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CN-122011180-A - Targeting Pirin nano antibody and application thereof

CN122011180ACN 122011180 ACN122011180 ACN 122011180ACN-122011180-A

Abstract

The invention provides a targeting Pirin nano antibody and application thereof, wherein the nano antibody comprises a CDR (complementary determining region) which is a CDR1 shown in SEQ ID NO.1 and a CDR3 shown in SEQ ID NO.2, or a CDR1 shown in SEQ ID NO. 10 and a CDR3 shown in SEQ ID NO. 11, and an HV2 with an amino acid sequence shown in SEQ ID NO. 3 and an HV4 with an amino acid sequence shown in SEQ ID NO. 4, or an HV2 shown in SEQ ID NO. 12 and an HV4 shown in SEQ ID NO. 13. The nanometer antibody KD value is in the range of 2.0-15nM, has antigen-antibody binding force and affinity close to nanomolar level, can be regarded as high-affinity antibody, and can be developed into a high-specificity diagnostic reagent, used as a therapeutic drug or used as an 'immune sensitizer'.

Inventors

  • LI ZENGPENG
  • CHEN MINGLIANG

Assignees

  • 自然资源部第三海洋研究所

Dates

Publication Date
20260512
Application Date
20251028

Claims (9)

  1. 1. A targeting Pirin nanobody is characterized in that the nanobody comprises complementarity determining regions CDR1 shown in SEQ ID NO. 1 and CDR3 shown in SEQ ID NO. 2, or CDR1 shown in SEQ ID NO. 10 and CDR3 shown in SEQ ID NO. 11, and HV2 shown in SEQ ID NO. 3 and HV4 shown in SEQ ID NO. 4, or HV2 shown in SEQ ID NO. 12 and HV4 shown in SEQ ID NO. 13.
  2. 2. The nanobody of claim 1, wherein the framework region of the nanobody comprises FR1, FR2, FR3 and FR4, the amino acid sequence of FR1 is shown as SEQ ID No. 5 or SEQ ID No. 14, the amino acid sequence of FR2 is shown as SEQ ID No. 6 or SEQ ID No. 15, the amino acid sequence of FR3 is shown as SEQ ID No. 7 or SEQ ID No. 16, and the amino acid sequence of FR4 is shown as SEQ ID No. 8 or SEQ ID No. 17.
  3. 3. A nanobody of claim 1 which targets Pirin, wherein the nanobody comprises a nanobody having the amino acid sequence shown in SEQ ID No. 9 or SEQ ID No. 18.
  4. 4. A nucleic acid molecule encoding the Pirin-targeting nanobody of any one of claims 1-3.
  5. 5. A construct comprising the nucleic acid molecule of claim 4.
  6. 6. The use of the nano-antibody of targeting Pirin in the preparation of antitumor drug according to claim 1.
  7. 7. The use of the nanobody of targeting Pirin according to claim 1 for the preparation of a tumor diagnosis/prognosis kit or for the preparation of a diagnosis/prognosis kit.
  8. 8. The use of the nano-antibody of claim 1 for targeting Pirin for preparing tumor immunity sensitizer.
  9. 9. A pharmaceutical composition comprising the nanobody of claim 1 targeted to Pirin.

