Antibodies targeting detrastuzumab and uses thereof

Abstract

The invention provides an antibody targeting detrastuzumab or an antigen-binding fragment thereof, a nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof, a carrier containing the nucleic acid molecule, a host cell containing the nucleic acid molecule or the carrier, a detection reagent containing the antibody or the antigen-binding fragment thereof, and a kit for detecting the antibody or the antigen-binding fragment thereof. A detection kit comprising the antibody or the antigen-binding fragment thereof, and uses thereof.

Description

Technical Field

[0001] The present application generally relates to the fields of genetic engineering and antibody medicine; specifically, the present application relates to antibodies targeting trastuzumab and their uses. Background Art

[0002] Antibody-drug conjugates (ADCs) are drugs formed by linking an antibody and a small molecule drug via a specific linker. ADCs combine the high specificity of antibody drugs with the high cytotoxic activity of small molecules, improving tumor targeting and reducing toxic side effects. ADCs accurately recognize their targets and release highly active cytotoxins within tumor tissue, thereby exerting targeted efficacy on tumor lesions while minimizing toxic side effects on normal tissues.

[0003] Small molecule cytotoxic drugs are an important component of ADCs. Common small molecule cytotoxic drugs include MMAE, DM1, and DXd. DXd (a derivative of ixotecan) is an inhibitor of DNA topoisomerase I (TOP I).

[0004] Trastuzumab deruxtecan for injection (trade name: ENHERTU; generic name: Fam-Trastuzumab deruxtecan (T-DXd); medical name: ENHERTU; research code: DS-8201) is a HER2-targeting antibody-drug conjugate. It was officially approved for marketing in China by the China National Medical Products Administration in February 2023. Its indications include: HER2-positive breast cancer, gastric cancer, gastroesophageal junction cancer, HER2-low-expressing breast cancer, and non-small cell lung cancer. Trastuzumab deruxtecan, through its unique combination of the DNA topoisomerase I inhibitor DXd and a stable linker, has demonstrated excellent efficacy and a favorable safety profile in clinical applications.

[0005] As the clinical application of trastuzumab continues to expand, there is a need to detect the drug's content in the blood to conduct pharmacokinetic analysis of the drug and monitor the production of anti-drug antibodies (ADA) to guide clinical rational drug use. Therefore, there is a need for testing trastuzumab in this field. Summary of the Invention

[0006] In a first aspect, the present application provides an antibody or antigen-binding fragment thereof that binds to trastuzumab, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and wherein according to the IMGT definition numbering, the amino acid sequence of the HCDR1 is as shown in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO: 4, the amino acid sequence of the HCDR3 is as shown in SEQ ID NO: 6, the amino acid sequence of the LCDR1 is as shown in SEQ ID NO: 10, the amino acid sequence of the LCDR2 is DTS, and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO: 14; or according to the Kabat definition numbering, the amino acid sequence of the HCDR1 is as shown in SEQ ID NO: 17, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO: 18, the amino acid sequence of the HCDR3 is as shown in SEQ ID NO: 19, the amino acid sequence of the LCDR1 is as shown in SEQ ID NO: 20, and the amino acid sequence of the LCDR2 is as shown in SEQ ID NO: ID NO: 21, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 22.

[0007] In a second aspect, the present application provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to the first aspect.

[0008] In a third aspect, the present application provides a vector comprising the nucleic acid molecule described in the second aspect.

[0009] In a fourth aspect, the present application provides a host cell comprising the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect.

[0010] In a fifth aspect, the present application provides a detection reagent or kit comprising the antibody or antigen-binding portion thereof described in the first aspect.

[0011] In a sixth aspect, the present application provides use of the antibody or antigen-binding portion thereof according to the first aspect for detecting trastuzumab in a biological sample of a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Shown is the layout of exemplary samples for antibody subtype characterization in a 96-well plate.

[0013] Figure 2 Shown is a set of results from the trastuzumab-coated plate ELISA assay during screening of positive master clones in 96-well plates.

[0014] Figure 3 The results of SDS-PAGE detection of the purified 26E5 antibody are shown.

[0015] Figure 4 The binding of purified 26E5 antibody to trastuzumab and human IgG was detected by ELISA.

[0016] Figure 5 The binding of the purified 26E5 antibody to trastuzumab after pre-incubation with Dxd was detected by ELISA. The results showed that Dxd could effectively block the binding of clone 26E5 to trastuzumab.

[0017] Sequence Description

[0018] SEQ ID NOs: 1, 3, 5 and 7 show the amino acid sequences of FR1, FR2, FR3 and FR4, respectively, in the heavy chain variable region of antibody clone 26E5.

[0019] SEQ ID NOs: 2, 4 and 6 show the amino acid sequences of HCDR1, HCDR2 and HCDR3, respectively, in the heavy chain variable region of antibody clone 26E5, numbered according to the IMGT definition.

[0020] SEQ ID NO: 8 shows the amino acid sequence of the heavy chain variable region of antibody clone 26E5.

[0021] SEQ ID NOs: 9, 11, 13 and 15 show the amino acid sequences of FR1, FR2, FR3 and FR4, respectively, in the light chain variable region of antibody clone 26E5.

[0022] SEQ ID NOs: 10, 12 and 14 show the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively, in the light chain variable region of antibody clone 26E5, numbered according to the IMGT definition.

[0023] SEQ ID NO: 16 shows the amino acid sequence of the light chain variable region of antibody clone 26E5.

[0024] SEQ ID NOs: 17-19 respectively show the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of antibody clone 26E5, numbered according to the Kabat definition.

[0025] SEQ ID NOs: 20-22 show the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively, in the light chain variable region of antibody clone 26E5, numbered according to the Kabat definition. DETAILED DESCRIPTION

[0026] ADC-related pharmacokinetic (PK) studies can be performed using ligand binding assays (LBA) or LC-MS / MS to analyze free small molecules, bound antibodies, and total antibodies. Antibodies against small molecule drugs can be used as capture antibodies to bind to the target for LBA analysis, or to enrich ADC samples during LC-MS / MS analysis.

