GAA monoclonal antibody and application thereof

Abstract

The invention relates to the field of antibodies, in particular to an anti-acid alpha-glucosidase (GAA) antibody, a kit containing the same, a method and application of the antibody, and particularly application of the antibody to detection of anti-drug antibodies and detection of the state of cross-immunoreactive substances (CRIM) of patients suffering from the Pompe disease.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and particularly to anti-acid α-glucosidase (GAA) antibodies, kits and methods comprising the same, and their uses, especially for the detection of drug-resistant antibodies and the detection of the cross-reacting immunological material (CRIM) status in patients with Pompe disease. Background Art

[0002] Pompe disease is a rare type II glycogen storage genetic disease caused by the deficiency of acid α-glucosidase (GAA). With the clinical application of enzyme replacement therapy and gene therapy drugs carrying the GAA gene, it is necessary to monitor the changes of drug-resistant antibodies (i.e., anti-GAA protein antibodies) in patients to evaluate whether anti-GAA antibodies that affect the efficacy of drugs are produced. However, there is currently no clinically applicable anti-GAA antibody detection kit on the market. In the development process of drug-resistant antibody detection kits, good positive control antibodies are one of the important detection materials, which play a very important role in verifying the detection performance of the kit during the development process and quality control after listing. However, the commercially available GAA antibodies currently on the market cannot meet the development requirements of anti-GAA drug-resistant antibody detection kits. Summary of the Invention

[0003] To solve the above technical problems, the inventors have developed a novel anti-GAA antibody and its application in Pompe disease-related clinical detection through in-depth research. The anti-GAA antibody of the present invention can be used as a positive quality control antibody for the detection of anti-GAA antibodies in patients with Pompe disease; and can be used as a detection antibody for GAA antigen detection and the determination of the CRIM status of patients with Pompe disease. Compared with commercially available anti-GAA antibodies, when the antibody of the present invention is used as a positive quality control product for the detection of drug-resistant antibodies in patients with Pompe disease and as a detection tool for the detection of the CRIM status of patients with Pompe disease, obvious detection advantages are achieved, and it has higher sensitivity, specificity, accuracy and reliability. Thus, the inventors have established the anti-GAA antibody of the present invention, detection kits and methods comprising the anti-GAA antibody of the present invention, and their applications in various Pompe disease clinical detections.

[0004] Therefore, in a first aspect, the present invention provides an anti-acid α-glucosidase (GAA) antibody comprising three HCDR sequences (HCDR1, HCDR2, HCDR3) of the heavy chain variable region of SEQ ID NO: 1 and three LCDR sequences (LCDR1, LCDR2 and LCDR3) of the light chain variable region of SEQ ID NO: 2. The present invention also provides nucleic acids encoding the antibodies of the present invention, vectors and host cells comprising the nucleic acids, methods for preparing the antibodies of the present invention, and immunoconjugates and compositions comprising the antibodies of the present invention.

[0005] In a second aspect, the present invention provides an anti-drug antibody (ADA) detection kit, wherein the kit comprises the anti-GAA antibody of the present invention as a positive control antibody. In some embodiments, the detection kit further comprises reagents for performing ADA detection. In some embodiments, the ADA detection comprises a screening test and / or a confirmation test for ADA detection. In some embodiments, the screening test is an indirect ELISA assay. In some embodiments, the confirmation test is a competitive ELISA assay.

[0006] In a third aspect, the present invention provides an anti-drug antibody (ADA) detection method, comprising: using the GAA antibody of the present invention as a positive control antibody, performing ADA detection on a sample to be tested and the positive control antibody, and determining the presence or amount of ADA in the sample. In some embodiments, the ADA detection comprises a screening test and / or a confirmation test for ADA detection. In some embodiments, the screening test is an indirect ELISA assay. In some embodiments, the confirmation test is a competitive ELISA assay. In some embodiments, the sample is from a human individual, particularly an individual with Pompe disease, and preferably the sample is a serum or plasma sample from the individual. In some embodiments, the method of the present invention is used to monitor changes in anti-GAA protein antibodies in an individual, wherein the individual is an individual who has received, is receiving, or will receive exogenous GAA protein or exogenous nucleic acid expressing GAA. In some embodiments, the individual is an individual who has received, is receiving, or will receive GAA treatment. In some embodiments, the GAA treatment comprises enzyme replacement therapy by administering exogenous GAA protein to the individual; in other embodiments, the GAA treatment comprises gene therapy by administering exogenous nucleic acid expressing GAA to the individual, for example, a vector (such as an AAV viral vector) comprising and expressing a GAA-encoding nucleic acid. In some cases, the presence or amount of ADA in the sample can be used as an auxiliary reference for evaluating the drug efficacy of GAA-treated patients.

[0007] In a fourth aspect, the present invention provides a kit and method for detecting the CRIM status of an individual, which comprises using an anti-GAA antibody or an antigen-binding fragment thereof, or an immunoconjugate thereof according to the present invention. In some embodiments, the detection comprises: using an anti-GAA antibody or an antigen-binding fragment thereof according to the present invention, or an immunoconjugate according to the present invention, to detect the presence or amount of one or any combination of the precursor, intermediate, and mature forms of acid α-glucosidase in a sample from an individual. In one embodiment, the individual is a patient with Pompe disease, particularly an infantile-onset Pompe disease (IOPD) patient. In some embodiments, the detection comprises: contacting an antibody or antigen-binding fragment (labeled or unlabeled) according to the present invention or an immunoconjugate according to the present invention with the sample, and detecting the complex formed by the antibody or antigen-binding fragment or immunoconjugate and acid α-glucosidase. In some embodiments, the detection is performed by western blot.

[0008] In a fifth aspect, the present invention provides a method for detecting acid α-glucosidase in a sample, which comprises: using an antibody or an antigen-binding fragment thereof according to the present invention as a capture antibody, and detecting the presence or amount of the captured acid α-glucosidase. Preferably, the method is performed by a sandwich ELISA assay.

[0009] In a sixth aspect, the present invention provides the use of an antibody or an antigen-binding fragment thereof according to the present invention or a kit according to the present invention for performance verification or product quality control of a Pompe disease anti-GAA drug antibody detection kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Shows the results of screening hybridoma cell supernatants using an indirect ELISA assay.

[0011] Figure 2 Shows the SDS-PAGE gel electrophoresis results of the purified antibody obtained from hybridoma cell line 4B1.

[0012] Figure 3 Shows the comparison of the sensitivity of anti-GAA murine monoclonal antibody and rabbit monoclonal antibody binding to antigen GAA using an indirect ELISA assay.

[0013] Figure 4 Shows the comparison of the performance of anti-GAA murine monoclonal antibody and rabbit monoclonal antibody as positive control products in a competitive ELISA assay.

[0014] Figure 5 Shows the results of detecting the CRIM status of positive samples (PC) and negative samples (NC1 and NC2) by Western Blot using anti-GAA murine monoclonal antibody and rabbit monoclonal antibody.

[0015] Figure 6 It shows the results of detecting antigen GAA in a sandwich ELISA assay using an anti-GAA murine monoclonal antibody as the capture antibody and an anti-GAA rabbit monoclonal antibody as the detection antibody. Detailed implementation mode

[0016] Unless otherwise defined herein, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the present invention will be apparent from this specification, the drawings, and the appended claims.

[0017] Definition

[0018] As used herein, the term "about", when used in conjunction with a numerical value, is intended to cover numerical values within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value. The term is also intended to cover values within ±1%, ±0.5%, or ±0.1% of the specified number.

[0019] As used herein, the expression "and / or" is used to indicate any one of the listed related items, or any and all possible combinations of multiple of the listed related items.

