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Application of radiolabeled anti-nano antibody in prognosis and diagnosis of cancer

A nanobody and radionuclide technology, applied in the field of biomedicine or biological imaging, can solve the problem of lack of nanobody

Active Publication Date: 2019-03-19
NANOMAB TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there is currently a lack of satisfactory nanobodies against PD-L1 in the field

Method used

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  • Application of radiolabeled anti-nano antibody in prognosis and diagnosis of cancer
  • Application of radiolabeled anti-nano antibody in prognosis and diagnosis of cancer
  • Application of radiolabeled anti-nano antibody in prognosis and diagnosis of cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0151] Example 1 Nanobody expression and purification in host bacteria Escherichia coli

[0152] (1) Electrotransform the corresponding plasmid of the nanobody (sequence shown in SEQ ID NO.: 1-7) into Escherichia coli WK6, and spread it on a culture plate containing ampicillin and glucose in LA+glucose , and cultured overnight at 37°C.

[0153](2) Pick a single colony and inoculate it in 5 mL of LB culture solution containing ampicillin, and cultivate overnight at 37° C. on a shaker.

[0154] (3) Inoculate 1 mL of the overnight strain into 330 mL of TB culture medium, and culture on a shaking table at 37°C. When the OD value reaches 0.6-1, add IPTG, and culture on a shaking table at 28°C overnight.

[0155] (4) Bacteria were collected by centrifugation, and crude antibody extract was obtained by osmosis method.

[0156] (5) Using the infiltration method to obtain the antibody crude extract.

[0157] (6) The nanobody with a purity of more than 90% is prepared by nickel colum...

Embodiment 2

[0159] Embodiment 2 Enzyme-linked immunoassay (ELISA) identifies the affinity of Nanobody

[0160] (1) Antigen protein PD-L1 and IgG coated: 0.5 μg (5 μg / mL, 100 μL) per well, coated with NaHCO 3 (100mM, Ph8.2) was used as blank control, overnight at 4°C.

[0161] (2) The next day, wash with PBST 3 times, add 200 μL of 1% BSA to block for 2 hours at room temperature.

[0162] (3) Dilute each purified nanobody to 10 μg / mL, take 100 μL and incubate with coated PD-L1, PD-L2 and blank control group, and react at room temperature for 1 hour.

[0163] (4) Wash off unbound antibody with PBST, add 100 μL mouse anti-HA tag antibody (diluted 1:2000), and let stand at room temperature for 1 hour.

[0164] (5) Wash off unbound antibody with PBST, add anti-mouse alkaline phosphataseconjugate (diluted 1:2000), and let stand at room temperature for 1 hour.

[0165] (6) Wash off the unbound antibody with PBST, add alkaline phosphatase chromogenic solution, and read the absorbance at a wave...

Embodiment 3

[0171] Example 3 Flow cytometry to detect whether the nanobody has the effect of blocking the binding of PD-1 and PD-L1

[0172] (1) Take 1×10 6 A HEK293F cell transiently expressing the full-length human PD-L1 protein was resuspended in 0.5% BSA-PBS buffer, 10 μg of anti-PD-L1 nanobody was added, and positive control, negative control and blank group (PBS) were set at the same time, and all samples were added 5μg hPD-1-Fc-Biotin, incubated at 4°C for 20min.

[0173] (2) Wash cells twice with PBS, add eBioscience SA-PE, incubate at 4°C for 20 min, wash cells twice with PBS, and detect with flow cytometer.

[0174] The results are shown in Table 1, and the results show that the Nanobodies of the present invention cannot block the binding effect of PD-1 and PD-L1.

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Abstract

The invention discloses application of radiolabeled anti-nano antibody in prognosis and diagnosis of cancer, and particularly discloses an immunoconjugate for detecting PD-L1 molecules. The immunoconjugate comprises VHH chain of specific anti-PD-L1 nano antibody and radionuclide and can be used for noninvasive detection of PD-L1 expression of a to-be-detected object. The immunoconjugate is small in size, high in specificity, suitable for whole body detection targeting primary and metastatic tumor, high in accuracy and small in radiation dosage.

Description

technical field [0001] The invention relates to the technical field of biomedicine or biological imaging, and more specifically relates to the application of radiolabeled anti-nanobodies in the prognosis and diagnosis of cancer. Background technique [0002] PD-1 and its ligand PD-L1 are important targets of tumor immunity. The use of monoclonal antibodies to block PD-1 and PD-L1 pathways has attracted widespread attention in recent years. PD-1 and PD-L1 are a pair of immunosuppressive molecules, which are important components of the immune system to prevent autoimmune diseases. The activation of their pathways can inhibit tumor immune response and induce tumor-specific T cell apoptosis. development is closely related. PD-1 (CD279) is a type I transmembrane protein that belongs to the immunoglobulin superfamily and is mainly expressed on activated CD4+ T cells, CD8+ T cells and B cells and other immune cells. Its ligand PD-L1 (also known as B7-H1, CD274) is a member of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28G01N33/68A61K45/00A61P35/00
CPCA61K45/00G01N33/6872C07K16/2827C07K2317/56A61P35/00A61K51/1045G01N33/532C07K2317/569C07K2317/22C07K2317/92C07K2317/76G01N33/534G01N2333/70532A61K47/6849A61K49/16A61K51/088
Inventor 丁航海黄仲廉
Owner NANOMAB TECH LTD
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