[0027] As the scope of clinical application of trastuzumab continues to expand, it is necessary to detect the content of the drug in the blood and conduct pharmacokinetic analysis of the drug; monitor the production of anti-drug antibodies (ADA) to guide rational clinical drug use. There is a need for detecting trastuzumab in this field. The present application achieves this need by providing an antibody or an antigen-binding fragment thereof targeting trastuzumab, thereby promoting the clinical application of trastuzumab. After testing, the antibody or antigen-binding fragment thereof targeting trastuzumab in the present application can specifically bind to trastuzumab, thereby providing support for accurately detecting the content of trastuzumab in the blood and conducting pharmacokinetic analysis of the drug.

[0028] definition

[0029] The practice of the present application employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology within the skill of the art.

[0030] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by one of ordinary skill in the art.The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0032] Although the numerical ranges and parameter approximations shown in the broad scope of this application, the numerical values shown in the specific embodiments are recorded as accurately as possible. However, any numerical value is necessarily contained in a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, a range of "1 to 10" recorded should be considered to include any and all sub-ranges between a minimum of 1 and a maximum of 10 (including endpoints); that is, all sub-ranges starting with a minimum of 1 or greater, such as 1 to 6.1, and sub-ranges terminating with a maximum of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0033] In a broad sense, the term "antibody" refers to an immunoglobulin molecule that is capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule, and thus encompasses intact antibodies / full-length antibodies, single antibody chains, or any antigen-binding fragment of an antibody (also referred to as an "antigen-binding portion"), such as Fab, Fab', F(ab')2, Fv, scFv, Fd fragments, single domain antibodies, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule comprising an antigen-recognition site of desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. When "antibody" and "antigen-binding fragment / antigen-binding portion" appear in the same context, "antibody" can be understood as the intact body relative to the "antigen-binding fragment / antigen-binding portion", and both correspond to the broad concept of an antibody.

[0034] Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and the first, second and third constant regions (CH1, CH2 and CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL). A full-length antibody can be any class of antibody, such as IgD, IgE, IgG, IgA or IgM (or subclasses of the above), but antibodies do not need to belong to any particular class. Based on the antibody amino acid sequence of the heavy chain constant domain, immunoglobulins can be assigned to different classes. Typically, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are called 、 、 、 ,as well as The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

[0035] As used herein, the terms "antigen-binding fragment or antigen-binding portion" are used interchangeably and refer to a portion or region of an intact antibody molecule that is responsible for binding to an antigen. The antigen-binding domain can comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of the VH and VL typically contains three complementary determining regions (CDR1, CDR2, and CDR3).

[0036] For a general antibody, examples of its antigen-binding fragment include, but are not limited to: (1) a Fab fragment, which can be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a F(ab')2 fragment, which can be a bivalent fragment having two Fab' fragments connected by a disulfide bridge in the hinge region (i.e., a dimer of Fab'); (3) an Fv fragment having the VL and VH domains of a single arm of an antibody; (4) a single-chain Fv (scFv), which can be a single polypeptide chain composed of a VH domain and a VL domain via a peptide connector; and (5) (scFv)2, which can comprise two VH domains connected by a peptide connector and two VL domains, wherein the two VL domains are combined with the two VH domains via a disulfide bridge.

[0037] In some specific embodiments of the present application, “antigen-binding fragment” includes but is not limited to Fab fragment, Fab′ fragment, F(ab′)2 fragment, Fv fragment, scFv fragment, Fd fragment or single domain antibody.

[0038] As used herein, the term "single-chain antibody (scFv)" refers to an antibody with a single-chain structure, typically constructed using genetic engineering techniques, comprising a single polypeptide chain consisting of a heavy chain variable region (VH) and a light chain variable region (VL). A flexible linker is typically designed between the heavy and light chain variable regions to facilitate folding of the heavy and light chain variable regions into the correct conformation for antigen binding.

[0039] As used herein, the term "Fab (fragment antigen binding) fragment", "Fab portion" or similar terms refers to an antibody fragment capable of binding to an antigen produced by treating an intact antibody with papain, comprising a complete light chain (VL-CL), a heavy chain variable region and a CH1 fragment (VH-CH1).

[0040] As used herein, the term "Fd fragment", "Fd portion" or similar terms refers to the heavy chain portion of the Fab fragment of an antibody, including the heavy chain variable region and the CH1 fragment (VH-CH1).

[0041] As used herein, the terms "Fc fragment," "Fc domain," and "Fc portion" are used interchangeably and refer to a portion of the constant region of an antibody heavy chain, including the hinge region, the CH2 fragment, and the CH3 fragment of the heavy chain constant region, and are determined with reference to the EU numbering of a human IgG1 antibody.

[0042] As used herein, the term "single-domain antibody," also known as a VHH antibody or nanobody, can be defined as an amino acid sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. FR1-FR4 refer to framework regions 1-4, respectively, and CDR1-CDR3 refer to complementarity-determining regions 1-3, respectively. "VHH" refers to the variable antigen-binding domain of heavy chain antibodies from the Camelidae family (camels, dromedaries, llamas, alpacas, etc.).

[0043] It is well known to those skilled in the art that the complementarity determining regions (CDRs, generally CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of an antibody. There are several common definitions of the CDR amino acid sequences of VH or VL, including the IMGT definition, the Kabat definition, the AbM definition, and the Chothia definition. See, for example, Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). In embodiments of the present application, the IMGT or Kabat definition is used to determine the CDR amino acid sequences in the antibody VH and VL amino acid sequences.