[0020] As used herein, the term "comprising" or "including" means including the recited elements, integers, or steps, or groups of elements, integers, or steps, but does not exclude any other elements, integers, or steps, or other groups of elements, integers, or steps. As used herein, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the case consisting of the recited elements, integers, or steps. For example, when referring to a polypeptide / protein "comprising" a specific sequence, it is also intended to cover a polypeptide / protein consisting of that specific sequence.

[0021] As used herein, "Pompe disease", also known as acid α-glucosidase deficiency or glycogen storage disease type II (GSD II), refers to a systemic lysosomal storage disease. In many cases, the disease mainly affects the muscles and also affects the central nervous system. In affected individuals, there is a lack of functional acid α-glucosidase (GAA) in lysosomes, resulting in the inability of glycogen to be converted into glucose for utilization, causing glycogen to accumulate in the cells of the patient and leading to the disease. Pompe disease can be diagnosed by detecting the amount and / or activity of α-glucosidase in a sample from an individual.

[0022] In this text, "enzyme replacement therapy" is also referred to as ERT, which refers to a treatment method that administers a protein drug with enzymatic activity to a patient to replace the lacking enzymatic activity in the patient's body. For example, for a patient with Pompe disease due to the lack of lysosomal α-glucosidase, a solution containing functional GAA enzyme can be administered (such as intravenous (IV) infusion) for enzyme replacement therapy. However, it has been shown that individual patients receiving ERT treatment can have quite different responses. One of the factors contributing to this response variability is considered to be related to the formation of high-titer anti-drug antibodies (i.e., anti-GAA antibodies). Studies in animals and humans have also shown that antibodies formed against the GAA enzyme can reduce the efficacy of ERT. Therefore, it is necessary to monitor the changes in anti-GAA antibodies in the body of ERT-treated patients to evaluate whether anti-GAA antibodies that affect the drug efficacy have been produced.

[0023] In this text, "gene therapy" refers to a method of using exogenous genes to treat, prevent, or cure diseases or medical conditions. Generally, gene therapy includes: providing a patient with a new functional gene copy of their damaged gene, or replacing a defective or missing gene in the patient's body with a functional form of the gene. After a patient receives gene therapy, with the in vivo expression of the exogenous gene, anti-drug antibodies against the exogenous gene product may appear, thereby affecting the efficacy of gene therapy.

[0024] In this text, "anti-drug antibody" is also referred to as ADA, which refers to an antibody produced in an individual's body against a therapeutic drug. In this text, anti-drug antibody specifically refers to an antibody against the GAA protein contained in the therapeutic drug or the GAA protein expressed from the GAA-encoding nucleic acid in the therapeutic drug, and is also called anti-GAA anti-drug antibody in some cases of the present invention. In some cases, after an individual is exposed to a therapeutic drug, the immune system will induce a humoral response, thereby producing anti-drug antibodies (ADA). This specific immune response may lead to a reduction in the amount of available drug, thereby resulting in a decrease in clinical efficacy in some cases. In addition, the appearance of anti-drug antibodies (ADA) may also cause adverse side effects. For example, it has been observed in preclinical and clinical studies that the production of drug-induced ADA may lead to allergic reactions or even anaphylactic shock. Therefore, for biopharmaceuticals, it is necessary to detect anti-drug antibodies of the drug.

[0025] In this text, the terms "acid α-glucosidase", "acid glucosidase", or "GAA" are used interchangeably and refer to: a lysosomal enzyme that can hydrolyze α-1-4 bonds in maltose and other linear oligosaccharides to degrade excessive glycogen in lysosomes. An example of human GAA can be found in the Unipro database accession number UniProtKB-P10253. When synthesized in the human body, GAA forms mature GAA enzymes (76 kDa, 70 kDa) through a precursor (110 kDa) and intermediate states (100 kDa, 95 kDa). In this text, unless explicitly stated to the contrary or otherwise specified, when referring to GAA, the expression encompasses all these forms of GAA enzyme proteins.

[0026] In this text, the term "antibody" refers to a polypeptide containing immunoglobulin variable regions of the light chain and the heavy chain, which specifically recognize and bind to an antigen. This term encompasses various antibody structures, including, but not limited to, monoclonal antibodies, single-chain antibodies or multi-chain antibodies, mono-specific or multi-specific antibodies (such as bispecific antibodies), murine antibodies, chimeric antibodies or humanized antibodies, full-length antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0027] In this text, "whole antibody" (which can be used interchangeably with "full-length antibody", "complete antibody", and "intact antibody") refers to an antibody containing at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this text) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this text) and a light chain constant region. The light chain constant region consists of one domain, CL. The variable region is the domain in the heavy or light chain of the antibody that participates in the binding of the antibody to its antigen. The constant region does not directly participate in the binding of the antibody to the antigen. The light chain of an antibody can be classified into one of two types (called kappa (κ) and lambda (λ)) based on the amino acid sequence of its constant domain. The heavy chain of an antibody can be divided into five main different types depending on the amino acid sequence of its heavy chain constant region: IgA, IgD, IgE, IgG, and IgM, and several of these types can be further divided into subclasses. For example, mouse IgG antibodies have subtypes IgG1, IgG2a, IgG2b, IgG2c, and IgG3. The term "isotype" refers to the type of antibody determined by the heavy chain constant region of the antibody. See, for example, Fundamental Immunology, Ch. 7 (edited by Paul, W., 2nd edition, Raven Press, N.Y. (1989)) (which is hereby incorporated by reference in its entirety for all purposes).

[0028] As used herein, the term "IgG-type antibody" refers to an immunoglobulin molecule of the IgG isotype consisting of two heavy chains and two light chains, wherein each heavy chain consists of a heavy chain variable region (VH) and heavy chain constant regions (CH1, CH2, and CH3), and each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).

[0029] As used herein, the term "antigen-binding fragment" of an antibody refers to a molecule that is not a full antibody and that contains the portion of the full antibody that is used to bind the antigen to which the full antibody binds. As will be understood by those skilled in the art, the antigen-binding portion of an antibody typically contains amino acid residues from "complementary determining regions" or "CDRs". Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of a full antibody. In some embodiments of the invention, the antigen-binding fragment is selected from: Fab, scFab, Fab', F(ab')2, Fab'-SH, Fv, single-chain Fv, diabody, triabody, tetrabody, and minibody.

[0030] As used herein, the term "chimeric antibody" refers to an antibody that contains sequences derived from different species. As used herein, a chimeric antibody particularly refers to an antibody in which the CDR sequences of the variable regions are derived from a murine antibody, while at least some of the other antibody sequences can be derived from an antibody of another species.

[0031] As used herein, an "isolated" antibody is an antibody that has been separated from the components in its natural environment. In some embodiments, the antibodies of the invention are isolated antibodies that are purified to greater than 95% or 99% purity. The purity of an antibody can be determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0032] As used herein, "sequence identity" means the degree of sequence identity on a nucleotide-by-nucleotide or amino acid-by-amino acid basis in a comparison window. The "percent sequence identity" can be calculated by comparing two optimally aligned sequences in the comparison window, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) are present in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to yield the percent sequence identity. The optimal alignment for determining the percent sequence identity can be achieved in a variety of ways known in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or within the target sequence region of interest.

[0033] As used herein, for antibody sequences, the percent amino acid sequence identity is determined by optimally aligning the candidate antibody sequence with the reference antibody sequence, in a preferred embodiment according to the Kabat numbering convention. As used herein, in the absence of specifying a comparison window (i.e., the target antibody region to be compared), the alignment will be applicable over the full length of the reference antibody sequence.

