Antibodies targeting denosumab and uses thereof

Abstract

The present application provides an antibody or antigen-binding fragment thereof targeting denosumab, a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, a vector comprising the nucleic acid molecule, a host cell comprising the nucleic acid molecule or vector, a detection reagent comprising the antibody or antigen-binding fragment thereof, a detection kit comprising the antibody or antigen-binding fragment thereof, and uses thereof.

Description

TECHNICAL FIELD

[0001] The present application relates generally to the field of genetic engineering and antibody medicine; in particular, the present application relates to antibodies targeting trastuzumab deruxtecan and uses thereof. BACKGROUND

[0002] An antibody-drug conjugate (ADC) is a drug formed by the connection of an antibody and a small molecule drug through a specific linker. ADC combines the high specificity of antibody drugs and the high cytotoxic activity of small molecule drugs, which can improve tumor targeting and reduce toxic side effects. ADC drugs accurately recognize target points and can release highly active cytotoxins inside tumor tissues, thereby exerting targeted drug efficacy on tumor lesions and reducing 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, etc. DXd (exatecan derivative) is a DNA topoisomerase I (TOP I) inhibitor.

[0004] Trastuzumab deruxtecan for injection (trade name: Kyowa Kirin; generic name: Fam-Trastuzumab deruxtecan (T-DXd); medical name: trastuzumab deruxtecan (ENHERTU); research code: DS-8201) is an antibody-drug conjugate targeting HER2. It has been officially approved by the China National Medical Products Administration for marketing in China in February 2023. Its indications include: HER2-positive breast cancer, gastric cancer, gastroesophageal junction cancer, HER2-low breast cancer, non-small cell lung cancer, etc. Trastuzumab deruxtecan shows excellent efficacy and good safety in clinical application by using a unique DNA topoisomerase I inhibitor DXd and a stable linker.

[0005] As the clinical application of trastuzumab deruxtecan continues to expand, it is necessary to detect the content of the drug in the blood, conduct pharmacokinetic analysis of the drug, monitor the production of anti-drug antibodies (ADA), and guide clinical rational drug use. Therefore, there is a need in the art to detect trastuzumab deruxtecan. SUMMARY

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

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

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

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

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

[0012] Figure 1 Exemplary sample layouts on 96-well plates in antibody subtype identification are shown.

[0013] Figure 2 A set of results from a daratumumab-coated plate ELISA detection screen for positive master clone selection in 96-well plates is shown.

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

[0015] Figure 4 Results of detection of the binding of the purified 26E5 antibody to trastuzumab and human IgG, respectively, by ELISA are shown.

[0016] Figure 5 Results of detection of the binding of the purified 26E5 antibody to trastuzumab after pre-incubation with Dxd by ELISA, showing that Dxd effectively blocks 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 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 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] Pharmacokinetic (PK) studies related to ADCs 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 act as capture antibodies to bind the analyte in PK analysis for LBA, or they can help enrich ADC samples in LC-MS / MS analysis.

[0027] As the clinical applications of trastuzumab continue to expand, there is a need to detect the level of this drug in the blood for pharmacokinetic analysis and to monitor the production of anti-drug antibodies (ADAs) to guide rational clinical drug use. There is a need in this field to detect trastuzumab. This application addresses this need by providing an antibody targeting trastuzumab or its antigen-binding fragment, thereby promoting the clinical application of trastuzumab. Testing has shown that the antibody targeting trastuzumab or its antigen-binding fragment in this application can specifically bind to trastuzumab, thus supporting the accurate detection of trastuzumab levels in the blood and facilitating pharmacokinetic analysis.

[0028] definition

[0029] Unless otherwise specified, this application is implemented using conventional molecular biology, microbiology, cell biology, biochemistry and immunology techniques in 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 terms used herein have the same meaning as understood by one of ordinary skill in the art. The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0032] Although the numerical ranges and parameter approximations shown in the broad scope of this application are intended to be as accurate as possible in the specific embodiments, any numerical value inherently contains a certain degree of error due to the standard deviation present in their respective measurements. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges contained therein. For example, the stated range “1 to 10” should be considered to include any and all subranges between the minimum value of 1 and the maximum value of 10 (inclusive); that is, all subranges beginning with a minimum value of 1 or greater, such as 1 to 6.1, and subranges ending with a maximum value of 10 or less, such as 5.5 to 10. Additionally, any references marked “incorporated herein” should be understood to be incorporated herein in their entirety.

[0033] In a broad sense, the term "antibody" refers to an immunoglobulin molecule capable of specific 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, antibody single chains or any antigen binding fragment (also referred to as "antigen binding portion") of an antibody, 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 antibody, multi-specific antibodies (e.g. bi-specific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of an antibody, amino acid sequence variants of an antibody and covalently modified antibodies. When "antibody" and "antigen binding fragment / antigen binding portion" appear in the same context, "antibody" is understood to be the complete entity in relation to "antigen binding fragment / antigen binding portion", both together corresponding to the broad concept of an antibody.