[0044] For a given antibody variable region amino acid sequence, the CDR amino acid sequence in the variable region amino acid sequence can be analyzed in a variety of ways, for example, it can be determined using the online software Abysis (http: / / www.abysis.org / ).

[0045] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope.

[0046] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor individuals.

[0047] As used in this article, the term "trastuzumab deruxtecan" refers to an antibody-drug conjugate targeting HER2, with the trade name "Euhertu", the generic name "Fam-Trastuzumab deruxtecan (T-DXd), the medical name "ENHERTU", and the scientific research code name "DS-8201". It was officially approved for marketing in China by the China National Medical Products Administration in February 2023.

[0048] The term "identity / homology / identity" with respect to amino acid or nucleic acid sequences is defined as the percentage of identical residues in amino acid or nucleotide sequence variants after alignment and introduction of gaps, if necessary, to achieve the maximum percentage identity. Methods and computer programs for alignment are well known in the art.

[0049] In a first aspect, the present application provides an antibody or antigen-binding fragment thereof that binds to trastuzumab, the antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and wherein according to the IMGT definition numbering, the amino acid sequence of the HCDR1 is as shown in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO: 4, the amino acid sequence of the HCDR3 is as shown in SEQ ID NO: 6, the amino acid sequence of the LCDR1 is as shown in SEQ ID NO: 10, the amino acid sequence of the LCDR2 is DTS, and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO: 14; or according to the Kabat definition numbering, the amino acid sequence of the HCDR1 is as shown in SEQ ID NO: 17, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO: 18, the amino acid sequence of the HCDR3 is as shown in SEQ ID NO: 19, the amino acid sequence of the LCDR1 is as shown in SEQ ID NO: 20, and the amino acid sequence of the LCDR2 is as shown in SEQ ID NO: ID NO: 21, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 22.

[0050] In some embodiments of the first aspect, the antibody or antigen-binding fragment thereof specifically binds to trastuzumab or a fragment of trastuzumab, such as the heavy chain, light chain, heavy chain variable region, light chain variable region and / or constant region of trastuzumab.

[0051] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8. Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 8.

[0052] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 16. Preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 16.

[0053] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region may differ from the amino acid sequence shown in SEQ ID NO:8 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and / or additions.

[0054] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:8 can be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still maintaining similar function of the heavy chain variable region.

[0055] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 8, and the resulting amino acid sequence still retains similar function of the heavy chain variable region.

[0056] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 8, as long as the altered amino acid sequence substantially maintains similar function of the heavy chain variable region.

[0057] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region may differ from the amino acid sequence shown in SEQ ID NO: 16 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and / or additions.

[0058] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 16 can be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still maintaining similar function of the light chain variable region.

[0059] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 16, and the resulting amino acid sequence still retains similar function of the light chain variable region.

[0060] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 16, as long as the altered amino acid sequence substantially maintains the similar function of the light chain variable region.

[0061] In some embodiments of the first aspect, the antigen-binding portion is selected from a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a scFv fragment, a Fd fragment, or a single domain antibody. Preferably, the antigen-binding fragment is in the form of a Fab fragment.

[0062] In some embodiments of the first aspect, the antibody is selected from a murine antibody, a human antibody, and a humanized antibody. Preferably, the antibody is a murine antibody.

[0063] In some embodiments of the first aspect, the antibody may be a monoclonal antibody.

[0064] In a second aspect, the present application provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to the first aspect.

[0065] In preferred embodiments, nucleic acids as described herein can be codon-optimized nucleic acids suitable for expression in host cells. For example, based on the degeneracy of codons, they still encode the same protein. Methods for codon optimization according to the host cell used are well known to those skilled in the art.

[0066] In some embodiments of the second aspect, the nucleic acid molecule may include a DNA molecule and an RNA molecule. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.

[0067] In some embodiments of the second aspect, the nucleic acid molecule is operably connected to a regulatory nucleotide sequence for expression in a host cell. In some embodiments, the nucleic acid molecule is operably connected to a regulatory nucleotide sequence, and the regulatory nucleotide sequence is contained in a vector (e.g., a plasmid) for expression in a host cell. The host cell can be from a mammal, such as a mouse, rat, dog, cat, sheep, cattle, monkey, camel, llama, the human being, etc. The host cell can be cultivated in a culture medium to express the antibody or its Fab, and then recovered to harvest the antibody or its Fab.

[0068] The present application provides a combination of polynucleotides, comprising a polynucleotide encoding the light chain of the antibody or antigen-binding portion thereof of the present application and a polynucleotide encoding the heavy chain of the antibody or antigen-binding portion thereof of the present application.

[0069] In a third aspect, the present application provides a vector comprising the nucleic acid molecule described in the second aspect.

[0070] In some embodiments of the third aspect, the vector is an expression vector. The vector (e.g., expression vector) may comprise the nucleic acid molecule or polynucleotide combination described in the second aspect. In some embodiments, the expression vector of the present application comprises the nucleic acid molecule or polynucleotide combination described herein, operably linked to regulatory sequences that permit expression of the encoded polypeptide in a host cell or cell-free expression system. The choice of expression vector depends on the choice of host cell and can be selected to have the desired expression and regulatory characteristics in the selected host cell.

[0071] An "expression vector" is a vector that includes one or more expression control sequences, which are DNA sequences that control and regulate the transcription and / or translation of another DNA sequence.

[0072] The nucleic acid in the vector can be operably connected to one or more expression control sequences. As used herein, "operably connected" means incorporated into a genetic construct so that the expression control sequence effectively controls the expression of the target coding sequence. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence composed of a region of a DNA molecule generally within 100 nucleotides upstream of the transcription start point (generally near the start site of RNA polymerase II). In order to place the coding sequence under the control of the promoter, the translation start site of the polypeptide translation reading frame must be positioned between 1 and about 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, position, and level. Unlike promoters, enhancers can work when located at different distances from the transcription site. Enhancers can also be located downstream of the transcription start site. When RNA polymerase is able to transcribe the coding sequence into mRNA, and then the mRNA can be translated into the protein encoded by the coding sequence, the coding sequence is "operably connected" to the expression control sequence in the cell and is "under the control" of the expression control sequence.