[0034] As used herein, "complementary determining region" or "CDR region" or "CDR" refers to the amino acid regions in the variable regions of an antibody that are primarily responsible for binding to an epitope. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located within the variable domain of the heavy chain of an antibody are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of the light chain of an antibody are referred to as LCDR1, LCDR2, and LCDR3. A variety of methods are known in the art for determining the CDR sequences in a given VH or VL amino acid sequence. For example, the Kabat complementary determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Another example is the Chothia method (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)) and the IMGT method (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), which are also used to determine CDR sequences. For a given antibody, those skilled in the art will readily identify the CDRs defined by these methods. Moreover, the correspondence between different methods is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). As used herein, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be determined according to various CDR definition methods known in the art. In some embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably determined by the IMGT, Kabat, or Chothia methods.

[0035] As used herein, "variable region" refers to the domain in the heavy or light chain of an antibody that participates in binding of the antibody to its antigen. The heavy chain variable region (VH) and the light chain variable region (VL) can be further subdivided into hypervariable regions (HVRs, also known as complementarity determining regions (CDRs)), which are interspersed with more conserved regions (i.e., framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. It is known in the art that one or more residues in one or both of the two variable regions (i.e., VH and / or VL) can be altered (e.g., residue deletions, insertions, and / or substitutions, particularly conservative residue substitutions, in one or more CDR regions and / or in one or more framework regions), without substantially altering at least one desired biological property of the antibody molecule (e.g., antigen binding ability). In some cases, the antigen binding properties or other functional properties of the mutated antibody can be evaluated in in vitro or in vivo assays. CDR grafting is another known method of modifying antibody variable regions. Since the CDR sequences are responsible for most antibody-antigen interactions, recombinant antibody variants mimicking the properties of a known antibody can be constructed by CDR grafting. In such antibody variants, the CDR sequences from a known antibody are grafted onto the framework regions of a different antibody with different properties. Thus, in some embodiments, the present invention also relates to an anti-GAA antibody or antigen-binding fragment thereof that comprises the CDR sequences of the heavy and light chain variable regions according to the present invention, but has a different framework region sequence. The framework region sequences for replacement can be obtained from public DNA databases, including germline antibody gene sequences, or from anti-GAA antibody sequences reported in the published literature.

[0036] Aspects of the invention are further described in detail below.

[0037] Antibody of the present invention

[0038] In a first aspect, the present invention provides an anti-GAA antibody or antigen-binding fragment thereof that binds to acid alpha-glucosidase (GAA), comprising: (i) the three LCDR sequences (LCDR1, LCDR2, and LCDR3) of the light chain variable region of SEQ ID NO:1; and (ii) the three HCDR sequences (HCDR1, HCDR2, HCDR3) of the heavy chain variable region of SEQ ID NO:2. In some embodiments, the HCDRs and LCDRs sequences are defined according to the IMGT, Kabat, or Chothia schemes.

[0039] In some embodiments, the anti-GAA antibody according to the present invention comprises:

[0040] (a) LCDR1 sequence of SEQ ID NO:3;

[0041] (b) LCDR2 sequence of SEQ ID NO:4;

[0042] (c) LCDR3 sequence of SEQ ID NO:5;

[0043] (d) HCDR1 sequence of SEQ ID NO:6;

[0044] (e) HCDR2 sequence of SEQ ID NO:7; and

[0045] (f) HCDR3 sequence of SEQ ID NO:8.

[0046] In some embodiments, the anti-GAA antibody according to the present invention comprises a light chain variable region. In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:1. In other embodiments, the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% or higher sequence identity with SEQ ID NO:1, preferably, an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% or higher sequence identity with SEQ ID NO:1.

[0047] In some embodiments, the anti-GAA antibody according to the present invention comprises a heavy chain variable region. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:2. In other embodiments, the heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% or higher sequence identity with SEQ ID NO:2, preferably, an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% or higher sequence identity with SEQ ID NO:2.

[0048] In some embodiments, the anti-GAA antibody according to the present invention comprises a light chain variable region and a heavy chain variable region. In some embodiments, the light chain variable region comprises or consists of SEQ ID NO:1; and the heavy chain variable region comprises or consists of SEQ ID NO:2.

[0049] In some embodiments, the anti-GAA antibody according to the present invention is an IgG-type antibody. In other embodiments, the anti-GAA antibody according to the present invention comprises (a) a kappa light chain constant region; and / or (b) an IgG heavy chain constant region, preferably an IgG2b heavy chain constant region. In some preferred embodiments, the anti-GAA antibody according to the present invention comprises a murine kappa light chain constant region and an IgG2b heavy chain constant region.

[0050] In some embodiments, the anti-GAA antibody according to the present invention is a murine monoclonal antibody, a chimeric antibody or a humanized antibody. In other embodiments, the antigen-binding fragment of the anti-GAA antibody according to the present invention is an antibody fragment selected from the following: Fab, Fab’, Fab’-SH, Fv, single-chain antibodies such as scFv, (Fab’)2 fragments, or linear antibodies.

[0051] In some embodiments, the anti-GAA antibody or its antigen-binding fragment according to the present invention can be labeled, for example, to facilitate detection purposes. The label that can be used to label the anti-GAA antibody or its antigen-binding fragment of the present invention can be readily determined by those skilled in the art according to the intended use of the antibody (such as the type of detection method used). Some examples of labels that can be mentioned include: labels or moieties that can be directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties that are detected indirectly, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions. Some other examples of labels that can be mentioned include, but are not limited to, radioactive isotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as fluorescein and its derivatives, luciferase, fluorescein, 2,3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, biotin / avidin, spin labels, phage labels, stable free radicals, and the like. When the antibody of the present invention is labeled with a non-peptide chemical compound such as a radioactive isotope or a fluorophore, such labeled antibodies are also referred to as immunoconjugates herein.

[0052] Based on the antibodies of the present invention, the present invention also provides, in other aspects, nucleic acids encoding the antibodies of the present invention, vectors and host cells comprising the nucleic acids, and methods for preparing the antibodies of the present invention. In some embodiments, the present invention provides nucleic acids encoding any of the above anti-GAA antibodies or fragments thereof. The nucleic acids may comprise nucleic acids encoding the amino acid sequences of the variable regions of the light and / or heavy chains of the antibody, or nucleic acids encoding the amino acid sequences of the light and / or heavy chains of the antibody. Nucleic acid sequences encoding the antibodies or antigen-binding fragments thereof of the present invention can be generated by de novo solid-phase DNA synthesis or by PCR methods using methods well known in the art. In some embodiments, the present invention provides one or more vectors comprising the nucleic acids of the present invention. In some embodiments, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, λ phages, or yeast artificial chromosomes (YACs). In some embodiments, the present invention provides host cells comprising the nucleic acids of the present invention or the vectors of the present invention. Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells. In some embodiments, the host cells are selected from yeast cells, mammalian cells (such as CHO cells or 293 cells). In other embodiments, the host cells are prokaryotic. In some embodiments, the present invention provides a method for preparing the antibodies of the present invention, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or the host cell culture medium). To recombinantly produce the antibodies of the present invention, the nucleic acids encoding the antibodies can be isolated and inserted into one or more vectors for further cloning and / or expression in host cells.

[0053] In still other aspects, the present invention provides immunoconjugates produced by conjugating the antibodies of the present invention to heterologous molecules. In some embodiments, in the immunoconjugate, the antibody (or its antigen-binding fragment) of the present invention is conjugated to a detectable or diagnostic agent. In some embodiments, the antibodies of the present invention can be conjugated to heterologous molecules in the form of full-length antibodies or antibody fragments. For example, conjugation can be carried out in the form of Fab fragments, Fab’ fragments, F(ab)’2 fragments, single-chain scFab antibodies, single-chain scFv, etc. Fragments. Linkers can be used to covalently link the different entities of the conjugate. Suitable linkers include chemical linkers or peptide linkers.