[0034] Generally, an intact or full length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and a 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). The full length antibody can be of any class, such as IgD, IgE, IgG, IgA or IgM (or a subclass thereof), although the antibody need not be of any particular class. The assignment of different subclasses of immunoglobulins to different classes is based on amino acid sequences of the constant domains of the heavy chains. Typically, immunoglobulins of the five major classes, IgA, IgD, IgE, IgG and IgM, are further divided into subclasses or isotypes, e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called , , , , and The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0035] The terms "antigen binding fragment or antigen binding portion" are used interchangeably herein and refer to a portion or region of an intact antibody molecule 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 VH and VL typically contains three complementarity determining regions, CDR1, CDR2 and CDR3.

[0036] For a general antibody, examples of antigen-binding fragments thereof 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 at the hinge region (i.e., a dimer of Fab’); (3) a Fv fragment having a VL and a VH domain of a single arm of an antibody; (4) a single chain Fv (scFv), which can be a single polypeptide chain consisting of a VH domain and a VL domain via a peptide linker; and (5) a (scFv)2, which can comprise two VH domains and two VL domains connected by a peptide linker, which are combined with the two VH domains via a disulfide bridge.

[0037] In some embodiments of the present application, the “antigen-binding fragment” includes, but is not limited to, 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.

[0038] The term “single chain antibody (scFv)” as used herein refers to an antibody of a single chain structure generally constructed using genetic engineering techniques, comprising a polypeptide chain of a heavy chain variable region (VH) and a light chain variable region (VL). A flexible linker is usually designed between the heavy chain variable region and the light chain variable region so that the heavy chain variable region and the light chain variable region can fold into a correct conformation capable of binding to an antigen.

[0039] The term “Fab (fragment antigen binding) fragment”, “Fab portion” or similar terms as used herein refer to an antibody fragment capable of binding to an antigen produced after treating an intact antibody with papain, including an intact light chain (VL-CL), a heavy chain variable region and a CH1 fragment (VH-CH1).

[0040] The term “Fd fragment”, “Fd portion” or similar terms as used herein refer to the heavy chain portion in the Fab fragment of an antibody, including a heavy chain variable region and a CH1 fragment (VH-CH1).

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

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

[0043] It is well known to the person skilled in the art that the complementarity determining regions (CDRs, typically CDR1, CDR2 and CDR3) are the regions in the variable region that have the most impact on the affinity and specificity of an antibody. There are several common definitions of CDR amino acid sequences of a VH or VL, including the IMGT definition, the Kabat definition, the AbM definition and the Chothia definition. See, e.g., 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 variable region amino acid sequence of an antibody, the CDR amino acid sequences in the variable region amino acid sequence can be analyzed in various ways, e.g. using the online software Abysis (http: / / www.abysis.org / ).

[0045] The term "specifically binds" as used herein refers to a non-random binding reaction between two molecules, e.g. an antibody to an epitope of an antigen.

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

[0047] The term "trastuzumab deruxtecan" as used herein refers to an antibody-drug conjugate targeting HER2 with the trade name of Enhertu, the generic name of Fam-Trastuzumab deruxtecan (T-DXd), the medical name of trastuzumab deruxtecan (ENHERTU), and the research code of DS-8201, which has been officially approved by the National Medical Products Administration of China for marketing in China in February 2023.

[0048] The term "identity / homology / identity" with respect to an amino acid or nucleic acid sequence is defined as the percentage of residues in the variant amino acid or nucleotide sequence that are identical after alignment and introduction of gaps, if necessary, to achieve the maximum percent 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 trastuzumab deruxtecan, 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, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and wherein the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 4, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 6, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR2 is DTS, and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 14, according to IMGT definition numbering; or the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 17, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 18, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 19, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 20, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 21, and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 22, according to Kabat definition numbering.

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

[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 with SEQ ID NO: 8. Preferably, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 8.

[0052] 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 with SEQ ID NO: 8. Preferably, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 8.

[0053] 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 with SEQ ID NO: 8. Preferably, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 8.

[0054] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence set forth 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 retaining similar functionality 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 also be added to the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 8, while the resulting amino acid sequence still retains similar functionality 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 can also be added to the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 8, while the resulting amino acid sequence still retains similar functionality of the heavy chain variable region.

[0057] 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 with SEQ ID NO: 8. Preferably, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 8.

[0058] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence of 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 retaining similar functionality 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 also be added to the C-terminal or N-terminal region of the amino acid sequence of SEQ ID NO: 16, while the resulting amino acid sequence still retains similar functionality 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 can also be added to or deleted from regions other than the C-terminal or N-terminal region of the amino acid sequence of SEQ ID NO: 16, as long as the altered amino acid sequence substantially retains similar functionality of the light chain variable region.

[0061] In some embodiments of the first aspect, the antigen binding moiety 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 moiety 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 can be a monoclonal antibody.

[0064] In a second aspect, the present application provides nucleic acid molecules encoding the antibody or antigen binding moiety thereof of the first aspect.

