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CN-122011188-A - Antibody capable of highly binding R9AP protein, antigen binding fragment thereof and application of antibody

CN122011188ACN 122011188 ACN122011188 ACN 122011188ACN-122011188-A

Abstract

The invention provides an antibody which is highly combined with R9AP protein, an antigen-binding fragment thereof and application thereof. The invention obtains the antibody or the antigen binding fragment thereof capable of specifically binding to the R9AP protein through research and screening, and the antibody or the antigen binding fragment thereof has higher affinity to the R9AP protein, can effectively detect the existence or the level of the R9AP protein in a sample, can be used for clinically assisting in detecting the EBV related tumor diseases, has the potential effect of preventing and treating the diseases caused by or related to the R9AP, and has wide application prospect.

Inventors

  • ZHONG QIAN
  • ZENG MUSHENG
  • Lin Ruobin
  • LI YAN
  • LIU YUANTAO
  • Deng Yizhang

Assignees

  • 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所）

Dates

Publication Date
20260512
Application Date
20260316

Claims (14)

  1. 1. An antibody or antigen-binding fragment thereof that specifically binds to an R9AP protein, wherein the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, and light chain complementarity determining regions CDR-L1, CDR-L2, and CDR-L3; The amino acid sequence of the CDR-H1 is shown as SEQ ID NO. 19, the amino acid sequence of the CDR-H2 is shown as SEQ ID NO. 20, and the amino acid sequence of the CDR-H3 is shown as SEQ ID NO. 21; The amino acid sequence of the CDR-L1 is shown as SEQ ID NO. 32, the amino acid sequence of the CDR-L2 is shown as SEQ ID NO. 33, and the amino acid sequence of the CDR-L3 is shown as SEQ ID NO. 34.
  2. 2. The antibody or antigen-binding fragment thereof of claim 1, further comprising a heavy chain framework region and/or a light chain framework region; Preferably, the heavy chain framework region is selected from an antibody heavy chain framework region of human, murine or rabbit origin, and/or the light chain framework region is selected from an antibody light chain framework region of human, murine or rabbit origin.
  3. 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO. 18, or Is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity and having the same function as shown in SEQ ID NO. 18, or An amino acid sequence which is substituted and/or deleted and/or added by one or more amino acids on the amino acid sequence shown in SEQ ID NO. 18 and has the same function, and/or, The amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof is shown as SEQ ID NO. 31, or Is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity and having the same function as shown in SEQ ID NO. 31, or An amino acid sequence with the same function and with the same function, wherein one or more amino acids are substituted and/or deleted and/or added on the amino acid sequence shown in SEQ ID NO. 31.
  4. 4. The antibody or antigen-binding fragment thereof of claim 1 or 2, further comprising a heavy chain constant region and/or a light chain constant region.
  5. 5. A protein comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4.
  6. 6. The protein of claim 5, further comprising additional functional molecules attached at the ends of the antibody or antigen-binding fragment thereof; The other functional molecules are functional molecules for assisting expression and/or purification, and preferably the other functional molecules are selected from at least one of signal peptide, protein tag, molecular chaperone and cofactor.
  7. 7. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-4, or the protein of any one of claims 5-6.
  8. 8. The biological material is characterized by being any one of the following (1) - (4): (1) A gene expression cassette comprising the nucleic acid molecule of claim 7; (2) A vector comprising the nucleic acid molecule of claim 7 or (1); (3) A host cell comprising the nucleic acid molecule of claim 7, (1) or (2); (4) A host cell expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the protein according to any one of claims 5 to 6.
  9. 9. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4 or the protein of any one of claims 5-6, and a conjugate; The conjugate is at least one of a detectable label, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.
  10. 10. The method for producing the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 4, or the protein according to any one of claims 5 to 6, comprising the step of culturing the host cell according to claim 8, and obtaining the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 4, or the protein according to any one of claims 5 to 6 from the culture.
  11. 11. The use of an antibody or antigen binding fragment thereof according to any one of claims 1 to 4, a protein according to any one of claims 5 to 6, a nucleic acid molecule according to claim 7, a biological material according to claim 8 or a conjugate according to claim 9, as follows: (1) Preferably, the detection reagent comprises an R9AP protein detection reagent and an EB virus related tumor disease detection reagent; (2) Preferably, the disease is caused by or related to R9AP protein.
  12. 12. A detection reagent comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the protein according to any one of claims 5 to 6, the nucleic acid molecule according to claim 7, the biological material according to claim 8 and/or the conjugate according to claim 9.
  13. 13. A method for detecting an R9AP protein, comprising the step of contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the protein according to any one of claims 5 to 6 with a sample to be tested.
  14. 14. A medicament comprising an antibody or antigen binding fragment thereof according to any one of claims 1 to 4, a protein according to any one of claims 5 to 6, a nucleic acid molecule according to claim 7, a biological material according to claim 8 and/or a conjugate according to claim 9.