[0054] Detection of anti-drug antibody (ADA)

[0055] Any biological agent administered to humans can be recognized by the immune system, thereby inducing a humoral response and generating anti-drug antibodies (ADA). In patients receiving disease treatment, ADA detection methods can be used to monitor the changes and persistence of ADA in patients during treatment, as well as its potential correlation with clinical outcomes. In addition, ADA analysis methods can also be used to evaluate the immunogenicity of biopharmaceuticals, assist in drug treatment decisions, or promote the development and product quality control of biopharmaceuticals, as well as drug performance evaluation. In the development, validation, and use of such anti-drug antibody detection methods, it is of great significance to provide positive control antibodies with good performance.

[0056] Based on the antibodies of the present invention, the present invention provides, in the second and third aspects, a detection kit and method comprising the antibodies or antigen-binding fragments of the present invention. As confirmed in the examples of this application, the antibodies of the present invention, when included as positive quality control products in anti-drug antibody detection, have shown significant advantages, including but not limited to, good sensitivity, specificity, accuracy, and stability.

[0057] In some embodiments, accordingly, the present invention provides an anti-drug antibody (ADA) detection kit, wherein the kit comprises the anti-GAA antibody of the present invention or its antigen-binding fragment as a positive control antibody, and wherein the anti-drug antibody is an anti-acid α-glucosidase antibody. In some embodiments, the kit according to the present invention further comprises reagents for ADA detection.

[0058] In other embodiments, the present invention also provides an anti-drug antibody (ADA) detection method, wherein the ADA is an anti-acid α-glucosidase (GAA) antibody, and the method comprises:

[0059] (a) using the antibody or its antigen-binding fragment of the present invention as a positive control antibody,

[0060] (b) performing ADA detection on the sample to be tested and the positive control antibody,

[0061] (c) determining the presence or amount of ADA in the sample.

[0062] As can be understood by those skilled in the art, the samples applicable to the detection kit and detection method of the present invention are not particularly limited. However, in some cases, they are preferably from individuals who have received, are receiving, or will receive GAA treatment, especially individuals with Pompe disease. In some embodiments, therefore, the samples used in the present invention are from human individuals, especially individuals with Pompe disease. In some embodiments, the individuals are those who have received, are receiving, or will receive exogenous GAA protein or exogenous nucleic acid expressing GAA. In some embodiments, the individuals are those who have received, are receiving, or will receive GAA treatment. In some embodiments, the GAA treatment includes enzyme replacement therapy by administering exogenous GAA protein to the individual; in other embodiments, the GAA treatment includes gene therapy by administering exogenous nucleic acid expressing GAA to the individual, for example, a vector (such as an AAV viral vector) containing and expressing GAA-encoding nucleic acid. In some embodiments, the detection kit and detection method of the present invention can be used to monitor the changes of anti-GAA protein antibodies in the body of the individual.

[0063] The type of sample for detection purposes can be determined according to the specific detection method and detection purpose used. For most drugs, adverse immune reactions are generally caused by immune responses mediated by the humoral immune mechanism. The sample for anti-drug antibody detection is preferably a serum or plasma sample from an individual in some embodiments.

[0064] The detection of anti-drug antibodies usually adopts a multi-level analysis method. First, a screening test is performed on all samples, then a confirmation test is performed on the specificity of the suspected antibody-positive samples, and then, according to the specific situation, a titer test and / or antibody neutralization activity detection are performed on the samples that have been confirmed to be antibody-positive. In this article, the mention of "screening test" refers to this screening test used in anti-drug antibody detection; the mention of "confirmation test" refers to this confirmation test used in anti-drug antibody detection.

[0065] The screening test is the first step in anti-drug antibody detection. This analysis method needs to have sufficient sensitivity to detect various types of anti-drug antibodies with low and high affinity in the sample. Since the sample (such as human serum or plasma) may contain components that affect the detection, in the screening test, corresponding negative samples (such as human serum or plasma from anti-GAA antibody-negative individuals) can be used to dilute or formulate positive control antibodies to produce positive quality control products to reduce the influence brought by confounding factors in the sample.

[0066] The confirmatory assay is typically the second step in anti-drug antibody detection, aiming to rule out false positive samples in the screening assay and determine the specificity of the binding of anti-drug antibodies to the drug in the sample. Typically, the confirmatory assay requires higher specificity and at least equivalent selectivity to the screening assay to identify false positive samples. Generally, in anti-drug antibody detection, a binding competition inhibition method is used for the confirmatory assay, in which an excess of the drug is added to the sample, and then the signal values of the samples with and without the added drug are detected simultaneously. If the sample contains anti-drug antibodies, the free drug will competitively bind to the anti-drug antibodies, resulting in a decrease in the detected signal value. The method and platform of the confirmatory assay can be the same as or different from those of the screening assay.

[0067] As demonstrated in the examples, the antibody of the present invention maintained good binding sensitivity and high specificity to the antigen GAA when formulated and diluted in human serum and plasma samples negative for anti-GAA antibodies. Therefore, the antibody of the present invention is suitable not only as a positive control antibody for the screening assay but also as a positive control antibody for the confirmatory assay. In some embodiments, therefore, the ADA detection kit and the ADA detection method according to the present invention include a screening assay and / or a confirmatory assay for ADA detection. In some embodiments, the screening assay uses the antibody of the present invention as a positive control antibody. In some embodiments, the confirmatory assay uses the antibody of the present invention as a positive control antibody. In some embodiments, both the screening assay and the confirmatory assay use the antibody of the present invention as a positive control antibody.

[0068] The screening assay and the confirmatory assay for the kit and method of the present invention can be carried out in a manner known in the art. In some embodiments, the screening assay employs an indirect ELISA assay. In other embodiments, the confirmatory assay employs a competitive ELISA assay. In still other embodiments, the ADA detection according to the present invention includes: (a) screening for positive samples suspected of containing ADA by an indirect ELISA assay; and / or (b) confirming positive samples containing ADA by a competitive ELISA assay.

[0069] In some embodiments, the indirect ELISA comprises the following steps: (i) contacting a sample to be tested with an ELISA plate on which antigen GAA is immobilized, and allowing an antigen-antibody complex to form; (ii) using a labeled secondary antibody to recognize the antigen-antibody complex bound to the ELISA plate, thereby generating a signal proportional to the amount of the complex. In some embodiments, the sample to be tested is diluted before step (i), preferably diluted by at least 100-fold. In some embodiments, the screening threshold is characterized by the antibody concentration before dilution of the sample to be tested. In some embodiments, the screening threshold is 100 ng / mL anti-GAA antibody, 90 ng / mL anti-GAA antibody, 80 ng / mL anti-GAA antibody, 70 ng / mL anti-GAA antibody, 60 ng / mL anti-GAA antibody, 50 ng / mL anti-GAA antibody, 45 ng / mL anti-GAA antibody, 40 ng / mL anti-GAA antibody, 35 ng / mL anti-GAA antibody, 30 ng / mL anti-GAA antibody, or 25 ng / mL anti-GAA antibody at a 100-fold dilution. In some embodiments, the sample to be tested is a sample from a patient (e.g., a serum or plasma sample) and a positive control prepared using the antibody of the present invention. In some embodiments, the positive control is prepared using a drug-resistant antibody-negative sample (e.g., a negative serum or plasma sample) equivalent to the patient sample.