[0065] In preferred embodiments, the nucleic acid described herein can be a codon-optimized nucleic acid suitable for expression in a host cell. For example, according to the degeneracy of the codons, it still encodes the same protein. Methods for codon optimization according to the host cell used are well known to the person skilled in the art.

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

[0067] In some embodiments of the second aspect, the nucleic acid molecule is operably linked to a regulatory nucleotide sequence for expression in a host cell. In some embodiments, the nucleic acid molecule is operably linked to a regulatory nucleotide sequence, which is included 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, cow, monkey, camel, llama, human, etc. The host cell can be cultured in a culture medium to express the antibody or antigen-binding fragment thereof, and in turn, be recovered to harvest the antibody or antigen-binding fragment thereof.

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

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

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

[0072] A nucleic acid in a vector can be operably linked to one or more expression control sequences. As used herein, "operably linked" means incorporated into a genetic construct in a manner which allows for expression control of a coding sequence of interest by the expression control sequence. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence that consists of a region of DNA, usually within 100 nucleotides upstream of the site where transcription starts (usually near the start site for RNA polymerase II), that initiates transcription by RNA polymerase. To place a coding sequence under the control of a promoter, the translational start site of the polypeptide-translation reading frame must be positioned between one and about 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcriptional start site. Enhancers can also be located downstream from the transcriptional start site. A coding sequence is "operably linked" to and "under the control" of an expression control sequence in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which can then be translated into a protein encoded by the coding 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 (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0074] An expression vector can include a tag sequence. A tag sequence is typically expressed as a fusion to the encoded polypeptide. Such tags can be inserted at 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, Flag™ tag (Kodak, New Haven, CT), maltose E binding protein, and protein A. In some embodiments, a nucleic acid molecule encoding an antibody of the application is present in a vector that contains nucleic acid encoding one or more domains of an Ig heavy chain constant region, e.g., corresponding to the hinge, CH2, and CH3 regions of a human immunoglobulin C gamma 1 chain (Fc fragment).

[0075] In a fourth aspect, the present application provides a host cell comprising the nucleic acid molecule of the second aspect or the vector of 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 E. coli (e.g., DH5a, BL21, and the like, which are well known in the art), Bacillus subtilis, Streptomyces, or Proteus mirabilis, and the like. The eukaryotic host cell can be a fungus such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, and the like, an insect cell such as Spodoptera frugiperda, and the like, a plant cell such as tobacco, and the like, a mammalian cell such as HEK293 cell, BHK cell, CHO cell, COS cell, myeloma cell, and the like. In some embodiments, the host cell of the present application is preferably a mammalian cell, more preferably a HEK293 cell, a BHK cell, a CHO cell, an NSO cell, or a COS cell.

[0076] In some embodiments, the present application also provides a method of producing the antibody or antigen-binding portion thereof of the first aspect, comprising:

[0077] a) culturing the host cell of the fourth aspect; and

[0078] b) recovering the antibody or antigen-binding portion thereof from the host cell or the culture supernatant of the host cell.

[0079] In some specific embodiments, the present application also discloses a method of producing an antibody that binds to trastuzumab, which can comprise: culturing a host cell under conditions such that the antibody that binds to trastuzumab is expressed; 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 material, e.g., a single band on SDS-PAGE electrophoresis, by a purification method including affinity purification based on trastuzumab, non-denaturing gel purification, HPLC or RP-HPLC, molecular exclusion, purification on a protein A column, or any combination of these techniques.

[0080] In a fifth aspect, the present application provides a detection reagent or kit comprising the antibody or antigen-binding fragment thereof of the first aspect.

[0081] In some embodiments of the fifth aspect, the detection reagent or kit detects trastuzumab based on conventional antibody detection techniques in the art, including but not limited to ELISA, Western Blotting, or electrochemiluminescence technology.

[0082] In some embodiments, the detection reagent or kit detects the dectraezumab based on ELISA technology. For example, in addition to the antibody or antigen-binding fragment thereof of the first aspect, the detection reagent or kit can further comprise a solid support, a secondary antibody, and / or a chromogenic substrate. In some specific embodiments, the antibody or antigen-binding fragment thereof of the first aspect can be coated on the solid support, for example, as a primary antibody, and after the primary antibody binds to the dectraezumab, the binding can be judged by the secondary antibody and the chromogenic substrate.

[0083] In some embodiments, the detection reagent or kit detects the dectraezumab based on Western Blotting technology. For example, in addition to the antibody or antigen-binding fragment thereof of the first aspect, the detection reagent or kit can further comprise SDS-PAGE electrophoresis reagents, a membrane (to which proteins are transferred), and the like reagents and equipment.

[0084] In some embodiments, the detection reagent or kit detects the dectraezumab based on electrochemiluminescence technology. For example, in addition to the antibody or antigen-binding fragment thereof of the first aspect, the detection reagent or kit can further comprise reagents and equipment required for electrochemiluminescence technology. In some exemplary embodiments, the electrochemiluminescence technology used in the present application is the MSD (Meso Scale Discovery) method, and the detection reagent or kit can further comprise an MSD plate coated with streptavidin and an MSD Read Buffer T working solution for electrochemiluminescence detection.