[0073] Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus, and adeno-associated virus. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (LaJolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0074] The expression vector may include a tag sequence. The tag sequence is generally expressed as a fusion with the encoded polypeptide. Such tags can be inserted into any position within the polypeptide, including the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, Fc fragments, polyhistidine, green fluorescent protein (GFP), glutathione S-transferase (GST), c-myc, hemagglutinin, FlagTM tag (Kodak, New Haven, CT), maltose E binding protein, and protein A. In some embodiments, the nucleic acid molecule encoding the antibody of the present application is present in a vector containing a nucleic acid encoding one or more domains of the Ig heavy chain constant region, such as the amino acid sequence corresponding to the hinge region, CH2 region, and CH3 region of the human immunoglobulin Cγ1 chain (Fc fragment).

[0075] In a fourth aspect, the present application provides a host cell containing the nucleic acid molecule described in the second aspect or the vector described in the third aspect. In certain embodiments of the present application, the host cell can be a prokaryotic host cell, a eukaryotic host cell or a bacteriophage. The prokaryotic host cell can be Escherichia coli (e.g., Escherichia coli cells well known in the art such as DH5α, BL21), Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic host cell can be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as S. frugiperda, plant cells such as tobacco, mammalian cells such as HEK293 cells, BHK cells, CHO cells, COS cells, myeloma cells. In some embodiments, the host cell described in the present application is preferably a mammalian cell, more preferably HEK293 cells, BHK cells, CHO cells, NSO cells or COS cells.

[0076] In some embodiments, the present application also provides a method for preparing the antibody or antigen-binding portion thereof according to the first aspect, comprising: a) cultivating the host cell according to the fourth aspect; and b) recovering the antibody or antigen-binding portion thereof from the host cell or the culture supernatant of the host cell.

[0077] In some embodiments, the present application also discloses a method for preparing an antibody that binds to trastuzumab. The method may include: culturing a host cell under expression conditions to express an antibody that binds to trastuzumab; and isolating and purifying the expressed antibody that binds to trastuzumab. Using the above method, a crude antibody can be obtained. The antibody that binds to trastuzumab is then purified to a substantially homogeneous substance, e.g., as a single band on SDS-PAGE electrophoresis, by a purification method including affinity purification based on trastuzumab, native gel purification, HPLC or RP-HPLC, size exclusion, purification on a protein A column, or any combination of these techniques.

[0078] In a fifth aspect, the present application provides a detection reagent or kit comprising the antibody or antigen-binding fragment thereof described in the first aspect.

[0079] In some embodiments of the fifth aspect, the detection reagent or kit is based on conventional antibody detection technology in the art, including but not limited to ELISA, Western Blotting or electrochemiluminescence technology to detect trastuzumab.

[0080] In some embodiments, the detection reagent or kit detects trastuzumab based on ELISA technology. For example, in addition to the antibody or antigen-binding fragment thereof described in the first aspect, the detection reagent or kit may further comprise a solid phase carrier, a secondary antibody, and / or a chromogenic substrate. In some specific embodiments, the antibody or antigen-binding fragment thereof described in the first aspect may be coated on the solid phase carrier, for example, as a primary antibody. After the primary antibody binds to trastuzumab, the binding status is determined using a secondary antibody and a chromogenic substrate.

[0081] In some embodiments, the detection reagent or kit is based on Western Blotting technology to detect trastuzumab. For example, in addition to the antibody or antigen-binding fragment thereof described in the first aspect, the detection reagent or kit may also include SDS-PAGE electrophoresis reagents, membranes (on which proteins are transferred), and other reagents and equipment.

[0082] In some embodiments, the detection reagent or kit detects trastuzumab based on electrochemiluminescence technology. For example, in addition to the antibody or antigen-binding fragment thereof described in the first aspect, the detection reagent or kit may further include reagents and equipment required for electrochemiluminescence technology. In some exemplary embodiments, the electrochemiluminescence technology used in this application is the MSD (Meso Scale Discovery) method, and the detection reagent or kit may further include an MSD plate coated with streptavidin and MSD Read Buffer T working solution for electrochemiluminescence detection.

[0083] Antibodies as described herein or their antigen-binding portion thereof can be combined with a detectable portion. Exemplary detectable portions include, but are not limited to, radioisotopes such as iodine-125, iodine-131, cesium-137, iridium-192 and cobalt-60, horseradish peroxidase, fluorescein isothiocyanate, biotin, alkaline phosphatase, chemiluminescent agents such as luminols, etc. Those skilled in the art can select suitable detectable portions to be combined with antibodies or their antigen-binding portion thereof of the present application as needed, thereby achieving different detection purposes.

[0084] In a sixth aspect, the present application provides use of the antibody or antigen-binding portion thereof according to the first aspect for detecting trastuzumab in a biological sample of a subject.

[0085] As used herein, the term "subject" refers to a mammal, including but not limited to primates, cattle, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice. Preferably, the mammal is a non-human primate or a human. A particularly preferred mammal is a human. As used herein, "patient" and "subject" are used interchangeably.

[0086] In some embodiments of the sixth aspect, a "biological sample" refers to any sample taken from a subject (e.g., a human or other animal), such as a human suffering from cancer or a human suspected of suffering from cancer, and containing trastuzumab. The biological sample can be a body fluid, such as blood, plasma, serum, urine, vaginal fluid, fluid from the scrotum (e.g., ascites from the testicles), vaginal washings, pleural fluid, ascites, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, discharge from the nipple, aspirated fluid from different parts of the body (e.g., thyroid, breast), intraocular fluid (e.g., aqueous humor), etc.