[0070] In some embodiments, the competitive ELISA comprises the following steps: (i) adding or not adding an excess of free antigen to the sample to be tested; (ii) contacting the sample to be tested with or without added antigen obtained in step (i) with an ELISA plate on which antigen GAA is immobilized; (iii) using a labeled secondary antibody to recognize the antigen-antibody complex bound to the ELISA plate, and simultaneously detecting the signal values caused by the sample with added antigen and the sample without added antigen. In some embodiments, the inhibition rate is determined by calculating the percentage decrease of the signal value of the former relative to the signal value of the latter; and a positive sample is confirmed based on whether the inhibition rate is greater than the confirmation threshold. In some embodiments, the sample to be tested is a suspected positive sample from a screening test (e.g., a serum or plasma sample) and a positive control prepared using the antibody of the present invention. In some embodiments, the positive control is prepared using a drug-resistant antibody-negative sample (e.g., a negative serum or plasma sample) equivalent to the suspected positive sample.

[0071] In other aspects, the present invention also contemplates the use of the antibody of the present invention as a positive control antibody in the following aspects.

[0072] (1) Performance verification: The positive control antibody of the present invention can be used to verify and monitor the performance of anti-drug antibody detection assays. By adding the positive control antibody of the present invention in the detection, it can be ensured that the detection runs as expected and produces accurate results.

[0073] (2) Quality control: The positive control antibody of the present invention can be used as part of the quality control process for anti-drug antibody detection assays. By adding the positive control antibody of the present invention in each detection run, it is possible to monitor the consistency and reliability of the detection assay over time.

[0074] (3) Assay standardization: The positive control antibody of the present invention can be used to standardize anti-drug antibody detection assays in different laboratories or detection platforms. By using the positive control antibody of the present invention as a reference standard, it is possible to ensure the comparability and consistency of results under different settings.

[0075] (4) Sensitivity establishment: As a key reagent for evaluating the performance of the methodology, the positive control antibody directly affects the sensitivity of the detection method. Sensitivity can generally be determined as follows: Gradiently dilute the positive control sample in a mixed blank biological matrix to obtain a series (at least 5) of known concentrations, and then determine or calculate the sensitivity.

[0076] (5) Troubleshooting: The positive control antibody of the present invention can be used to solve problems in anti-drug antibody detection assays. If the assay produces unexpected results, the positive control antibody of the present invention can be used to identify and solve potential sources of error in the detection.

[0077] CRIM status detection

[0078] In a fourth aspect, the present invention provides the use of the anti-GAA antibody of the present invention for detecting the cross-reactive immunologic material (CRIM) status of an individual treated with GAA.

[0079] According to the age of onset, affected organs, and disease progression rate, glycogen storage disease type II is divided into infantile onset Pompe disease (IOPD) and late-onset Pompe disease (LOPD). According to the age of onset, LOPD can be further divided into childhood-onset and adult-onset (onset after 18 years old). The ERT benefits of different IOPD children vary, which is related to the disease burden of children during treatment, the status of cross-reactive immunologic material (CRIM), etc. It has been reported that about 25% of IOPD children have no expression of GAA protein, resulting in CRIM negativity. Almost all CRIM-negative children and a small number of CRIM-positive children will produce anti-rhGAA IgG antibodies after receiving ERT treatment, but CRIM-negative children produce antibodies faster and have higher antibody titers. For CRIM-negative IOPD children, immune tolerance induction treatment can be given before receiving ERT, that is, by giving children immunosuppressants to avoid or reduce antibody production. See, for example, Diagnosis and Treatment of Glycogen Storage Disease Type II in Children, Chinese Expert Consensus, Chinese Journal of Pediatrics, Vol. 59, No. 6, June 2021.

[0080] In some embodiments, the antibodies of the present invention can be used for the assessment of the CRIM status of patients with Pompe disease.

[0081] In some embodiments, the present invention provides a kit for detecting the CRIM status of an individual, which comprises an antibody or an antigen-binding fragment thereof according to the present invention, or an immunoconjugate according to the present invention.

[0082] In some embodiments, the present invention provides a method for detecting the CRIM status of an individual, the method comprising: using an antibody or an antigen-binding fragment thereof according to the present invention, or an immunoconjugate according to the present invention, to detect the presence or amount of any one or any combination of the precursor, intermediate state, and mature form of acid α-glucosidase in a sample from the individual. In some embodiments, the precursor of acid α-glucosidase refers to the precursor form of acid α-glucosidase synthesized in the human body with a molecular weight of approximately 110 kDa; in some embodiments, the intermediate state of acid α-glucosidase refers to the intermediate form of acid α-glucosidase synthesized in the human body with a molecular weight less than 110 kDa but greater than 76 kDa, for example, intermediate states of 100 kDa and 95 kDa; in some embodiments, the mature acid α-glucosidase refers to the mature GAA enzyme synthesized in the human body with a molecular weight of approximately 76 kDa or 70 kDa.

[0083] In some embodiments, preferably, the methods and kits according to the present invention are used for detecting the CRIM status of an individual with suspected or confirmed Pompe disease, and more preferably the individual is an individual with suspected or confirmed infantile-onset Pompe disease (IOPD).

[0084] In some embodiments, the detection comprises: contacting an antibody or antigen-binding fragment (labeled or unlabeled) according to the present invention or an immunoconjugate according to the present invention with the sample, and detecting the complex formed by the antibody or antigen-binding fragment or immunoconjugate and acid α-glucosidase. In some embodiments, the detection is performed by western blot.

[0085] GAA antigen detection

[0086] In a fifth aspect, the present invention provides a method for detecting acid α-glucosidase in a sample, which comprises: using an antibody or an antigen-binding fragment thereof according to the present invention as a capture antibody, and detecting the presence or amount of the captured acid α-glucosidase. Preferably, the method is carried out by a sandwich ELISA assay.

[0087] Examples

[0088] Materials

[0089] The following commercially available materials were used in this example: RPMI1640 (abbreviation: R0) was purchased from Hyclone, catalog number: SH30809.01; fetal bovine serum was purchased from Hyclone, catalog number: SH30406.05; HAT (H: Hypoxanthine, A: Aminopterin, T: Thymidine) medium was purchased from Thermo, catalog number: 21060-017; 50% PEG1500 was purchased from Roche, catalog number: 10783641001; paraffin was purchased from Macklin, catalog number: P821317; horseradish peroxidase (HRP)-goat anti-mouse IgG and HRP-goat anti-rabbit IgG were purchased from Zhongshan Golden Bridge, catalog numbers were: ZB-2305, ZB-5301; HRP-goat anti-human IgG was purchased from Abcam, catalog number: ab6858; TMB (3,3',5,5'-tetramethylbenzidine) single-component chromogenic solution was purchased from Solarbio, catalog number: PR1200; ELISA termination solution was purchased from PHYGENE, catalog number: 20211110; Protein G packing material was purchased from GenScript, catalog number: L00209; high-sensitivity ECL chemiluminescent reagent was purchased from Sangon Biotech, catalog number: C500044; GAA rabbit monoclonal antibody (Clone#2489C) was purchased from B&D, catalog number: MAB8329-100; recombinant human GAA protein was purchased from Genzyme Ireland Limited, approval number: S20150049.

[0090] The following experimental samples were used in this example:

[0091] ELISA experimental samples were human plasma and serum negative for anti-GAA antibody, sourced from the sample library of Beijing Jinlan Gene Technology Co., Ltd.

[0092] CRIM detection samples were total proteins extracted from leukocytes, among which positive samples were proteins extracted from RAJI cells (human lymphoma cells); negative samples were proteins from B cells transformed from PBMCs of patients with Pompe disease, and the PBMC samples of the patients were collected after obtaining the informed consent of the patients.