[0085] The antibody or antigen-binding portion thereof described herein can be combined with a detectable moiety. Exemplary detectable moieties 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 luminol, and the like. A person skilled in the art can select a suitable detectable moiety to be combined with the antibody or antigen-binding portion thereof of the present application as needed to achieve different detection purposes.

[0086] In a sixth aspect, the present application provides use of the antibody or antigen-binding portion thereof of the first aspect for detecting dectraezumab in a biological sample of a subject.

[0087] The term "subject" as used herein refers to a mammal, including, but not limited to, primates, cows, 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. "Patient" and "subject" are used interchangeably herein.

[0088] In some embodiments of the sixth aspect, "biological sample" refers to any sample taken from a subject (e.g., a human or other animal), e.g., a human having cancer, or a human suspected of having cancer, and containing denosumab. The biological sample can be a body fluid, e.g., blood, plasma, serum, urine, vaginal fluid, fluid from the scrotum (e.g., ascites fluid from the testes), vaginal lavage fluid, pleural fluid, ascites fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, expressed fluid from the nipple, suctioned fluid from different parts of the body (e.g., thyroid, breast), intraocular fluid (e.g., aqueous humor), etc.

[0089] In this specification and in the claims, the words "comprising", "including", and "containing" mean "including but not limited to", and are not intended to (and are not to be construed as) mean that the other parts, additives, components, or steps are essential, or required.

[0090] It should be understood that features, aspects, components or steps described in a particular aspect, embodiment or example of the application can be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

[0091] The foregoing disclosure generally describes the application, with a further description being set forth in the Examples, which are not to be construed as limiting the application. The Examples do not include detailed descriptions of conventional methods, such as those for constructing vectors and plasmids, inserting genes encoding proteins into vectors and plasmids, or introducing plasmids into host cells, as such methods are well known to those of ordinary skill in the art and are described in many publications, e.g., see Sambrook, J., Fritsch, EF. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold spring Harbor Laboratory Press.

[0092] Examples

[0093] Example 1: Preparation of hybridomas producing anti-denosumab antibodies

[0094] 1.1 Antigen preparation (emulsification)

[0095] Dinutuximab (supplied by Linyi WuBei Business Trading Co., Ltd.; brand: Dinutuximab of First Dose) on the market was diluted to 0.1 mg / mL with physiological saline, 3.5 mL of diluted Dinutuximab (0.1 mg / mL) was mixed with an equal volume of Freund's complete adjuvant (Sigma, F5881) / Freund's incomplete adjuvant (Sigma, F5505), and then stirred evenly in an ice bath for 3-5 minutes using an adjuvant emulsifier (Boaolong, BDYQ1001) to fully emulsify the antigen, and then placed on ice for standby.

[0096] 1.2 Animal immunization

[0097] Prepare 6-8 week old Balb / c mice 6, inject the emulsified Dinutuximab subcutaneously at multiple points on the back, 0.2 mL per point, and inject 1 mL per mouse (about 50 μg per mouse). Immunize every 2-3 weeks, a total of 3 to 4 times, until the serum antibody titer test is positive.

[0098] 1.3 Cell fusion

[0099] Take the mouse that has completed the immunization process and whose serum antibody titer meets the requirements, and collect blood through the orbital venous plexus. After the blood is left at room temperature for 30 minutes, centrifuge at 3000g for 15 minutes to separate the serum, which is stored in a -80°C refrigerator for standby as a positive control.

[0100] Euthanize the mouse and soak it in 75% alcohol for disinfection. In a clean bench, sterilely remove the mouse spleen, grind and crush the spleen to release single spleen cells, and use incomplete medium to rinse the cells to prepare a single cell suspension. After lysing the red blood cells, filter the spleen cell suspension through a 40 μm cell filter, centrifuge and wash the cells with incomplete medium twice. Take a small amount of cells and count them using a CountStar cell counter.

[0101] Remove the mouse myeloma cells (Sp2 / 0 cells) in the logarithmic growth phase from the cell incubator, centrifuge to remove the old culture medium, wash the Sp2 / 0 cells with incomplete medium and count them.

[0102] Mix the mouse spleen cells and Sp2 / 0 cells at a ratio of 3:1 to 5:1 and use PEG / electrofusion instrument for fusion.

[0103] Resuspend the fused cells with HAT supplement (Gibco, Cat: 21060-017) and Hybridoma Feeder additive factor (Boaolong, Cat: CM-2001) in the conditioned medium, and evenly distribute them in a 96-well plate for culture.

[0104] After 10 to 14 days of culture, when the cell clones in the 96-well plate grow to more than 1 / 10 of the area of the plate well, the cell culture supernatant is identified by ELISA, and the positive clones are screened and transferred to the conditioned medium containing HT supplement (Gibco, Cat: 11067-030) for further culture.

