Search

CN-121991205-A - Nanometer antibody for detecting HPV 16E 6 protein and application thereof

CN121991205ACN 121991205 ACN121991205 ACN 121991205ACN-121991205-A

Abstract

HPV16 is the main cause of malignant tumors such as cervical cancer, and E6 protein thereof becomes a key therapeutic and diagnostic target. In the prior art, HPV16 detection antibodies have the problems of poor stability, dependence on cold chain transportation, low preparation efficiency and the like. The invention obtains the nanometer antibody sequence with high affinity (dissociation constant KD is less than or equal to 1 multiplied by 10 ‑7 M) and stable at room temperature through the total synthesis library construction and phage assisted directed evolution (PACE) screening technology. The activity of the antibody is maintained to be more than or equal to 95% after the antibody is stored for 30 days at 25 ℃, the antibody can be directly used for household test paper, the accuracy rate is 98%, and the antibody can be expanded to medical uses such as CAR-T cell treatment. Compared with the traditional antibody, the invention has the beneficial effects of short preparation period, low cost, wide application scene and the like, and provides a breakthrough solution for early screening of cervical cancer.

Inventors

  • WANG CHENGZHI
  • GAO YUAN
  • Cao Shuaihua
  • CHEN HE
  • WANG QIUJUN
  • WANG XIN

Assignees

  • 北京智源深澜科技有限公司

Dates

Publication Date
20260508
Application Date
20260121

Claims (10)

  1. 1. An isolated nanobody comprising three complementarity determining regions CDR1, CDR2 and CDR3, The amino acid sequence of the CDR1 is selected from one of the amino acid sequences shown in SEQ ID NO. 7-22, The amino acid sequence of CDR2 is selected from one of the amino acid sequences shown in SEQ ID NO. 23-25, The amino acid sequence of the CDR3 is at least one of the amino acid sequences shown in SEQ ID NO. 26-46; preferably, the amino acid sequence of the nanobody has more than or equal to 90% sequence identity and retains variants that bind HPV 16E 6 protein function; Preferably, the amino acid sequence of the nano antibody is selected from one of the amino acid sequences shown in SEQ ID NO. 1-6; Preferably, the dissociation equilibrium constant (KD) of the HPV 16E 6 protein is less than or equal to 1X 10 -8 M, the association rate constant (kon) is less than or equal to 5E3, and the dissociation rate constant (kdis) is less than or equal to 1E-4; Preferably, the dissociation equilibrium constant (KD) of the HPV 16E 6 protein is less than or equal to 1X 10 -9 M, the association rate constant (kon) is less than or equal to 6E3, and the dissociation rate constant (kdis) is less than or equal to 1E-5; preferably, the binding activity is maintained at 90% or more after storage for 7 days at 25 ℃ and the activity is maintained at 95% or more after storage for 30 days at 4 ℃.
  2. 2. A method of making the nanobody of claim 1 comprising: s1, constructing a nano antibody library in a total synthesis mode; S2, screening positive clones by phage assisted directed evolution (PACE); S3, expressing and purifying the nano antibody in a host cell; Preferably, in the step of screening positive clones by phage assisted directed evolution (PACE), 10% -30% of arabinose is used for induction, the induction temperature is 37 ℃, the induction time is 8-12 hours, and the rotating speed is 800-1200rpm; preferably, the host cell is escherichia coli S2060, and the induction is performed when the bacterial liquid OD600 reaches 0.4-0.6 in the culture process; Preferably, a wash buffer containing 20-40mM imidazole and an elution buffer containing 250-300mM imidazole are used in the purification step.
  3. 3.A polynucleotide, characterized in that, encoding the nanobody of claim 1.
  4. 4. An expression vector comprising the polynucleotide of claim 3.
  5. 5. A host cell comprising the polynucleotide of claim 3 or the vector of claim 11.
  6. 6. A protein conjugate comprising the nanobody of claim 1 or an active fragment thereof, and a ligand selected from the group consisting of a fluorophore or radioisotope, a fluorophore and/or a delivery vehicle.
  7. 7. A kit for detecting HPV16E6 protein, comprising the nanobody of any one of claims 1-5; preferably, the kit is used for home detection and comprises room temperature stable detection test paper.
  8. 8. The use of the nanobody of claim 1 in the preparation of a kit for detecting HPV subtype 16 or in the preparation of a medicament for treating or preventing a disease associated with HPV subtype 16; The disease includes at least one of cervical cancer, anal canal cancer, tonsil cancer, oral cancer, laryngeal cancer, intranasal cancer, esophageal cancer, baume's disease, basal cell carcinoma, pahucho's disease, squamous cell carcinoma, and flat wart.
  9. 9. A chimeric antigen receptor comprising the nanobody of claim 1, wherein the chimeric antigen receptor further comprises a hinge region and an intracellular signaling domain; The hinge region is selected from a CD 8a hinge region or an IgG1 hinge region; The intracellular signaling domain comprises a cd3ζ chain and a 4-1BB co-stimulatory domain; The chimeric antigen receptor is used to transduce immune cells, which are T cells, NK cells or macrophages.
  10. 10. Use of the nanobody of claim 1, the chimeric antigen receptor of claim 9 in the manufacture of a medicament for treating a disease associated with HPV16 infection, wherein the disease comprises at least one of cervical cancer, anal canal cancer, tonsil cancer, oral cancer, laryngeal cancer, intranasal cancer, esophageal cancer, bauwinia, basal cell carcinoma, pahucho disease, squamous cell carcinoma, and verruca plana.

