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Anti-CTLA-4 nanobody Nb30 as well as preparation method and application thereof

A CTLA-4, nanobody technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, nanotechnology for materials and surface science, etc. higher question

Active Publication Date: 2016-10-26
GUANGXI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There are many problems in the application of antibody drugs, such as long research and development cycle, high production cost, difficulty in large-scale production, poor stability, easy degradation, high storage cost, easy contamination, high maintenance cost, and immunogenicity, etc. Clinical application range

Method used

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  • Anti-CTLA-4 nanobody Nb30 as well as preparation method and application thereof
  • Anti-CTLA-4 nanobody Nb30 as well as preparation method and application thereof
  • Anti-CTLA-4 nanobody Nb30 as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1. Preparation of Nanobody

[0094] The present invention provides four kinds of nanobodies derived from camel, their names are Nb16, Nb30, Nb36 and Nb91. Among them, the amino acid sequence of Nb16 is shown in SEQ ID No. 1 in the sequence listing and is encoded by the nucleotide sequence of SEQ ID No. 2; the amino acid sequence of Nb30 is shown in SEQ ID No. 3 in the sequence listing and is encoded by SEQ ID The nucleotide sequence of No. 4 is encoded; the amino acid sequence of Nb36 is shown in SEQ ID No. 5 in the sequence listing, and is encoded by the nucleotide sequence of SEQ ID No. 6; the amino acid sequence of Nb91 is shown in SEQ ID No in the sequence listing. As shown in .7, it is encoded by the nucleotide sequence of SEQ ID No.8.

[0095] Replace the DNA fragment between the PstI and NotI recognition sequences of the vector pComb3 (Biovector product) with the DNA molecule shown in SEQID No. 2, and the other sequences are unchanged, to obtain the recombina...

Embodiment 2

[0108] Example 2. Determination of the binding rate of Nanobody and CTLA-4

[0109] 1. Determination of the binding rate of Nanobody and CTLA-4 (direct method)

[0110] T cells were separated from human peripheral blood cells of healthy volunteers, cultured in 96-well plates, 10μg / mL PHA (sigma, L9017) was added to T cells after co-cultivation for 72h, and Nb16 (1μg) of Example 1 was added to 1×10 6 Incubate each of the above T cells in the dark at 4°C for 30 minutes. After washing with PBS three times, add 1μg PE anti-HA tag antibody (abcam, Clone: ​​16B12) and incubate at 4°C for 30 minutes. After washing three times with PBS, put the sample on the BACKMAN flow cytometer , The result is Figure 5 Shown in A.

[0111] According to the above method, replace Nb16 with Nb30, Nb36, and Nb91, and the other steps are the same, to obtain the combined results of Nb30, Nb36, and Nb91 with CTLA-4, respectively, as Image 6 Middle A, Figure 7 Middle A, Figure 8 Shown in A.

[0112] 2. Determi...

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Abstract

The invention discloses an anti-CTLA-4 nanobody Nb30 as well as a preparation method and application thereof. The nanobody provided by the invention comprises a complementarity-determining region and a framework region; the complementarity-determining region of the nanobody consists of CDR1, CDR2 and CDR3; the amino acid sequence of CDR1 is that of a 26th-35th amino acid of SEQ ID No. 3 in a sequence table; the amino acid sequence of CDR2 is that of a 51st-58th amino acid of SEQ ID No. 3 in the sequence table; the amino acid sequence of CDR3 is that of a 97th-115th amino acid of SEQ ID No. 3 in the sequence table. The nanobody Nb30 can be combined to T cells and CTLA-4, can be used for research and development of a CTLA-4 molecular detection reagent, and can be used for preparing a tumor inhibitor or a tumor cell inhibitor and preparing drugs for inhibiting CTLA-4 activity and promoting proliferation of the T cells.

Description

Technical field [0001] The invention relates to an anti-CTLA-4 nanobody Nb30 in the field of biomedicine, and a preparation method and application thereof. Background technique [0002] Cytotoxic T lymphocytic associated antigen 4 (CTLA-4 / CTLA4), also known as CD152, is a leukocyte differentiation antigen and a transmembrane glycoprotein molecule expressed on the surface of activated T cells. It is an immunoglobulin Superfamily members play a negative regulatory role on T cell proliferation and play an important regulatory role in immune response. CTLA-4Ig can effectively and specifically inhibit cellular and humoral immune responses in vivo and in vitro, and has significant therapeutic effects on transplant rejection, tumors and various autoimmune diseases. It has extremely low toxic and side effects and is currently considered to be more promising New immunosuppressive drugs. [0003] There are many problems in the application of antibody drugs, such as long research and develo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00B82Y30/00
CPCA61K39/00B82Y30/00C07K16/2818C07K2317/565
Inventor 赵永祥卢小玲
Owner GUANGXI MEDICAL UNIVERSITY
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