[0105] After 2 to 3 rounds of cloning, positive monoclonal cell strains are obtained.

[0106] Example 2: Identification of the binding activity of antibodies in hybridoma cell culture supernatant and clone screening

[0107] 2.1 Coating

[0108] Prepare the coating reagent working solution: dilute the deertuzumab and human IgG with 1x PBS to a concentration of about 1 μg / mL of coating working solution.

[0109] Add the prepared coating working solution to the 96-well plate at 100 μL / well, and coat overnight at 2-8°C.

[0110] 2.2 Blocking

[0111] Wash the plate with 1 x PBST at no less than 300 μL / well for 3 times, and dry on clean paper. Add blocking solution (1% (w / v) BSA in 1 x PBST) to the enzyme-labeled plate wells at 300 μL / well, and incubate in a 37±5°C incubator for 120 minutes to 130 minutes. Then wash the plate with 1 x PBST at 350 μL / well for 3 times, and dry on clean paper for standby.

[0112] 2.3 Sample preparation

[0113] Dilute the mouse positive serum according to the dilution ratio in the following table as a positive control:

[0114]

[0115] That is, dilute the mouse positive serum 1000 times in two steps as a working solution: first, add 45ul PBS and 5ul serum stock solution to tube A and mix well; second, add 3960ul PBS and 40ul A tube stock solution to tube B and mix well. The table below regarding the dilution ratio is understood similarly to this example.

[0116] Take about 120 μL of culture supernatant from the 96-well cell culture plate of the outgrowth clone as the hybridoma supernatant sample for standby.

[0117] 2.4 Sample addition

[0118] Add PC and the supernatant sample of the hybridoma cell to be detected in the enzyme-labeled plate coated with dectuzumab and human IgG, 50 μL / well, seal the plate and slightly shake, incubate in the 37℃ incubator for 60-70 min.

[0119] 2.5 Preparation of detection reagent working solution

[0120] Dilute the detection reagent with diluent, and the specific dilution ratio is as follows according to the dilution plate diagram:

[0121]

[0122] Wash the plate with 1 x PBST washing solution, 300 μL / well at least, and dry on clean paper; add the corresponding detection reagent working solution to the enzyme-labeled plate, 100 μL / well, and incubate in the 37℃ incubator for 60-70 min.

[0123] 2.6 Color development

[0124] Wash the plate with 1 x PBST washing solution, 300 μL / well at least, and dry on clean paper. Add color developing substrate TMB solution to the enzyme-labeled plate, 100 μL / well, and incubate at room temperature for 15-20 min.

[0125] 2.7 Detection, data analysis, and selection of positive clones

[0126] Add 1M H2SO4 to the enzyme-labeled plate, 50 μL / well, and slightly shake to ensure that there is no orange-yellow phenomenon at the edge of each well. Place the enzyme-labeled plate into the plate reader within 5 min for detection, and the detection wavelength is 450 nm-630 nm. Clones with OD450 response value greater than 1 in the dectuzumab-coated plate and OD450 response value less than 0.5 in the human IgG-coated plate are selected as candidate positive clones, which are further cloned and screened, or stored for later use.

[0127] Figure 2 A set of results in the process of detecting and screening positive master clones in the 96-well plate by ELISA of the dectuzumab-coated plate is shown, in which A1 well is a positive serum sample (positive control), and other darkly labeled wells are positive candidate master clones.

[0128] Example 3: Cloning and screening of monoclonal antibodies

[0129] Count the positive candidate master clones, and adjust the cell density to 1×10 3 / mL to 1×10 5 / mL with culture medium.

[0130] Take out 130 positive master clone cells and add to 6.5 mL complete culture medium, about 20 cells / mL, mix well, and then add to 96-well plate A, B, C three rows of plate wells at 100 μL / well, about 2 cells per well. Add 2.9 mL complete culture medium to the remaining 2.9 mL cell suspension to make the cell number about 10 / mL, and then add to D, E, F three rows of plate wells at 100 μL / well, about 1 cell per well. Add 2.2 mL complete culture medium to the remaining 2.2 mL cell suspension to make the cell number about 5 / mL, and then add to G, H two rows at 100 μL / well, about 0.5 cells per well.

[0131] After 4-5 days of culture, small clones can be observed under an inverted microscope.

[0132] On the 8th-9th day, obvious clonal growth can be observed under a microscope, and some can even be observed with the naked eye. ELISA detection is performed in time.

[0133] The positive monoclonal is picked out, and then subsequent screening and verification work is carried out.

[0134] 2-3 rounds of cloning are carried out until a suitable positive monoclonal cell is screened. For the first time, the hybridoma cells are added with HT supplement (Gibco, Cat: 11067-030) in complete culture medium.

[0135] A positive hybridoma cell clone is screened in this example, which is named clone 26E5.

