by any statement made by any examiner or any other officer or employee of the Patent and Trademark Office, unless such statement is specifically and without qualification or reservation expressly adopted in the responsive document by the applicant.

[0095] The terms and expressions employed are used as terms of description and not of limitation, and in the use of such terms and expressions there is no intention to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, although the invention has been specifically disclosed by preferred embodiments and optional features, it is understood that modifications and variations of the concepts disclosed herein may be employed by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims and the description of the invention.

Claims

1. An anti-HER2 monoclonal antibody or its conjugated fragment comprising a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 95% identity thereto, and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 95% identity thereto.

2. An anti-HER2 monoclonal antibody or a conjugated fragment comprising a heavy chain and a light chain, wherein (i) the heavy chain comprises three CDR regions having the amino acid sequences of SEQ ID NOs. 5, 6, and 7, or amino acid sequences having at least 95% identity thereto, and (ii) the light chain comprises three CDR regions having the amino acid sequences of SEQ ID NOs. 8, 9, and 10, or amino acid sequences having at least 95% identity thereto.

3. The anti-HER2 monoclonal antibody or its conjugated fragment according to claim 1 or 2, wherein the antibody is conjugated to a detection agent.

4. A composition comprising the anti-HER2 antibody and carrier according to claim 1 or claim 2.

5. A method for detecting a HER2 polypeptide or a fragment thereof in a test sample, the method being: a) The step of contacting the anti-HER2 monoclonal antibody or its conjugated fragment according to claim 1 or 2 with a test sample under conditions that enable the formation of a polypeptide / antibody complex; b) A step of detecting the polypeptide / antibody complex of a), wherein the detection of the polypeptide / antibody complex is an indicator of the presence of the HER2 polypeptide in the sample. A method that includes this.

6. A method for monitoring a HER2 polypeptide or a fragment thereof in a sample from a subject, wherein the method is: a) The step of contacting the sample with at least one anti-HER2 monoclonal antibody or its conjugated fragment according to claim 1 or 2, under conditions that enable the formation of a polypeptide / antibody complex; b) A step of detecting the polypeptide / antibody complex of a), wherein the detection of the polypeptide / antibody complex indicates the presence of the HER2 polypeptide or a fragment thereof in the subject; c) A step of performing steps a) and b) at multiple time points to monitor the HER2 polypeptide or its fragments in the subject over time, A method that includes this.

7. The method according to claim 5, further comprising contacting the sample of a) with a second anti-HER2 antibody or fragment thereof, which includes a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence of at least 95% identity, a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence of at least 95% identity, or an anti-HER2 antibody or fragment thereof comprising a heavy chain and a light chain, wherein (i) the heavy chain comprises three CDR regions having the amino acid sequences of SEQ ID NO: 11, 12, 13 or an amino acid sequence of at least 95% identity, and (ii) the light chain comprises three CDR regions having the amino acid sequences of SEQ ID NO: 14, 15, 16 or an amino acid sequence of at least 95% identity.

8. The method according to claim 5, wherein the sample is, in a): i) Capture antibody or its binding fragment, and ii) A method of contact with the detection antibody or its bound fragment.

9. The method according to claim 8, wherein the capture antibody and the detection antibody are bound to HER2.

10. The method according to claim 8, wherein the capture antibody is immobilized.

11. The method according to claim 8, wherein the detection antibody comprises a detection agent.

12. The method according to claim 6, wherein the subject is being treated with a therapeutic agent.

13. The method according to claim 12, wherein the therapeutic agent is trastuzumab, trastuzumab emtansine, pembrolizumab, pertuzumab, nivolumab, atezolizumab, or a combination thereof.

14. The method according to claim 6, wherein the subject is treated with trastuzumab, trastuzumab emtansine, pembrolizumab, pertuzumab, nivolumab, atezolizumab, or a combination thereof, and the trastuzumab, trastuzumab emtansine, pembrolizumab, pertuzumab, nivolumab, atezolizumab, or a combination thereof does not interfere with or only moderately interferes with the binding of the capture antibody and / or detection antibody or its binding fragment.

15. The method according to claim 5, wherein the sample is lymph node or tissue aspirate (e.g., breast), serum, whole blood, plasma, urine, saliva, tears, cerebrospinal fluid, supernatant from normal cell lysates, supernatant from preneoplastic cell lysates, supernatant from neoplastic cell lysates, and / or supernatant from cancer cell lines maintained in tissue culture.

16. The method according to claim 5, wherein the detection of b) is performed using a lateral flow assay.