[0087] Throughout the specification and claims, the words “comprises,” “comprising,” and “including” mean “including but not limited to,” and are not intended to exclude other parts, additives, components, or steps.

[0088] It will be appreciated that features, characteristics, components, or steps described in conjunction with a particular aspect, embodiment, or example of the present application may be applicable to any other aspect, embodiment, or example described herein unless incompatible therewith.

[0089] The foregoing disclosure generally describes the present application. The examples are provided to further illustrate the present application and should not be construed as limiting the present application. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, insert protein-encoding genes into vectors and plasmids, or introduce plasmids into host cells. Such methods are well known to those skilled in the art and are described in numerous publications, for example, see Sambrook, J., Fritsch, EF. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press.

[0090] Example

[0091] Example 1: Preparation of hybridomas producing anti-trastuzumab antibodies

[0092] 1.1 Antigen preparation (emulsification)

[0093] Commercially available trastuzumab (supplier: Linyi Wubei Trading Co., Ltd.; brand: Daiichi Sankyo's trastuzumab) was diluted to 0.1 mg / mL using normal saline. 3.5 mL of the diluted trastuzumab (0.1 mg / mL) was mixed with an equal volume of Freund's complete adjuvant (Sigma, F5881) / Freund's incomplete adjuvant (Sigma, F5505). The mixture was then stirred in an ice bath using an adjuvant emulsifier (Biolong, BDYQ1001) for 3-5 minutes to fully emulsify the antigen, and then placed on ice for later use.

[0094] 1.2 Animal immunization

[0095] Prepare six 6-8 week old Balb / c mice and subcutaneously inject emulsified trastuzumab at multiple sites on the back, 0.2 mL per site, 1 mL per mouse (approximately 50 μg per mouse). Immunize every 2-3 weeks for a total of 3-4 times until serum antibody titers are positive.

[0096] 1.3 Cell fusion

[0097] Blood was collected from mice that had completed the immunization process and whose serum antibody titers met the requirements through the orbital venous plexus. The blood was allowed to rest at room temperature for 30 minutes, then centrifuged at 3000g for 15 minutes to separate the serum. The serum was then stored at -80°C until use as a positive control.

[0098] Mice were euthanized and disinfected by soaking in 75% alcohol. Spleens were aseptically removed in a clean bench and ground to release individual splenocytes. Single-cell suspensions were prepared by rinsing the cells with incomplete culture medium. After lysing red blood cells, the splenocyte suspension was filtered through a 40 μm cell strainer, centrifuged, and washed twice with incomplete culture medium. A small number of cells were removed and counted using a CountStar cell counter.

[0099] Mouse myeloma cells (Sp2 / 0 cells) in the logarithmic growth phase were taken out from the cell culture incubator, the old culture medium was removed by centrifugation, and the Sp2 / 0 cells were washed with incomplete culture medium and counted.

[0100] Mouse spleen cells and Sp2 / 0 cells were mixed at a ratio of 3:1 to 5:1 and fused using a PEG / electric fusion instrument.

[0101] The fused cells were gently resuspended in conditioned medium containing HAT supplement (Gibco, Cat: 21060-017) and Hybridoma Feeder supplement factor (Boaolong, Cat: CM-2001), and evenly plated in 96-well plates for culture.

[0102] After 10 to 14 days of culture, when the cell clones in the 96-well plate grew to more than 1 / 10 of the plate well area, the cell culture supernatant was identified by ELISA, and positive clones were screened and transferred to conditioned medium containing HT supplement (Gibco, Cat: 11067-030) for further culture.

[0103] After 2 to 3 rounds of cloning, a positive monoclonal cell line is obtained.

[0104] Example 2: Identification of binding activity of antibodies in hybridoma cell culture supernatants and clone screening

[0105] 2.1 Coating

[0106] Prepare coating reagent working solution: dilute trastuzumab and human IgG with 1x PBS to a concentration of approximately 1 μg / mL coating working solution.

[0107] The prepared coating working solution was added into 96-well plates at 100 μL / well and coated at 2-8°C overnight.

[0108] 2.2 Closure

[0109] Wash the plate three times with at least 300 μL / well of 1x PBST plate wash buffer and pat dry on a clean tissue. Add 300 μL / well of blocking buffer (1% (w / v) BSA in 1x PBST) to each well of the ELISA plate and block in a 37 ± 5°C incubator for 120 to 130 minutes. Then, wash the plate three times with 350 μL / well of 1x PBST plate wash buffer and pat dry on a clean tissue.

[0110] 2.3 Sample preparation

[0111] Dilute the mouse positive serum according to the dilution ratio in the following table as a positive control:

[0112] Specifically, mouse positive serum was diluted 1000-fold in two steps to prepare the working solution: First, 45 μl of PBS and 5 μl of the stock serum solution were added to tube A and mixed thoroughly; second, 3960 μl of PBS and 40 μl of the stock solution from tube A were added to tube B and mixed thoroughly. The table below regarding dilution ratios should be interpreted similarly to this example.

[0113] About 120 μL of culture supernatant was taken from the 96-well cell culture plate where clones were grown as the hybridoma supernatant sample to be tested.

[0114] 2.4 Sample addition

[0115] PC and hybridoma cell supernatant samples to be tested were added to the ELISA plate coated with trastuzumab and human IgG in sequence, 50 μL / well, sealed and gently shaken, and incubated in a 37°C incubator for 60-70 minutes.

[0116] 2.5 Preparation of detection reagent working solution

[0117] Dilute the test reagent with diluent. The specific dilution ratio is as follows according to the dilution plate chart:

[0118] Wash the plate three times with 1 x PBST at a volume of at least 300 μL / well and pat dry on a clean tissue. Add 100 μL / well of the corresponding detection reagent working solution to the ELISA plate and incubate in a 37°C incubator in the dark for 60-70 min.