[0136] Example 4: Antibody purification

[0137] After the positive monoclonal cells are expanded and cultured, 200 mL of cell culture supernatant is collected, centrifuged at 3000 rpm for 20 minutes, and the residual cells and debris are removed. The supernatant is filtered with 0.45 μm and 0.22 μm filter membranes in sequence. Then the filtered cell supernatant is loaded onto a protein G affinity chromatography column which is pre-equilibrated, the column is washed with phosphate buffer to equilibrate, and then eluted with 50 mM citric acid buffer (pH 3.0) at a flow rate of 5 mL / min, the complete elution peak is collected, and finally the eluate is adjusted to pH 7.4 with 1 M Tris HCL buffer (pH 9.0). The purified antibody is quantified using a NanoDrop microspectrophotometer, and then is aliquoted.

[0138] Example 5: SDS-PAGE detection of purified antibody

[0139] According to the quantitative concentration of the antibody, 10 μg of sample was taken and added to PBS so that the volume did not exceed 30 μL, and the loading buffer was added in proportion, and treated in a boiling water bath for 5 minutes. The protein precast gel (Genscript, Cat: M00659) was fixed in the electrophoresis tank, and the electrophoresis buffer was added, and the sample was loaded in turn. Electrophoresis was carried out at a voltage of 150 V for 1 hour. After electrophoresis, the gel was removed and rinsed with deionized water once, and then placed in 0.1% Coomassie brilliant blue staining solution for 1 hour. After staining, the gel was transferred to the decolorizing solution for decolorizing, and the decolorizing solution was replaced every 30 to 60 minutes until the protein bands on the gel were clearly visible.

[0140] The results of SDS-PAGE detection of antibody clone 26E5 are shown in FIG. 6, wherein the N column of bands represents 26E5 antibody under non-reducing conditions, and the R column of bands represents 26E5 antibody under reducing conditions. Figure 2

[0141] Example 6: Antibody subtype identification

[0142] The antibody subtype identification was performed using the Pierce Rapid ELISA Mouse Monoclonal Isotyping Kit (Invitrogen, Cat: 37503).

[0143] 50 mL of 10×TBS was added to 450 mL of ultrapure water to dilute it into 1×TBS for standby. 30 mL of 30×wash buffer was added to 870 mL of ultrapure water to prepare 1×wash buffer for standby.

[0144] Each sample to be tested was prepared with 450 μL for subtype identification. Specifically, for hybridoma cell supernatant, the hybridoma cell supernatant was diluted 50-fold with 1×TBS by adding 20 μL of hybridoma cell supernatant to 980 μL of 1×TBS; for ascites, first 1 μL of ascites was added to 5 mL of 1×TBS (1:5000), and then 67 μL of the dilution obtained in the previous step was added to 933 μL of 1×TBS, so that the sample was diluted 75,000-fold with 1×TBS; for purified antibody, the purified antibody was diluted to 250 ng / mL with 1×TBS.

[0145] The TMB substrate and the plate strip coated with the capture antibody were equilibrated at room temperature. As shown in FIG. 8, the plate strip was washed with 1×wash buffer for 5 minutes, and then 100 μL of sample was added to each well, and incubated at room temperature for 1 hour. Figure 1 ​As shown, 50 μL of diluted sample to be tested was added into each well of the strip A-H, 8 wells per strip. 50 μL of goat anti-mouse (IgG+IgA+IgM)-HRP was added into each of the 8 wells of the strip. The strip was gently tapped to mix. After the plate was sealed with a sealing film, it was incubated at room temperature for 1 hour. The solution in the wells was discarded. 1x washing buffer was added into the wells using a washing bottle, and the strip was washed 3 times and patted dry on a paper towel. 75 μL of TMB substrate, which had been equilibrated to room temperature, was added into each well. After 1 minute, the positive wells turned blue. The color development time and color intensity varied depending on the antibody concentration and subtype. After 5-15 minutes, the reaction was stopped by adding 75 μL of stop solution into each well. The color of the wells changed from blue to yellow at random. The light intensity at 450 nm wavelength was detected by a spectrophotometer. Alternatively, the judgment was made directly by the naked eye.

[0146] According to the results of the detection as in Figure 1 The subtype of the antibody to be tested was determined according to the judgment of the layout. There was only one positive well (heavy chain typing) in the A-F wells of each sample, and there was only one positive well (light chain typing) in the G-H wells.

[0147] As detected in this example, the antibody clone 26E5 belongs to the IgG3 subtype, and its light chain is a κ chain.

[0148] The sequence of the antibody clone 26E5 was identified by sequencing the antibody genes of the hybridoma cells producing the antibody clone 26E5, and the sequence of the variable region is shown in the following table:

[0149]

[0150] The CDR sequence of the antibody clone 26E5 is shown as follows:

[0151]

[0152] Example 7: Identification of the binding activity of the purified antibody

[0153] 7.1 Coating

[0154] Trastuzumab and human IgG were diluted with 1x PBS to a concentration of about 1 μg / mL to prepare a coating working solution.

[0155] The prepared coating working solution was sequentially added into a 96-well plate, 100 μL / well, and coated at 2-8°C overnight.