[0093] Example 1 Antibody Preparation

[0094] In this example, anti-GAA monoclonal mouse antibodies were prepared, and the specific steps are as follows:

[0095] 1) Immunization

[0096] Eight-week-old female Balb / c mice were immunized with recombinant human GAA protein by multiple subcutaneous injections in the abdomen and intraperitoneal injection. The mice were given a primary immunization, two booster immunizations, and an activation immunization, for a total of 4 immunizations. The dose for the first three immunizations was 100 μg, administered by multiple subcutaneous injections in the abdomen; the dose for the activation immunization was 80 μg, administered by intraperitoneal injection. The primary immunization was a mixture of recombinant human GAA protein and an equal volume of Freund's complete adjuvant, which was fully emulsified before injection; the two booster immunizations were carried out 14 days and 28 days later, respectively, using a mixture of recombinant human GAA protein and an equal volume of Freund's incomplete adjuvant, which was fully emulsified before injection; the activation immunization was carried out 5 days before cell fusion, and recombinant human GAA protein dissolved in PBS was directly injected. Blood was collected from the orbital sinus before each immunization, and serum was collected. The production of antibodies in the serum was detected by the indirect ELISA method, using recombinant human GAA protein as the antigen for coating and goat anti-mouse-HRP diluted 1:25000 as the secondary antibody for detection.

[0097] 2) Obtain monoclonal cell lines that stably produce antibodies

[0098] The experimental operation steps were carried out according to the requirements of the PEG and HAT reagent instructions.

[0099] Culture medium preparation: R20: R0 containing 20% fetal bovine serum; R10: R0 containing 10% fetal bovine serum.

[0100] Cell fusion: The spleen cells were ground, the red blood cells were lysed, and the cells were counted. Mouse myeloma cells SP2 / 0 cells equal to 1 / 3 of the number of spleen cells were mixed with the spleen cells, and the supernatant was removed by centrifugation. The 50 ml centrifuge tube containing the mixed cells was placed in warm water at 37 °C, 1 ml of PEG was added to the tube, and the cell mass was gently loosened with a pipette tip after 1 minute. Then, 9 ml of pre-warmed R0 was added to terminate the action of PEG, and the mixture was centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded. The mixed cells were aliquoted into a 96-well cell culture plate coated with feeder cells and cultured in an incubator at 37 °C and 5% CO2. Half of the culture medium was replaced starting from the 3rd day, and the culture medium was R20 containing HAT. The growth of hybridoma cells was observed regularly, and the supernatant was aspirated for antibody detection when the clones in the wells were obvious.

[0101] Cell screening and subcloning screening: The cell supernatants in the cell fusion plate were screened by indirect ELISA. Recombinant human GAA protein was used as the antigen for coating, and goat anti-mouse-HRP diluted 1:25000 was used as the secondary antibody. The detection wavelength of the microplate reader was 450 nm. The wells with higher OD values were retested with duplicate wells the next day. The wells with higher retested OD values were subcloned: 100 cells were counted and evenly plated onto a 96-well cell culture plate pre-coated with feeder cells, and the culture medium was R20. One week later, the subcloned cells were screened by ELISA using the same method. Subcloning was performed continuously 3 times to obtain monoclonal cells, which were cultured and domesticated until they could be cultured with R10 medium. A total of 3 positive antibodies, 4B1, 4H3, and 4C2, were screened out, all of which could specifically bind to the antigen. The results are as Figure 1 shown. The supernatants collected from the monoclonal cells of 4B1, 4H3, and 4C2 all showed binding to recombinant human GAA protein in the indirect ELISA assay. One antibody, 4B1, with the highest OD value, was selected for subsequent antibody preparation, and this antibody is the GAA mouse monoclonal antibody described in the present invention.

[0102] 3) Preparation of ascites and purification of antibodies

[0103] Inject 0.5 ml of sterile paraffin into female Balb / c mice over 8 weeks old by intraperitoneal injection. One week later, inject 0.5 - 1E7 monoclonal cells into the peritoneal cavity of the mice. When the abdomen of the mice becomes round, sacrifice the mice and collect the ascites.

[0104] Perform the experimental operation steps according to the requirements of the Protein G affinity chromatography packing instructions.

[0105] Load the Protein G packing into the column. After equilibrating the column with the equilibration buffer, load the filtered ascites sample onto the column; after the sample is loaded onto the column, elute the miscellaneous proteins with the equilibration buffer; elute the antibody with the elution buffer and balance the pH of the elution buffer with the neutralization buffer; load the collected antibody into a dialysis bag and dialyze it with PBS buffer, and then store the antibody after completion.

[0106] 4) Detection of antibody purity and concentration:

[0107] The obtained antibody was detected for purity by SDS-PAGE gel electrophoresis. The molecular weight of the heavy chain of the antibody was 50 - 75 KD, and the molecular weight of the light chain was about 25 KD. The results are as Figure 2 shown. The SDS-PAGE band size of the purified 4B1 antibody was consistent with the expected value, indicating successful antibody purification, and the antibody purity was above 95%, which could be used in subsequent experiments. The antibody concentration was detected by a micro-spectrophotometer to be 1 mg / mL.

[0108] 5) Antibody sequencing:

[0109] The collected monoclonal cells were sent to a sequencing company for sequencing, and the results are as follows:

[0110] Antibody subtype: kappa for the light chain and IgG2b for the heavy chain.

[0111] Variable region and CDR amino acid sequences (CDR sequences are determined according to the IMGT numbering scheme):

[0112] Variable region of the light chain:

[0113] DIVMSQSPSSLAVSAGEKVTMNCKSSQSLVNSRIRKNYLAWYQQKPGQSPKLLIYWASTRE SGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLFSFGGGTKLELK (SEQ ID NO:1)

[0114] CDR-L1: QSLVNSRIRKNY;

[0115] CDR-L2: WAS;

[0116] CDR-L3: KQSYNLFS

[0117] Variable region of the heavy chain:

[0118] EVKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQSPERRLEWVASINTGGSTYYPD SVKGRFTISRDNARNILYLQMSSLRSEDTAMYSCARGDPFAYWGQGTLVTVSA (SEQ ID NO:2)

[0119] CDR-H1: GFTFSTYA;

[0120] CDR-H2: INTGGST;

[0121] CDR-H3: ARGDPFAY.

[0122] Application of the anti-GAA antibody in the detection of anti-drug antibodies (ADA) in Example 2

[0123] According to the requirements of the "Technical Guidelines for the Study of Drug Immunogenicity", the detection of anti-drug antibodies needs to be carried out in sequence: 1. Screening test: initially screen the sample as negative / positive; 2. Confirmatory test: exclude false positive samples among the initially screened positive samples; 3. Titer test: determine the antibody titer of the positive sample. Before using the detection method for anti-drug antibody detection, a positive control should be used to verify the performance of the detection method and determine the corresponding threshold for positive sample determination or ADA titer determination. When detecting clinical samples, a fixed positive control is required to control the quality of the experiment to ensure that the experiment is carried out correctly and the experimental data is reliable. For the positive control used in ADA detection method verification and ADA sample detection, typically, a negative serum / plasma is used to dilute the positive control antibody to a certain concentration for preparation.

[0124] In the detection of ADA, the screening test and titer test are usually carried out by indirect ELISA experiment. By performing serial dilution of the sample, the maximum sample dilution that can produce a positive result is determined, and thus the antibody titer of the sample is determined. To ensure the reliable detection of low-concentration ADA samples, the control antibody used as the positive control should have sufficient sensitivity and stability to reflect the detection ability of the detection method for low-concentration antibody levels. Using different positive control antibodies will result in different sensitivities of the detection method. The higher the sensitivity, the lower the lower limit of detection, and the samples containing a smaller amount of positive antibodies can be detected. The sensitivity of the positive control antibody is crucial for accurately setting the lower limit of detection of the detection test.