Description

Antibody capable of highly binding R9AP protein, antigen binding fragment thereof and application of antibody Technical Field The invention belongs to the technical field of antibodies, and particularly relates to an antibody capable of highly binding R9AP protein, an antigen binding fragment thereof and application thereof. Background R9AP is an anchoring protein for G protein signaling 9 (RGS 9) that anchors the regulator of the Gtpase Acceleration Protein (GAP) complex of RGS9 to the outer disc membrane, both stabilizing the GAP complex against proteolysis and enhancing its activity by directing it to the appropriate subcellular location, thus mediating GPCR signaling. Our recent studies found that R9AP is a key common receptor for Epstein-Barr virus (EBV) infection of epithelial and B cells, which plays an irreplaceable core role in the viral invasion process in both cell types, providing a completely new explanation for the molecular mechanism of EBV spread across cell types. In B cells, R9AP forms a synergistic complex with the gp42 protein of EBV and MHC class II molecules on the surface of the host cell. The gp42 protein of EBV is firstly combined with HLA II on the surface of B cells and undergoes conformational change, the conformational change can relieve the spatial restriction of gp42 on a virus gH/gL complex, so that gH/gL is exposed and is specifically combined with an N-terminal extracellar structural domain of R9AP, and the gp42-HLA II-R9AP complex formed by the three can further trigger the conformational activation of a virus fusion protein gB, and finally mediate the fusion of a virus envelope and a B cell membrane to complete the intracellular delivery of a virus genome. In contrast, in epithelial cells, R9AP adopts a different synergistic pattern due to the lack of HLA II molecules on the cell surface and the inhibition of epithelial cell infection by gp 42. It recognizes the gH/gL complex of EBV together with the EPHA2 receptor on the surface of the host cell, forming a "gH/gL-EPHA2-R9AP" ternary complex. Wherein, R9AP is combined with gH/gL through the N-terminal 1-50 amino acid region with high affinity, while EPHA2 is interacted with gH/gL through another combining site, the two synergistically enhance the conformational stability of gH/gL, thereby activating the membrane fusion activity of gB and promoting the fusion process of the virus envelope and epithelial cell membrane. In addition, R9AP can also bind directly to the EBV glycoprotein gH/gL complex, thereby initiating gH/gL-gB mediated membrane fusion. EBV, also known as human herpesvirus type 4, is a known DNA oncogenic virus. The current research shows that EB virus is closely related to the occurrence of various malignant tumors, including nasopharyngeal carcinoma, infectious mononucleosis, hodgkin's lymphoma, non-Hodgkin's lymphoma, burkitt's lymphoma, and various epithelial cell cancers including gastric cancer. We also found that R9AP was strongly expressed in nasopharyngeal carcinoma, EBV-positive gastric cancer, B-cell lymphoma, and highly co-localized in tissue sections with EBV infection markers EBERs (EBV-encoded non-coding RNA) -immunohistochemical results showed that the overlap ratio of the R9AP positive region to the EBERs positive region in EBV-positive tumor tissue was >80%, whereas no R9AP expression was detected in normal gastric mucosa, liver tissue. This finding suggests that R9AP may also serve as a diagnostic marker for EBV-associated tumors. Therefore, the development of high affinity antibodies against R9AP can specifically detect the expression of R9AP protein in EBV-associated tumor tissue, and assist in clinical diagnosis. And due to the key function of R9AP in EBV invasion, the R9AP high affinity antibody also has the function of potentially preventing and inhibiting the infection transmission of EBV. However, there is still a lack of high affinity antibodies to R9AP in the art. Disclosure of Invention Based on this, the present invention aims to provide an antibody which highly binds to R9AP protein, an antigen-binding fragment thereof and uses thereof. In order to achieve the above purpose, the present invention adopts the following technical scheme. In a first aspect the present invention provides an antibody or antigen binding fragment thereof that specifically binds to an R9AP protein, said antibody or antigen binding fragment thereof comprising heavy chain complementarity determining regions CDR-H1, CDR-H2 and CDR-H3, and light chain complementarity determining regions CDR-L1, CDR-L2 and CDR-L3; The amino acid sequence of the CDR-H1 is shown as SEQ ID NO. 19, the amino acid sequence of the CDR-H2 is shown as SEQ ID NO. 20, and the amino acid sequence of the CDR-H3 is shown as SEQ ID NO. 21; The amino acid sequence of the CDR-L1 is shown as SEQ ID NO. 32, the amino acid sequence of the CDR-L2 is shown as SEQ ID NO. 33, and the amino acid sequence of the CDR-L3 is shown as SEQ ID NO. 34. In so