[0119] 2.6 Color Rendering

[0120] Wash the plate three times with 1x PBST at a volume of at least 300 μL / well and pat dry on a clean tissue. Add 100 μL / well of the chromogenic substrate TMB solution to the plate and incubate at room temperature in the dark for 15-20 minutes.

[0121] 2.7 Detection, data analysis, and selection of positive clones

[0122] Add 1M H2SO4 (50 μL / well) to the ELISA plate and gently shake to ensure there is no orange-yellow coloration around the edges of the wells. Within 5 minutes, place the plate in a plate reader and analyze at a wavelength of 450-630 nm. Clones with an OD450 response greater than 1 in the trastuzumab-coated plate and less than 0.5 in the human IgG-coated plate will be designated as candidate positive clones for cloning and subsequent screening, or frozen for future use.

[0123] Figure 2 The figure shows a set of results during the screening of positive master clones in a 96-well plate using ELISA on a trastuzumab-coated plate, wherein well A1 is a positive serum sample (positive control) and the other dark-marked wells are positive candidate master clones.

[0124] Example 3: Cloning and screening of monoclonal antibodies

[0125] The cells of the positive candidate main clone were counted and the cell density was adjusted to 1×10 3 / mL to 1×10 5 / mL.

[0126] Remove 130 positive primary clone cells and add them to 6.5 mL of complete culture medium at a concentration of approximately 20 cells / mL. Mix thoroughly and add 100 μL / well to wells in rows A, B, and C of a 96-well plate, seeding approximately 2 cells per well. Add 2.9 mL of complete culture medium to the remaining 2.9 mL of cell suspension to a cell count of approximately 10 cells / mL. Add 100 μL / well to wells in rows D, E, and F, seeding approximately 1 cell per well. Add 2.2 mL of complete culture medium to the remaining 2.2 mL of cell suspension to a cell count of approximately 5 cells / mL. Add 100 μL / well to wells in rows G and H, seeding approximately 0.5 cells per well.

[0127] After 4-5 days of culture, small colonies can be seen growing under an inverted microscope.

[0128] On the 8th to 9th day, obvious clustered clones can be seen growing under the microscope, and some are even visible to the naked eye. ELISA testing should be performed in time.

[0129] Pick out the positive monoclonal clones and then proceed to subsequent screening and verification.

[0130] Perform 2-3 rounds of cloning until suitable positive monoclonal cells are screened. For the hybridoma cells cloned initially, add HT supplement (Gibco, Cat: 11067-030) to the complete culture medium.

[0131] In this example, a positive hybridoma cell clone was screened and named clone 26E5.

[0132] Example 4: Purification of antibodies

[0133] After expanding the positive monoclonal cells, 200 mL of cell culture supernatant was collected and centrifuged at 3000 rpm for 20 minutes to remove residual cells and debris. The supernatant was filtered through 0.45 μm and then 0.22 μm filters. The filtered cell supernatant was then loaded onto a pre-equilibrated protein G affinity chromatography column. The column was rinsed with phosphate buffer until equilibrium was reached, and then eluted with 50 mM citrate buffer (pH 3.0) at a flow rate of 5 mL / min. The entire elution peak was collected, and the eluate was finally adjusted to pH 7.4 with 1 M Tris HCl buffer (pH 9.0). The purified antibody was quantified using a NanoDrop micro-spectrophotometer and aliquoted for use.

[0134] Example 5: SDS-PAGE detection of purified antibodies

[0135] Based on the antibody quantitative concentration, 10 μg of sample was taken and added to PBS to a volume of no more than 30 μL. Sample loading buffer was added proportionally and the sample was treated in a boiling water bath for 5 minutes. A protein precast gel (GenScript, Cat: M00659) was fixed in an electrophoresis tank, electrophoresis buffer was added, and the samples were loaded one by one. Electrophoresis was performed at 150 V for 1 hour. After the electrophoresis, the gel was removed, rinsed with deionized water, and then stained in 0.1% Coomassie Brilliant Blue solution for 1 hour. After staining, the gel was transferred to a destaining solution for destaining, and the destaining solution was changed every 30 to 60 minutes until the protein bands on the gel were clearly visible.

[0136] The SDS-PAGE test results of antibody clone 26E5 are as follows Figure 2 As shown, the N column strips represent the 26E5 antibody under non-reducing conditions, and the R column strips represent the 26E5 antibody under reducing conditions.

[0137] Example 6: Antibody subtype identification

[0138] Antibody subtype identification was performed using the Pierce Rapid ELISA Mouse Monoclonal Antibody Typing Kit (Invitrogen, Cat: 37503).

[0139] Add 50 mL of 10× TBS to 450 mL of ultrapure water to make 1× TBS. Add 30 mL of 30× Wash Buffer to 870 mL of ultrapure water to make 1× Wash Buffer.

[0140] Prepare 450 μL of each sample for subtype identification. Specifically, for hybridoma supernatant, dilute the hybridoma supernatant 50-fold with 1× TBS by adding 20 μL of hybridoma supernatant to 980 μL of 1× TBS. For ascites, first add 1 μL of ascites to 5 mL of 1× TBS (1:5000), then add 67 μL of the dilution from the previous step to 933 μL of 1× TBS, diluting the sample 75,000-fold with 1× TBS. For purified antibodies, dilute the purified antibodies to 250 ng / mL with 1× TBS.