Description

Nanometer antibody for detecting HPV 16E 6 protein and application thereof Technical Field The invention belongs to the field of biological diagnosis and medicine, and in particular relates to a nano antibody for detecting HPV16E6 protein. Background Cervical cancer is a malignant tumor with higher morbidity and mortality in women, and is continuously infected with high-risk Human Papilloma Virus (HPV), especially HPV type 16, which is the main cause. The E6 protein acts as an early expression protein of HPV16 and plays a key carcinogenesis role in tumorigenesis. Thus, the expression level of E6 protein has become an important marker for cervical cancer screening and early diagnosis. The existing detection means such as PCR and immunohistochemistry are sensitive but depend on laboratory equipment, complex operation flow and professionals, so that home detection or large-scale crowd self-screening is difficult to realize. The nanometer antibody (Nanobody), i.e. single domain antibody, is engineered from camelid-derived heavy chain antibody, has the advantages of small molecular weight, strong stability, high expression, easy engineering transformation and the like, and is suitable for being used as a core raw material of a detection kit with convenient construction, low cost and high sensitivity. In the prior art, although HPV16E6 nano antibodies exist, the antibody prepared by animal immunization has large batch difference and poor stability (storage at-20 ℃) and cannot be used as home detection of non-professionals, and the traditional phage display screening efficiency is low, so that high-affinity antibodies are difficult to obtain. In addition, the prior art wants to obtain the nano antibody which is stable at room temperature and can be monitored at home, and has the technical obstacle that the expression level of HPV16E6 protein is low, the sensitivity of the traditional antibody is insufficient, and the detection at home requires the antibody to be stable for a long time at room temperature, so that the prior art cannot meet the requirement. Therefore, there is an urgent need for a nanobody with high specificity and high affinity, which can also be used to construct a rapid, portable detection method or kit suitable for home use for HPV 16E 6 protein. Disclosure of Invention According to one aspect of the application, a nano antibody for HPV 16E 6 protein is provided, and an application based on the nano antibody, in particular a convenient detection scheme is provided, so that home autonomous detection of cervical cancer in early stage is realized, and the accessibility and compliance of early screening and early diagnosis are improved. In order to achieve the above purpose, the present invention provides the following technical solutions: Obtaining a nano antibody library through a total synthesis mode, and screening and evolving nano antibodies which are high in affinity and specifically identify HPV 16E 6 proteins by adopting a phage assisted directed evolution technology; sequencing and expressing positive clones to obtain purified functional nano antibodies; the nano antibody has good stability at room temperature or in a wide temperature range, and is suitable for home detection equipment/kits without cold chain transportation. In particular, the invention provides an isolated nanobody comprising three complementarity determining regions CDR1, CDR2 and CDR3, The amino acid sequence of the CDR1 is selected from one of the amino acid sequences shown in SEQ ID NO. 7-22, The amino acid sequence of CDR2 is selected from one of the amino acid sequences shown in SEQ ID NO. 23-25, The amino acid sequence of the CDR3 is at least one of the amino acid sequences shown in SEQ ID NO. 26-46; preferably, the amino acid sequence of the nanobody has more than or equal to 90% sequence identity and retains variants that bind HPV 16E 6 protein function; As a specific scheme, the amino acid sequence of the nano antibody is selected from one of the amino acid sequences shown in SEQ ID NO. 1-6. Alternatively, the dissociation equilibrium constant (KD) of the HPV 16E 6 protein is less than or equal to 1X 10 -8 M, the association rate constant (kon) is less than or equal to 5E3, and the dissociation rate constant (kdis) is less than or equal to 1E-4; Preferably, the dissociation equilibrium constant (KD) for binding to HPV 16E 6 protein is 1X 10 -9 M, the binding rate constant (kon) is 6E3, and the dissociation rate constant (kdis) is 1E-5. Alternatively, the nanobody maintains a binding activity of 90% or more after storage for 7 days at 25 ℃. The activity can be maintained to be more than or equal to 95% after 30 days of storage at 4 ℃. According to still another aspect of the present application, there is provided a method for nanobody as set forth in any one of the above, comprising: s1, constructing a nano antibody library in a total synthesis mode; S2, screening positive clones by phage assisted directed evoluti