[0125] In the anti-GAA detection of Pompe disease, the sample to be tested is usually a human serum / human plasma sample collected from a patient, which contains components such as lipids, fibrin, anticoagulants, etc., which may affect the detection test and lead to false positives. Therefore, for the initially screened suspected positive samples obtained through the screening test, a confirmation experiment is required to exclude false positives. The confirmation test is typically carried out by a competitive ELISA experiment. In this confirmation test, in order to confirm that the component causing the positive signal in the initially screened suspected positive sample is the drug-resistant antibody rather than the confounding component, a control group and a competition group are usually set up. The control group consists of the sample or sample diluent (the same as in the screening test), while the competition group consists of the sample or sample diluent added with free antigen. If the initially positive signal is caused by the confounding component, the antigen added to the sample will not form a competitive mechanism and thus will not cause or will not significantly cause a decrease in the signal intensity on the ELISA plate. On the contrary, if the positive signal is caused by the specific ADA antibody, the antigen added to the sample will form a competitive mechanism and thus will cause a significant decrease in the signal intensity on the ELISA plate. In the art, the degree of decrease in the signal intensity of the competition group relative to the control group, also known as the inhibition rate of the sample, is usually expressed as a percentage. When the inhibition rate of the sample reaches the confirmation threshold, the sample will be confirmed as positive. In order to ensure that the competitive ELISA test accurately and reliably detects true ADA positive samples, the positive control sample used for the competitive ELISA should have sufficient specificity and stability to reflect the performance of the detection method used to distinguish specific ADA from non-specific confounding components.

[0126] In this example, a commercially available rabbit anti-GAA monoclonal antibody (abbreviation: rabbit mAb) and a prepared mouse anti-GAA monoclonal antibody (abbreviation: mouse mAb) were used for sensitivity comparison and competitive ELISA experiments.

[0127] 1) The specific steps for sensitivity comparison are as follows:

[0128] Coat an ELISA plate with recombinant human GAA protein at a concentration of 2 μg / ml, 50 μL / well, and incubate overnight at 2 - 8°C; block it by incubating with 5% defatted milk at 37°C for 2 h. Use human plasma negative for anti-GAA antibody as a quality control diluent, and serially dilute mouse monoclonal antibody (0.5 mg / ml) and rabbit monoclonal antibody (0.5 mg / ml) into positive quality control products with concentrations of 800.0 ng / ml, 400.0 ng / ml, 200.0 ng / ml, 100.0 ng / ml, 50.0 ng / ml, 25 ng / ml, and 12.5 ng / ml respectively. According to the requirements for sensitivity investigation in the guiding principles, before ELISA detection, dilute each concentration point of the quality control product 100-fold with sample diluent (5% defatted milk). The blank control is the sample diluent. Take 50 μL of the diluted quality control product and the blank control respectively and add them to the ELISA plate, and incubate at 37°C for 1 h. The secondary antibodies used for detection are HRP-goat anti-mouse IgG (abbreviated as mouse secondary antibody), HRP-goat anti-rabbit IgG (abbreviated as rabbit secondary antibody), and anti-human antibody (abbreviated as human secondary antibody). Specifically, add the human-mouse mixed secondary antibody diluted with sample diluent to the wells containing mouse monoclonal antibody quality control product and blank control; add the human-rabbit mixed secondary antibody diluted with sample diluent to the wells containing rabbit monoclonal antibody quality control product and blank control for secondary antibody incubation, where the dilution factors of mouse secondary antibody and rabbit secondary antibody in the mixed secondary antibody are both 5000-fold, and the dilution factor of human secondary antibody is 25000-fold, and the addition amount of the secondary antibody mixture is 50 μL / well. After adding the secondary antibody, incubate the ELISA plate at 37°C for 1 h for color development. Then use the termination solution to terminate the color reaction, and read the plate at a wavelength of 450 nm on an ELISA reader. The results are as Figure 3 shown, where the abscissa is the antibody concentration in the quality control product, and the ordinate is the optical density (OD) value obtained by actually adding the sample after diluting the quality control product 100-fold in the ELISA detection. As Figure 3 can be seen, both anti-GAA mouse monoclonal antibody and rabbit monoclonal antibody can specifically bind to GAA antigen, and as the antibody concentration increases, the OD value increases; however, the rabbit monoclonal antibody can reach the screening threshold when the antibody concentration is above 100 ng / mL, and the mouse monoclonal antibody can reach the screening threshold when the antibody concentration is above 25 ng / mL, which indicates that the mouse monoclonal antibody has higher method sensitivity when used as a positive control.

[0129] 2) Application of anti-GAA mouse monoclonal antibody in the competitive ELISA confirmation test

[0130] In the competitive ELISA confirmation test for detecting the production of anti-GAA antibody in the serum or plasma of Pompe disease patients, the performance of the anti-GAA mouse monoclonal antibody of the present invention as a positive quality control product was investigated and compared with a commercial anti-GAA rabbit monoclonal antibody.

[0131] Anti-GAA rabbit monoclonal antibody positive control and anti-GAA mouse monoclonal antibody positive control were prepared separately. Briefly, rabbit monoclonal antibody (0.5 mg / ml) and mouse monoclonal antibody (0.5 mg / ml) were separately mixed into negative samples (human plasma negative for anti-GAA antibody) for preparation to obtain control products with an antibody concentration of 1 μg / mL. Recombinant human GAA protein was coated on an ELISA plate at a concentration of 2 μg / ml, 50 μL / well, and blocked by incubating with 5% skim milk at 37 °C for 2 h. The prepared high-concentration rabbit monoclonal antibody and mouse monoclonal antibody control products were diluted 100-fold using a diluent (5% skim milk) and a diluent containing recombinant human GAA protein, respectively, to obtain a control group (PC) without GAA protein and a competitive group (cPC) containing GAA protein. After sample dilution, it was incubated at 37 °C for 1 hour. 50 μL of the mixture was added to the ELISA plate. According to the method described in (1) above, the secondary antibody was added, a color reaction was carried out, and the signal intensity generated on the ELISA plate was detected at a wavelength of 450 nm using an ELISA reader. Through the adjustment and optimization of experimental conditions, the concentration of GAA protein used for the rabbit monoclonal antibody competitive group was 10 ng / μL; the concentration of GAA protein used for the rabbit monoclonal antibody competitive group was 8 ng / μL. The results are as Figure 4 shown. When the rabbit monoclonal antibody was used for a competition experiment with 500 ng / well of antigen, the inhibition rate was 25.08%, while when the mouse monoclonal antibody was used for a competition experiment with 400 ng / well of antigen, the inhibition rate could reach 89.27%. This indicates that the competitive binding effect of the rabbit monoclonal antibody with the GAA antigen is not obvious and cannot meet the requirements of the guiding principles; while the competitive binding effect of the mouse monoclonal antibody with the GAA antigen is obvious and is suitable as a positive control for the competitive ELISA confirmation test.

[0132] Example 3. Application of anti-GAA antibody in CRIM detection

[0133] Detection of the cross-reactive immunologic material (CRIM) status is a test item for Pompe disease patients before treatment. Whether there is endogenous GAA protein in the patient's body can be judged through the test results. CRIM negative indicates that the patient has no endogenous GAA protein. If CRIM-negative patients directly receive treatment, the patient's immune system will recognize GAA enzyme as a foreign antigen and induce the body to produce high-titer antibodies, thus seriously affecting the treatment efficacy and causing harm to the patient. For CRIM-negative patients, before receiving GAA treatment, immune tolerance induction (ITI) can be used to avoid or reduce the production of anti-GAA antibodies (i.e., drug-resistant antibodies). Therefore, CRIM status detection has an important guiding role in selecting the treatment plan for patients.