[0141] The TMB substrate and capture antibody coated strips were equilibrated at room temperature. Figure 1As shown, add 50 μL of diluted test sample to each well of strips A to H, 8 wells per strip. Add 50 μL of goat anti-mouse (IgG+IgA+IgM)-HRP to each of the 8 wells of the strip. Gently tap the strip to mix. Seal the plate with a film sealer and incubate at room temperature for 1 hour. Discard any remaining solution. Use a wash bottle to add 1x wash buffer to the wells, wash three times, and pat the strip dry on absorbent paper. Add 75 μL of TMB substrate equilibrated to room temperature to each well. After 1 minute, observe if positive wells turn blue. The development time and color intensity vary depending on the antibody concentration and isotype. After 5-15 minutes, add 75 μL of stop solution to each well to terminate the reaction. The wells will randomly change color from blue to yellow. Light intensity at 450 nm can be measured using a spectrophotometer or visually.

[0142] According to Figure 1 The subtype of the antibody to be tested is determined by typesetting. For each sample, there is one and only one positive well in wells AF (heavy chain typing), and one and only one positive well in wells GH (light chain typing).

[0143] As tested in this example, the antibody clone 26E5 belongs to the IgG3 subtype, and its light chain is a kappa chain.

[0144] The sequence of antibody clone 26E5 was identified by sequencing the antibody gene of the hybridoma cells producing antibody clone 26E5. The sequence of its variable region is shown in the following table:

[0145] The CDR sequences of antibody clone 26E5 are shown below:

[0146] Example 7: Identification of Binding Activity of Purified Antibodies

[0147] 7.1 Coating

[0148] Dilute trastuzumab and human IgG with 1x PBS to a coating working solution with a concentration of approximately 1 μg / mL.

[0149] The prepared coating working solution was added into the 96-well plate in sequence, 100 μL / well, and coated at 2-8°C overnight.

[0150] 7.2 Closure

[0151] Wash the plate three times with 1x PBST plate washer at a volume of at least 300 μL / well and pat dry on a clean tissue paper. Add blocking solution (1% (w / v) BSA in 1x PBST) to the enzyme-labeled wells at a volume of 300 μL / well and block in a 37±5°C incubator for 120-130 minutes. Wash the plate three times with 1x PBST plate washer at a volume of 350 μL / well and pat dry on a clean tissue paper. Store unused plates in sealed containers at 2-8°C until ready for use.

[0152] 7.3 Sample preparation

[0153] Dilute the mouse positive serum according to the dilution ratio in the table below as PC (positive control):

[0154] The purified antibody was diluted with PBS in a gradient ratio of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, and 1:640, mixed and set aside.

[0155] 7.4 Sample addition

[0156] All pre-treated samples (positive control and purified antibody dilution) were added to the ELISA plate in sequence, 100 μL / well, sealed and gently shaken, and incubated at 37°C for 60-70 minutes.

[0157] 7.5 Preparation of detection reagent working solution

[0158] Dilute the test reagent with diluent. The specific dilution ratio is as follows according to the dilution plate chart:

[0159] Wash the plate three times with 1 x PBST plate washer at a volume of at least 300 μL / well and pat dry on a clean tissue paper. Add the corresponding detection reagent working solution to the ELISA plate at a volume of 100 μL / well and incubate in a 37°C incubator in the dark for 60-70 min.

[0160] 7.6 Color Rendering

[0161] Wash the plate three times with 1x PBST at a volume of at least 300 μL / well and pat dry on a clean tissue. Add 100 μL / well of the chromogenic substrate TMB solution to the plate and incubate at room temperature in the dark for 15-20 minutes.

[0162] 7.7 Testing, Data Storage and Result Analysis

[0163] Add 1M H2SO4 to the ELISA plate, 50 μL / well, and shake gently to ensure that there is no orange-yellow color at the edge of each well. Place the ELISA plate in a plate reader for detection within 5 minutes at a wavelength of 450 nm-630 nm.

[0164] The experimental results are as follows Figure 4 As shown, the 26E5 antibody was able to bind to trastuzumab, and as the concentration of the 26E5 antibody increased, the binding of the 26E5 antibody to trastuzumab increased, while the 26E5 antibody had only very weak and almost negligible binding to human IgG.

[0165] Example 8: Competitive binding activity detection of purified antibodies

[0166] 8.1 Coating

[0167] Dilute trastuzumab with 1xPBS to a coating working solution with a concentration of approximately 1 μg / mL.

[0168] The prepared coating working solution was added into the 96-well plate in sequence, 100 μL / well, and coated at 2-8°C overnight.

[0169] 8.2 Closure

[0170] Wash the plate three times with 1x PBST plate washer at a volume of at least 300 μL / well and pat dry on a clean tissue paper. Add blocking solution (1% (w / v) BSA in 1x PBST) to the enzyme-labeled wells at a volume of 300 μL / well and block in a 37±5°C incubator for 120-130 minutes. Wash the plate three times with 1x PBST plate washer at a volume of 350 μL / well and pat dry on a clean tissue paper. Store unused plates in sealed containers at 2-8°C until ready for use.

[0171] 8.3 Sample preparation

[0172] 8.3.1 Dilute five purified antibodies from the same clone 26E5 in PBS at a ratio of 1:20.

[0173] 8.3.2 Dxd working solution: Dilute Dxd to 5.0 μg / mL with PBS.

[0174] 8.4 Adding samples

[0175] 8.4.1 Divide the diluted purified antibody sample into two equal portions: mix one portion with equal volumes of Dxd working solution (150 μL:150 μL), and mix the other portion with equal volumes of PBS. Incubate at 37°C for 60-70 minutes.

[0176] 8.4.2 Add the mixed samples after incubation to the ELISA plate in sequence, 100 μL / well, seal the plate, shake gently, and incubate at 37°C for 60-70 minutes.

[0177] 8.5 Preparation of detection reagent working solution

[0178] Dilute the test reagent with diluent. The specific dilution ratio is as follows according to the dilution plate chart:

[0179] Wash the plate three times with 1 x PBST at a volume of at least 300 μL / well and pat dry on a clean tissue. Add 100 μL / well of the corresponding detection reagent working solution to the ELISA plate and incubate in a dark incubator at 37°C for 60-70 min.