[0156] 7.2 Blocking

[0157] Wash the plate with 1 x PBST no less than 300 μL / well for 3 times, and pat dry on clean paper; add blocking solution (1% (w / v) BSA in 1 x PBST) to the enzyme-labeled wells, 300 μL / well, and seal in a 37±5°C incubator for 120-130 minutes. Wash the plate with 1 x PBST no less than 350 μL / well for 3 times, and pat dry on clean paper for standby use. The unused plate is sealed and stored at 2-8°C for standby use.

[0158] 7.3 Sample preparation

[0159] Dilute the mouse positive serum according to the dilution ratio in the following table as PC (positive control):

[0160]

[0161] Dilute the purified antibody with PBS in sequence according to the dilution ratio of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, and 1:640, and mix well for standby use.

[0162] 7.4 Sample addition

[0163] Add all the pretreated samples (positive control and purified antibody diluent) to the enzyme-labeled plate in sequence, 100 μL / well. After sealing the plate, slightly shake, and incubate in a 37°C incubator for 60-70 minutes.

[0164] 7.5 Preparation of detection reagent working solution

[0165] Dilute the detection reagent with diluent. The specific dilution ratio is shown in the dilution plate diagram as follows:

[0166]

[0167] Wash the plate with 1 x PBST no less than 300 μL / well for 3 times, and pat dry on clean paper; add the corresponding detection reagent working solution to the enzyme-labeled plate, 100 μL / well, and incubate in a 37°C incubator for 60-70 minutes.

[0168] 7.6 Color development

[0169] Wash the plate with 1 x PBST no less than 300 μL / well for 3 times, and pat dry on clean paper. Add color developing substrate TMB solution to the enzyme-labeled plate, 100 μL / well, and incubate at room temperature for 15-20 minutes.

[0170] 7.7 Detection, data storage, and result analysis

[0171] Add 1M H2SO4, 50 μL / well, shake slightly, make sure there is no orange-yellow phenomenon on the edge of each well, and put the enzyme-labeled plate into the plate reader within 5 min for detection, with the detection wavelength of 450 nm-630 nm.

[0172] The experimental results are shown in Figure 4 The antibody 26E5 can bind to Dxd, and the binding of the antibody 26E5 to Dxd is enhanced with the increase of the concentration of the antibody 26E5, while there is only very weak and almost negligible binding to human IgG.

[0173] Example 8: Detection of the competitive binding activity of the purified antibody

[0174] 8.1 Coating

[0175] Dilute Dxd with 1xPBS to a coating working solution with a concentration of about 1 μg / mL.

[0176] Add the prepared coating working solution into the 96-well plate in turn, 100 μL / well, and coat overnight at 2-8℃.

[0177] 8.2 Blocking

[0178] Wash the plate with 1 x PBST at no less than 300 μL / well for 3 times, and dry on clean paper; add the blocking solution (1% (w / v) BSA in 1x PBST) into the enzyme-labeled wells, 300 μL / well, and block in the 37±5℃ incubator for 120-130 min; wash the plate with 1 x PBST at 350 μL / well for 3 times, and dry on clean paper for standby, and seal the plate not in use at 2-8℃ for standby.

[0179] 8.3 Sample preparation

[0180] 8.3.1 Dilute 5 purified antibodies from the same clone 26E5 with PBS at a ratio of 1:20.

[0181] 8.3.2 Dxd working solution: dilute Dxd with PBS to 5.0 ug / mL.

[0182] 8.4 Sample addition

[0183] 8.4.1 Divide the diluted purified antibody sample into two equal parts: mix one part with the Dxd working solution (150 μL: 150 μL) in equal volume, and mix the other part with PBS in equal volume, and incubate in the 37℃ incubator for 60-70 min.

[0184] 8.4.2 Add the mixed sample after completion of incubation into the enzyme-labeled plate in turn, 100 μL / well, shake slightly after sealing the plate, and incubate in the 37℃ incubator for 60-70 min.

[0185] 8.5 Preparation of detection reagent working solution

[0186] The detection reagent was diluted with diluent, and the dilution ratio was determined according to the dilution plate map as follows:

[0187]

[0188] The plate was washed with 1 x PBST washing solution at not less than 300 μL / well for 3 times, and dried on clean paper; the corresponding detection reagent working solution was added to the enzyme-labeled plate at 100 μL / well, and incubated at 37°C in the dark for 60-70 min.

[0189] 8.6 Color development

[0190] The plate was washed with 1 x PBST washing solution at not less than 300 μL / well for 3 times, and dried on clean paper. The color developing substrate TMB solution was added to the enzyme-labeled plate at 100 μL / well, and incubated at room temperature in the dark for 15-20 min.

[0191] 8.7 Detection, data storage and result analysis

[0192] 1M H2SO4 was added to the enzyme-labeled plate at 50 μL / well, and gently shaken to ensure that there was no orange-yellow phenomenon at the edge of each well. The enzyme-labeled plate was placed in the plate reader for detection within 5 min, and the detection wavelength was 450 nm-630 nm.

