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Anti-TIGIT nanobody and application thereof

A nanobody, expression vector technology, applied in the field of biomedicine or biopharmaceuticals

Active Publication Date: 2020-09-29
SHANGHAI NOVAMAB BIOPHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, no less than 12 TIGIT antibodies have entered the clinic, but no TIGIT antibody has been approved for marketing in the world, so it is still necessary to develop new, significantly differentiated TIGIT antibodies that are more suitable for clinical applications, especially TIGIT nanobodies

Method used

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  • Anti-TIGIT nanobody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0187] Example 1: Human TIGIT extracellular protein expression and camel immune library construction

[0188] Use mammalian cell HEK293F to transiently express human TIGIT extracellular domain protein: mix the pFUSE-IgG recombinant plasmid that has cloned human TIGIT extracellular domain gene with transfection reagent PEI, and then transfect into HEK293F cells; 37°C, 6% CO 2 Cultivate in a shaker incubator for 5 days; then collect the cell supernatant and combine with Protein A beads at room temperature for 1 hour; wash the beads with phosphate buffer solution pH 7.0, and elute the protein with 0.1M pH 3.0 glycine solution; The eluted protein was ultrafiltered into PBS solution, and after the yield was measured, samples were taken for SDS-PAGE detection. Test results such as figure 1 As shown, the expressed and purified hTIGIT(ECD)-Fc protein has a purity greater than 90%, which can be used for camel immunization and antibody screening.

[0189] The purified hTIGIT(ECD)-Fc p...

Embodiment 2

[0191] Example 2: Screening of blocking TIGIT Nanobodies by flow cytometry

[0192] Cloning the Nanobody genes with different sequences from the above strains into the pFUSE-IgG vector, and then mixing the recombinant plasmid with the transfection reagent PEI 1:3, transfected into HEK293F cells; 37°C, 6% CO 2 Cultivate in a shaker incubator for 5 days; then collect the cell supernatant and combine with Protein A beads at room temperature for 1 hour; wash the beads with phosphate buffer, pH 7.0, and elute the protein with 0.1M, pH 3.0 glycine solution; The eluted protein was then ultrafiltered into PBS solution for candidate functional activity research.

[0193] Aliquot the cultured CHOZEN / TIGIT stably transfected cells into 96-well plates, 3E5 cells per well, centrifuge at 3000rpm for 3min to remove the supernatant, add diluted antibodies (Nb5, Nb10, Nb14, Nb16, Nb49, Nb66 , Nb72, Nb86, Nb96, Nb100) and CD155-Biotin protein were incubated for 20 min, and the antibody concent...

Embodiment 3

[0194] Example 3: Detection of binding activity of TIGIT nanobody to cell surface antigen

[0195] Antibody binding function verification was performed using CHOZN / TIGIT stably transfected cells with high expression of TIGIT: the cultured CHOZN / TIGIT cells were digested with trypsin and neutralized with complete medium, and the cells were washed once with PBS, and then the cells were collected; In the 96-well plate, add the diluted antibody (the dilution concentration of each group of antibodies is 40ug / mL, 20ug / mL, 10ug / mL, 5ug / mL, 2.5ug / mL, 1.25ug / mL, 0.625ug / mL, 0.313 ug / mL, 0.156ug / mL, 0.078ug / mL, 0.039ug / mL, 0.019ug / mL) were incubated at 4°C for 20min; after centrifugation, the cells were washed once with PBS, and then goatanti human IgG-FITC was added (according to 1:200 Dilute), incubate at 4°C for 20min; wash the cells once with PBS, centrifuge at 3000rpm, 4°C for 4min, discard the supernatant, add 200uL PBS / well, resuspend the cells, transfer to a flow tube, and detec...

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Abstract

The invention discloses an anti-TIGIT nanobody and application thereof, and particularly provides an anti-TIGIT nanobody and a sequence thereof. The invention also provides a coding sequence encodingthe nanobody or a VHH chain thereof, a corresponding expression vector and a host cell capable of expressing the nanobody, as well as a production method of the nanobody. The nanobody can block the interaction between TIGIT and CD155 on the surface of CT26 cells, and can effectively bind to the TIGIT protein on the cell surface; the nanobody can recognize human and macaca fascicularis TIGIT; the blocking activity of the nanobody is significantly better than a control antibody Tiragolumab; and the nanobody has a significant activating effect on T cells, and the activation effect is significantly better than that of the control antibody Tiragolumab.

Description

technical field [0001] The invention relates to the technical field of biomedicine or biopharmaceuticals, and more specifically relates to an anti-TIGIT nanobody and its application. Background technique [0002] In recent years, TIGIT's immune checkpoint protein has become one of the hotspots in the field of cancer immunotherapy research and development. The full name of TIGIT is T cell immunoglobulin and ITIM domain protein (T cell immunoreceptor with Ig and ITIM domains). It is an inhibitory receptor expressed on the surface of many types of T cells. Many different activating and inhibitory receptors are expressed on the surface of T cells, and these receptors often pair up to fine-tune the activity of T cells. TIGIT and the activating receptor CD226 are a pair, and their common ligand is PVR (CD155). TIGIT inhibits the activation of T cells by competing with CD226 and binding to PVR, disrupting the activation of CD226 and other mechanisms. TIGIT is highly expressed i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K16/30C07K16/46C12N15/13A61K47/68A61K51/10
CPCA61K51/1027A61K47/6849C07K16/2803C07K16/30C07K16/461C07K2317/22C07K2317/24C07K2317/33C07K2317/35C07K2317/52C07K2317/565C07K2317/567C07K2317/569C07K2317/76
Inventor 万亚坤朱敏盖军伟李光辉乔鹏沈晓宁
Owner SHANGHAI NOVAMAB BIOPHARM CO LTD
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