[0180] 8.6 Color Rendering

[0181] Wash the plate three times with 1x PBST at a volume of at least 300 μL / well and pat dry on a clean tissue. Add 100 μL / well of the chromogenic substrate TMB solution to the plate and incubate at room temperature in the dark for 15-20 minutes.

[0182] 8.7 Testing, Data Storage and Result Analysis

[0183] Add 1M H2SO4 to the ELISA plate, 50 μL / well, and shake gently to ensure that there is no orange-yellow color at the edge of each well. Place the ELISA plate in a plate reader for detection within 5 minutes at a detection wavelength of 450 nm-630 nm.

[0184] The experimental results are as follows Figure 5 As shown, after preincubation with five purified antibodies from the same clone 26E5 (i.e., 26E5 was cloned after two rounds of cloning, resulting in five subclones, and then purified antibodies mAb 1, mAb 2, mAb 3, mAb 4, and mAb 5), Dxd was able to block the binding of clone 26E5 to trastuzumab (blocking efficiency ranging from 86.78% to 91.50%). The blocking efficiency was determined by the following formula: (OD value of the experimental group - OD value of the control group) / OD value of the control group x 100%, where the control group consisted of an equal volume mixture of each purified antibody and PBS from step 8.4. These results demonstrate that antibody clone 26E5 is capable of binding to the small molecule Dxd portion of trastuzumab.

[0185] Sequence information

[0186] SEQ ID NO: 1 (VH-FR1)

[0187] QVQLKESGPGLVAPSQSLSITCTVS

[0188] SEQ ID NO: 2 (VH-CDR1 - numbering according to IMGT definition)

[0189] GFSLTNFG

[0190] SEQ ID NO: 3 (VH-FR2)

[0191] VHWVRQPPGKGLEWLGV

[0192] SEQ ID NO: 4 (VH-CDR2 - numbering according to IMGT definition)

[0193] IWAGGST

[0194] SEQ ID NO: 5 (VH-FR3)

[0195] NYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYC

[0196] SEQ ID NO: 6 (VH-CDR3 - numbering according to IMGT definition)

[0197] VRDDYGSSYGEDY

[0198] SEQ ID NO: 7 (VH-FR4)

[0199] WGQGTTLTVSS

[0200] SEQ ID NO: 8 (VH)

[0201] QVQLKESGPGLVAPSQSLSITCTVSGFSLTNFGVHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCVRDDYGSSYGEDYWGQGTTLTVSS

[0202] SEQ ID NO: 9 (VL-FR1)

[0203] QIVLTQSPAIMSASPGEKVTMTCSAS

[0204] SEQ ID NO: 10 (VL-CDR1 - numbering according to IMGT definition)

[0205] SSVSY

[0206] SEQ ID NO: 11 (VL-FR2)

[0207] MHWYQQKSGTSPKRWIY

[0208] SEQ ID NO: 12 (VL-CDR2 - numbering according to IMGT definition)

[0209] DTS

[0210] SEQ ID NO: 13 (VL-FR3)

[0211] KLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC

[0212] SEQ ID NO: 14 (VL-CDR3 - numbering according to IMGT definition)

[0213] QQWSSNPPT

[0214] SEQ ID NO: 15 (VL-FR4)

[0215] FGGGTKLEIK

[0216] SEQ ID NO: 16 (VL)

[0217] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGGTKLEIK

[0218] SEQ ID NO: 17 (VH-CDR1 - numbering according to Kabat definition)

[0219] NFGVH

[0220] SEQ ID NO: 18 (VH-CDR2 - numbering according to Kabat definition)

[0221] VIWAGGSTNYNSALMS

[0222] SEQ ID NO: 19 (VH-CDR3 - numbering according to Kabat definition)

[0223] DDYGSSYGEDY

[0224] SEQ ID NO: 20 (VL-CDR1 - numbering according to Kabat definition)

[0225] SASSSVSYMH

[0226] SEQ ID NO: 21 (VL-CDR2 - numbering according to Kabat definition)

[0227] DTSKLAS

[0228] SEQ ID NO: 22 (VL-CDR3 - numbering according to Kabat definition)

[0229] QQWSSNPPT

[0230] All publications and patent documents cited in this specification are incorporated herein by reference, as if each publication or patent was expressly indicated to be incorporated herein by reference. Without departing from the true idea and scope of the present disclosure, various changes and equivalents may be made to each embodiment disclosed in the present application. Unless otherwise indicated in the context, any feature, step or embodiment of the embodiment of the present disclosure may be used in combination with any other feature, step or embodiment.

Claims

1. An antibody or antigen-binding fragment thereof that binds to trastuzumab, the antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and wherein according to IMGT definition numbering, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 4, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 6, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 10, the amino acid sequence of LCDR2 is DTS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 14; or According to the Kabat definition, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 17, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 18, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 19, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 20, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 21, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:

22.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 8, preferably the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 8, and The amino acid sequence of the light chain variable region has at least 90% sequence identity with SEQ ID NO: 16, and preferably the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

16.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the antigen-binding portion is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a scFv fragment, a Fd fragment and a single domain antibody.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is selected from the group consisting of a murine antibody, a human antibody and a humanized antibody.

5. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. A vector comprising the nucleic acid molecule according to claim 5, preferably an expression vector.

7. A host cell comprising the nucleic acid molecule of claim 5 or the expression vector of claim 6.

8. A detection reagent or kit comprising the antibody or antigen-binding portion thereof according to any one of claims 1 to 4.

9. The detection reagent or kit according to claim 8, wherein the detection reagent or kit detects trastuzumab based on ELISA, Western Blotting or electrochemiluminescence technology.

10. Use of the antibody or antigen-binding portion thereof of any one of claims 1 to 4 for detecting trastuzumab in a biological sample of a subject.

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