[0193] The experimental results are shown in Table 1. Figure 5 After pre-incubation with Dxd and 5 purified antibodies from the same clone 26E5 (i.e., 5 subclones were obtained after two rounds of cloning of 26E5, and then purified antibodies mAb 1, mAb 2, mAb 3, mAb 4 and mAb 5 were obtained from the 5 subclones), Dxd was able to block the binding of clone 26E5 to trastuzumab (with a blocking rate of between 86.78% and 91.50%). The blocking rate 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%, wherein the control group was an equal volume mixture of each purified antibody and PBS in step 8.4. The experimental results proved that the antibody clone 26E5 could bind to the small molecule Dxd part in trastuzumab.

[0194] Sequence information

[0195] SEQ ID NO: 1 (VH-FR1)

[0196] QVQLKESGPGLVAPSQSLSITCTVS

[0197] SEQ ID NO: 2 (VH-CDR1 - numbered according to IMGT definition)

[0198] GFSLTNFG

[0199] SEQ ID NO: 3 (VH-FR2)

[0200] VHWVRQPPGKGLEWLGV

[0201] SEQ ID NO: 4 (VH-CDR2 - numbered according to IMGT definition)

[0202] IWAGGST

[0203] SEQ ID NO: 5 (VH-FR3)

[0204] NYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYC

[0205] SEQ ID NO: 6 (VH-CDR3 - numbered according to IMGT definition)

[0206] VRDDYGSSYGEDY

[0207] SEQ ID NO: 7 (VH-FR4)

[0208] WGQGTTLTVSS

[0209] SEQ ID NO: 8 (VH)

[0210] QVQLKESGPGLVAPSQSLSITCTVSGFSLTNFGVHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCVRDDYGSSYGEDYWGQGTTLTVSS

[0211] SEQ ID NO: 9 (VL-FR1)

[0212] QIVLTQSPAIMSASPGEKVTMTCSAS

[0213] SEQ ID NO: 10 (VL-CDR1 - numbered according to IMGT definition)

[0214] SSVSY

[0215] SEQ ID NO: 11 (VL-FR2)

[0216] MHWYQQKSGTSPKRWIY

[0217] SEQ ID NO: 12 (VL-CDR2- according to IMGT definition numbering)

[0218] DTS

[0219] SEQ ID NO: 13 (VL-FR3)

[0220] KLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC

[0221] SEQ ID NO: 14 (VL-CDR3- according to IMGT definition numbering)

[0222] QQWSSNPPT

[0223] SEQ ID NO: 15 (VL-FR4)

[0224] FGGGTKLEIK

[0225] SEQ ID NO: 16 (VL)

[0226] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGGTKLEIK

[0227] SEQ ID NO: 17 (VH-CDR1- according to Kabat definition numbering)

[0228] NFGVH

[0229] SEQ ID NO: 18 (VH-CDR2- according to Kabat definition numbering)

[0230] VIWAGGSTNYNSALMS

[0231] SEQ ID NO: 19 (VH-CDR3- according to Kabat definition numbering)

[0232] DDYGSSYGEDY

[0233] SEQ ID NO: 20 (VL-CDR1- according to Kabat definition numbering)

[0234] SASSSVSYMH

[0235] SEQ ID NO: 21 (VL-CDR2 - numbered according to Kabat definition)

[0236] DTSKLAS

[0237] SEQ ID NO: 22 (VL-CDR3 - numbered according to Kabat definition)

[0238] QQWSSNPPT

[0239] All publications and patent documents cited in this specification are hereby incorporated by reference as if each individual publication or patent document were specifically and individually indicated to be incorporated by reference. Various changes and modifications can be made in the embodiments of the application disclosed without departing from the true spirit and scope of the application. Any feature, step or embodiment of the embodiments of the present disclosure can be used in combination with any other feature, step or embodiment, unless the context excludes it.

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 a HCDR1, a HCDR2, and a HCDR3, the light chain variable region comprises a LCDR1, a LCDR2, and a LCDR3, and wherein the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 4, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 6, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR2 is DTS, and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 14, according to IMGT definition numbering; or the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 17, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 18, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 19, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 20, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 21, and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 22, according to Kabat definition numbering. wherein the antigen-binding fragment is selected from a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a Fv fragment, or a scFv fragment.

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

16.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:

16.

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

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

6. A vector comprising the nucleic acid molecule of claim 5.

7. The vector of claim 6, wherein the vector is an expression vector.

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

9. A test reagent or kit comprising the antibody or antigen binding fragment thereof of any one of claims 1 4.

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

11. The antibody or antigen-binding fragment thereof of any one of claims 1-4 for use in detecting degrading of the antibody or antigen-binding fragment thereof in a biological sample from a subject for non-diagnostic purposes.

4. The antibody or antigen-binding fragment thereof for use in detecting degrading of the antibody or antigen-binding fragment thereof in a biological sample from a subject for non-diagnostic purposes.

Images