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CN-121975009-A - Human-mouse chimeric monoclonal antibody of anti-human LRP4 protein, application and detection method thereof

CN121975009ACN 121975009 ACN121975009 ACN 121975009ACN-121975009-A

Abstract

The invention discloses a human-mouse chimeric monoclonal antibody of an anti-human LRP4 protein, and application and a detection method thereof, belonging to the technical field of monoclonal antibody preparation. The invention provides an anti-human LRP4 human-mouse chimeric monoclonal antibody, and defines the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain and heavy chain variable regions of the monoclonal antibody. Compared with commercial LRP4 monoclonal/polyclonal antibody (mostly murine or rabbit), the invention is formed by constantly combining the variable region of the murine monoclonal antibody and the humanized antibody by genetic engineering technology, so that the high-efficiency binding capability of the chimeric antibody and antigen is maintained, the immunogenicity of the chimeric antibody can be obviously reduced, and the foundation is laid for the subsequent development of higher humanized antibodies and fully humanized antibodies.

Inventors

  • LI KE
  • YAN YAPING
  • HAO WENBIN
  • ZHANG YAJIAN
  • FAN XUE
  • Liu Ruiman

Assignees

  • 陕西脉元生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260209

Claims (10)

  1. 1. An anti-human LRP4 human murine chimeric monoclonal antibody comprising a light chain and a heavy chain; The light chain comprises a light chain variable region and a light chain constant region, wherein: The light chain variable region comprises three light chain complementarity determining regions, wherein the three light chain complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 respectively have amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, and the light chain constant region is a human light chain constant region, and the amino acid sequences of the light chain constant region are shown as SEQ ID NO. 14; The heavy chain comprises a heavy chain variable region and a heavy chain constant region, wherein: The heavy chain variable region comprises three heavy chain complementarity determining regions, wherein the three heavy chain complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 respectively have amino acid sequences shown as SEQ ID NO. 7, SEQ ID NO. 8/SEQ ID NO. 10 and SEQ ID NO. 9, and the heavy chain constant region is a human heavy chain constant region, and the amino acid sequences of the heavy chain constant region are shown as SEQ ID NO. 15.
  2. 2. The chimeric anti-human LRP4 human monoclonal antibody according to claim 1, wherein the amino acid sequence of the light chain variable region is shown in SEQ ID No.1, the nucleotide sequence encoding the light chain variable region is shown in SEQ ID No.11, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.2 or SEQ ID No.3, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No.12 or SEQ ID No. 13.
  3. 3. The anti-human LRP4 human murine chimeric monoclonal antibody according to claim 1, wherein said anti-human LRP4 human murine chimeric monoclonal antibody comprises an amino acid sequence having the amino acid sequence shown in (I), (II) or (III): (I) Comprising a light chain variable region sequence shown as SEQ ID NO.1, and a heavy chain variable region sequence shown as SEQ ID NO.2 or SEQ ID NO. 3; (II) an amino acid sequence having the same function by substituting, deleting or adding one or more amino acids based on the amino acid sequence shown in (I); (III) an amino acid sequence having at least 50% homology with the amino acid sequence shown in (I) and having sequence identity or similarity with (I).
  4. 4. The human murine chimeric monoclonal antibody against human LRP4 according to claim 3, wherein said amino acid sequence of (III) comprises a functional variant of human murine chimeric monoclonal antibody against human LRP4 protein, said functional variant being a protein having a significant or significant sequence identity or similarity to said human murine chimeric monoclonal antibody against human LRP4 protein.
  5. 5. A recombinant expression vector comprising a nucleotide sequence that expresses an anti-human LRP4 human murine chimeric monoclonal antibody of any one of claims 1-4.
  6. 6. A host cell, characterized in that, the host cell comprises the vector of claim 5.
  7. 7. Use of an anti-human LRP4 human murine chimeric monoclonal antibody of any one of claims 1-4, a recombinant expression vector of claim 5 or a host cell of claim 6 for the preparation of a product for one or more of the detection of LRP4 protein, the identification of LRP4 protein and the detection of LRP4 protein autoantibodies.
  8. 8. A product for detecting LRP4 protein, comprising: a detection reagent or kit comprising at least one of the anti-human LRP4 human murine chimeric monoclonal antibody of any one of claims 1-4, the recombinant expression vector of claim 5, and the host cell of claim 6.
  9. 9. The product for detecting LRP4 protein as claimed in claim 8, wherein the kit includes an immunofluorescence detection kit, a blotting detection kit, an ELISA detection kit, a flow sorting kit, or an IHC detection kit.
  10. 10. A method for detecting LRP4 protein or polypeptide, comprising contacting a biological sample with an anti-human LRP4 human murine chimeric monoclonal antibody of any one of claims 1-4, and detecting LRP4 protein or polypeptide in said sample; The biological sample comprises plasma, whole blood, serum, tissue, cells or lysates of tissue and cells comprising LRP4 proteins/polypeptides.