[0134] When GAA is synthesized inside the human body, it forms mature GAA enzymes (76 kDa, 70 kDa) through a precursor (110 kDa) and intermediate states (100 kDa, 95 kDa). During CRIM status detection, if any form of GAA is detected, it is determined as CRIM positive. CRIM status detection is usually carried out by Western blot method.

[0135] In this example, a commercial rabbit monoclonal antibody and a prepared mouse monoclonal antibody were used for the CRIM status detection experiment. The specific steps are as follows:

[0136] Respectively take 15, 10, 5, 2.5 μg of positive samples (PC, protein extracted from human RAJI cells) and 40 μg of negative samples (NC, protein extracted from B cells transformed by EBV from PBMC of Pompe disease patients), perform SDS-PAGE gel electrophoresis, and block after membrane transfer. Incubate overnight at 4°C with 2 μg / ml anti-GAA mouse monoclonal antibody and anti-GAA rabbit monoclonal antibody respectively. Add HRP-goat anti-rabbit IgG secondary antibody and HRP-goat anti-mouse IgG secondary antibody diluted 5000 times respectively, incubate at room temperature for 1 hour, wash the membrane and then develop and take pictures. The results are as Figure 5 shown. (a) is the experimental result of the mouse monoclonal antibody, and (b) is the experimental result of the rabbit monoclonal antibody. In the experiment, negative samples NC1 and NC2 from two different Pompe disease patients were used for detection. Five GAA target bands were observed for both anti-GAA monoclonal antibodies when the loading amount of the positive sample (PC) was above 10 μg; but for the negative control, the background observed when using the mouse monoclonal antibody of the present invention was cleaner and there were fewer non-specific bands, indicating that the mouse monoclonal antibody of the present invention has stronger specificity.

[0137] Example 4. Application of anti-GAA antibody in the detection of antigen GAA

[0138] In this example, a commercial rabbit monoclonal antibody and a prepared mouse monoclonal antibody were used to detect the GAA protein content in the sample by sandwich ELISA method.

[0139] The specific steps are as follows:

[0140] The experiment was a 50 μL system, and the anti-GAA mouse monoclonal antibody with a concentration of 2 μg / mL was used for coating. Different concentrations of recombinant human GAA protein were added and reacted at 37°C for 1 hour. 1 μg / mL of anti-GAA rabbit monoclonal antibody was added. Anti-rabbit secondary antibody-HRP diluted 1:10000 was added, 50 μL per well, and reacted at 37°C for 1 hour. The detection wavelength of the microplate reader was 450 nm. The results are as Figure 6As shown, the sandwich ELISA assay established with anti-GAA murine monoclonal antibody and anti-GAA rabbit monoclonal antibody can specifically detect GAA antigen, and as the concentration of GAA antigen increases, the OD value increases, indicating that this method can be applied to the detection of GAA protein concentration and the development of GAA protein quantitative kits, etc.

[0141] Overview of Sequence Listing

[0142]

Claims

1. An anti-GAA antibody or an antigen-binding fragment thereof that binds to acidic α-glucosidase (GAA), comprising: (i) Three LCDR sequences (LCDR1, LCDR2, and LCDR3) of the light chain variable region of SEQ ID NO:1; and (ii) Three HCDR sequences (HCDR1, HCDR2, HCDR3) of the heavy chain variable region of SEQ ID NO:2, wherein the HCDRs and LCDRs sequences are defined according to the IMGT, Kabat, or Chothia scheme.

2. The anti-GAA antibody or an antigen-binding fragment thereof according to claim 1, wherein the anti-GAA antibody comprises: (a) The LCDR1 sequence of SEQ ID NO:3; (b) The LCDR2 sequence of SEQ ID NO:4; (c) The LCDR3 sequence of SEQ ID NO:5; (d) The HCDR1 sequence of SEQ ID NO:6; (e) The HCDR2 sequence of SEQ ID NO:7; and (f) The HCDR3 sequence of SEQ ID NO:

8.

3. The anti-GAA antibody or an antigen-binding fragment thereof according to claim 1 or 2, wherein the anti-GAA antibody comprises a light chain variable region, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO:

1.

4. The anti-GAA antibody or an antigen-binding fragment thereof according to any one of claims 1-3, wherein the anti-GAA antibody comprises a heavy chain variable region, and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO:

2.

5. The anti-GAA antibody or an antigen-binding fragment thereof according to any one of claims 1-4, wherein the anti-GAA antibody comprises the light chain variable region of SEQ ID NO:1 and the heavy chain variable region of SEQ ID NO:

2.

6. The anti-GAA antibody or an antigen-binding fragment thereof according to any one of claims 1-5, wherein the anti-GAA antibody comprises: (a) A kappa light chain constant region; and / or (b) An IgG heavy chain constant region, preferably an IgG2b heavy chain constant region.

7. The anti-GAA antibody or an antigen-binding fragment thereof according to any one of claims 1-6, wherein: (i) The anti-GAA antibody is a murine monoclonal antibody, a chimeric antibody, or a humanized antibody; and / or (ii) The antigen-binding fragment is an antibody fragment selected from the following: Fab, Fab’, Fab’-SH, Fv, single-chain antibodies such as scFv, (Fab’)2 fragments, or linear antibodies.

8. An isolated nucleic acid encoding the anti-GAA antibody or an antigen-binding fragment thereof according to any one of claims 1-7.

9. A vector or a host cell comprising the nucleic acid of claim 8.

10. An immunoconjugate comprising the antibody or an antigen-binding fragment thereof according to any one of the foregoing claims 1-7.

11. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-7, or an immunoconjugate according to claim 10.

12. A kit for detecting an anti-drug antibody (ADA), wherein the kit comprises an anti-GAA antibody or antigen-binding fragment thereof according to any one of claims 1-7 as a positive control antibody, and wherein the anti-drug antibody is an anti-acid alpha-glucosidase antibody.

13. The kit according to claim 12, wherein the kit further comprises reagents for ADA detection.

14. A method for detecting an anti-drug antibody (ADA), wherein the ADA is an anti-acid alpha-glucosidase (GAA) antibody, and the method comprises: (a) using an antibody or antigen-binding fragment thereof according to any one of the preceding claims 1-7 as a positive control antibody, (b) performing ADA detection on a sample to be tested and the positive control antibody, (c) determining the presence or amount of ADA in the sample.

15. The method according to claim 14, wherein: The sample is from an individual who has received, is receiving, or will receive GAA treatment, particularly an individual with Pompe disease, and preferably the sample is a serum or plasma sample from the individual.

16. The kit according to claim 12 or 13 or the method according to claim 14 or 15, wherein the ADA detection comprises: (a) screening for positive samples suspected of having ADA by an indirect ELISA assay; and / or (b) confirming positive samples having ADA by a competitive ELISA assay.

17. A kit for detecting the CRIM status of an individual, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-7, or an immunoconjugate according to claim 10.

18. A method for detecting the CRIM status of an individual, the method comprising: Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-7, or an immunoconjugate according to claim 10, for detecting the presence or amount of any one or any combination of the precursor, intermediate, and mature forms of acid alpha-glucosidase in a sample from an individual, preferably, the individual is a patient with Pompe disease, more preferably the individual is a patient with infantile-onset Pompe disease (IOPD).

19. The method according to claim 18, wherein The detection is performed by western blot.

20. A method for detecting acid alpha-glucosidase in a sample, comprising: - using an antibody or antigen-binding fragment thereof according to any one of claims 1-7 as a capture antibody, and - detecting the presence or amount of captured acid alpha-glucosidase, preferably, the method is performed using a sandwich ELISA assay.

21. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-7, or a kit according to claims 12-13 or 16, for performance verification or product quality control of a kit for detecting anti-GAA anti-drug antibodies in Pompe disease.

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