Description

Targeting Pirin nano antibody and application thereof Technical Field The invention relates to the field of biological medicine, in particular to a targeting Pirin nanometer antibody and application thereof. Background Pirin (PIR) protein is a highly conserved, fe (II) -dependent functional nucleoprotein belonging to the cupin superfamily members and plays a central regulatory role in a variety of critical cellular processes, especially in tumor development and immunomodulation. One is transcriptional and apoptotic regulation Pirin, which acts as a transcriptional coactivator, is involved in regulating NFI/CTF1 and Bcl-3, thereby affecting DNA replication, transcriptional activation, and apoptosis signaling pathways. Its central role in tumors is as a "brake" of apoptosis. In tumors such as colorectal cancer Pirin inhibits FAS-mediated apoptosis by a delicate dual mechanism, on the one hand, it prevents binding of the nfkb 2 complex (p 52-RELB) to the FAS gene promoter, thus inhibiting FAS expression at the transcriptional level, and on the other hand, it inhibits upstream kinase NIK-mediated nfkb 2 activation and nuclear translocation. In addition Pirin can bind directly to FAS protein, retain it in the cytoplasm, prevent its transport to the cell membrane and assembly of the apoptotic signal complex. And the second is that Pirin has the activity of quercetin 2, 3-dioxygenase, and can catalyze and degrade natural flavonoid compound-quercetin with anticancer and anti-inflammatory activity. This function correlates Pirin with the regulation of intracellular redox homeostasis and PI3K/Akt signaling pathways. Thirdly, pirin shows high expression in various malignant tumors such as colorectal cancer, liver cancer, kidney cancer, glioma and the like, and the high expression level is usually related to poor prognosis. The core cancerogenic mechanism is to inhibit FAS-dependent apoptosis by constructing a survival path of PIR-NIK-NF kappa B2-FAS. Since FAS-mediated apoptosis is one of the major weapons for cytotoxic T lymphocytes (CD 8 + T cells) to kill tumor cells, the high expression of Pirin is equivalent to wearing a piece of "body armor" for tumor cells, enabling them to effectively evade the monitoring and clearance of the immune system. Given the key role Pirin plays in tumors, the development of nanobodies targeting Pirin has great potential to be a next generation tumor diagnosis and treatment tool. Disclosure of Invention In order to solve the above problems, the present invention provides a targeting Pirin nanobody and its application, which provides a molecular tool for developing new Pirin inhibitors. In order to achieve the above object, an embodiment of the present invention provides a nanobody targeting Pirin in the first aspect, the nanobody including complementarity determining regions CDR1 shown in SEQ ID NO. 1 and CDR3 shown in SEQ ID NO. 2, or CDR1 shown in SEQ ID NO. 10 and CDR3 shown in SEQ ID NO. 11, and HV2 having amino acid sequences shown in SEQ ID NO.3 and HV4 having amino acid sequences shown in SEQ ID NO. 4, or HV2 shown in SEQ ID NO. 12 and HV4 shown in SEQ ID NO. 13. According to the embodiment of the invention, the target Pirin nano antibody has an KD value within the range of 2.0-15nM, has antigen-antibody binding force and affinity close to nanomolar level, and can be regarded as a high-affinity antibody. Therefore, the nanobody can be developed into a high-specificity diagnostic reagent, and the expression level of Pirin in a tumor biopsy sample can be detected through an Immunohistochemistry (IHC) or Western Blotting (WB) technology. This can be used not only as an auxiliary diagnostic marker, but more importantly as a prognostic indicator. Pirin high expressing patients may have poorer prognosis and may be insensitive to apoptosis-inducing chemotherapeutics or certain immunotherapies. The ELISA kit developed based on the nano antibody can be used for quantitatively detecting Pirin in serum or tissue lysate due to high stability and production convenience, and provides a new tool for early screening and therapeutic response monitoring of tumors. Thus, the nano antibody can be directly used as a therapeutic drug. Since nanobodies can effectively enter the interface of interactions between proteins (including recessed epitopes) that are difficult for conventional antibodies to reach, they can be designed to specifically block Pirin binding to nfkb 2 or FAS proteins. This blocking will break Pirin the inhibition of the apoptotic pathway, reactivating FAS-mediated apoptosis programs, thereby directly killing cancer cells. Thus, the nano antibody can be used as an 'immune sensitizer'. In current tumor immunotherapy, one of the core challenges is that many "cold tumors" (i.e., tumors with little infiltration of immune cells, low immunogenicity) are unresponsive to immune checkpoint inhibitors (e.g., PD-1/PD-L1 antibodies). The targeted Pirin nanometer antibody can make the killing e