Description

Human-mouse chimeric monoclonal antibody of anti-human LRP4 protein, application and detection method thereof Technical Field The invention belongs to the technical field of monoclonal antibody preparation, and in particular relates to a human-mouse chimeric monoclonal antibody of an anti-human LRP4 protein, and application and a detection method thereof. Background Low density lipoprotein receptor-related protein 4 (LRP 4) is an important member of the LDL receptor family, and is widely expressed in neuromuscular junctions, hippocampus, pyriform cortex, astrocytes, bones, mammary glands, kidneys, etc., as originally found in gene trapping of mammalian cells by Nakayama et al. The human LRP4 gene is located in the 1 st short arm zone 1 zone 2 sub-zone of chromosome 11, contains 1905 amino acids, LRP4 is a single transmembrane protein, and the extracellular region contains LDLa repeats, EGF-like domains and β -propeller domains, which are critical for ligand binding to the extracellular domain of the protein. The intracellular domain is short, contains NPxY and PDZ binding motifs, and is involved in intracellular signaling and endocytic transport of membrane proteins. LRP4 also shares intracellular sequence motifs with other LRPs, including NPxY motifs, which play a very important role in receptor endocytosis, trafficking and intracellular signaling. In biology, LRP4 is involved in many processes including limb development, craniofacial organ development, osteogenesis, kidney development, and neural development. Mutations or dysfunctions of LRP4 are closely related to various diseases, and in limb development, LRP4 ensures normal formation of extremity structures such as phalangeal bones and metacarpal bones by regulating activities of important signaling pathways such as Wnt and Notch, and LRP4 dysfunction can cause serious deformities such as digitalis. LRP4 is also involved in the balance of bone metabolism, mutations of which can lead to hyperproliferative sclerosant bone. In kidney development, LRP4 deficiency can lead to renal hypoplasia or dysfunction. In particular, LRP4 functions most classically in the formation of neuromuscular junctions (Neuromuscular Junction, NMJ), which act as Agrin receptors, bind to MuSK to form complexes, activate downstream signals, leading to the aggregation of acetylcholine receptors (AChR) at postsynaptic membranes, which is the structural basis for the efficient transmission of nerve signals to muscle, and if this process is disrupted by autoantibodies, targeting LRP4 proteins affects the transmission of neuromuscular signals, leading to the occurrence of myasthenia gravis. The current research field for LRP4 is quite extensive, and besides the traditional role in genetic and neurological diseases, its role in cancer is becoming a new hotspot. To study LRP4 protein, the antibodies are often not separated, and the currently commercialized LRP4 antibodies are mostly rabbit-source or mouse-source monoclonal/polyclonal antibodies, although the preparation method is simple, standardized antibody reagents which cannot be compatible with a general detection platform (such as ELISA (enzyme-linked immunosorbent assay) using anti-human IgG secondary antibodies, immunofluorescence and the like), particularly chimeric or humanized antibodies, are difficult to meet the further research requirements of LRP4 protein in human related diseases, and the commercial mouse-source/rabbit-source monoclonal antibodies have smaller packaging specifications and are expensive. Disclosure of Invention In order to overcome the defects of the prior art, the invention aims to provide a human-mouse chimeric monoclonal antibody of anti-human LRP4 protein and application thereof, and the variable region of the murine antibody and the constant region of the human antibody are recombined by utilizing a genetic engineering technology, so that the invention has profound significance for research on the LRP4 protein and deep research on various diseases participated by the protein. In order to achieve the above purpose, the invention is realized by adopting the following technical scheme: the invention discloses an anti-human LRP4 human mouse chimeric monoclonal antibody, which specifically comprises a light chain and a heavy chain, wherein: The three CDR regions CDR1, CDR2 and CDR3 of the light chain have amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively, and the three CDR regions CDR1, CDR2 and CDR3 of the heavy chain have amino acid sequences shown as SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 10 and SEQ ID NO. 9 respectively. As one embodiment, in the anti-human LRP4 monoclonal chimeric antibody, the amino acid sequences of the three CDR regions of the light chain are shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively, and the amino acid sequences of the three CDR regions of the heavy chain are shown as SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, respe