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Anti-CTLA-4 nano antibody Nb91 and preparation method and application thereof

A technology of CTLA-4 and nano-antibody, which is applied in botany equipment and methods, biochemical equipment and methods, nanotechnology for materials and surface science, etc., can solve the problems of poor stability and easy degradation, limited application range, and production cost advanced questions

Active Publication Date: 2016-12-07
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 nano antibody Nb91 and preparation method and application thereof
  • Anti-CTLA-4 nano antibody Nb91 and preparation method and application thereof
  • Anti-CTLA-4 nano antibody Nb91 and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0093] Embodiment 1, the preparation of nanobody

[0094] The present invention provides four nanobodies derived from camels, whose names are respectively Nb16, Nb30, Nb36 and Nb91. Wherein, the amino acid sequence of Nb16 is shown in SEQ ID No.1 in the sequence listing, 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, encoded by SEQ ID No. The nucleotide sequence encoding of No.4; the amino acid sequence of Nb36 is as shown in SEQ ID No.5 in the sequence listing, encoded by the nucleotide sequence of SEQ ID No.6; the amino acid sequence of Nb91 is as shown in SEQ ID No in the sequence listing .7, 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 keep the other sequences unchanged to obtain the recombinant vector pComb3-Nb16. ...

Embodiment 2

[0108] Example 2, Determination of Nanobody and CTLA-4 Binding Rate

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

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

[0111] According to the above method, Nb16 was replaced by Nb30, Nb36 and Nb91 respectively, and other steps were kept unchanged, and the binding results of Nb30, Nb36 and Nb91 and CTLA-4 were obtained, respectively as follows 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 nano antibody Nb91 and a preparation method and an application thereof. The nano antibody provided by the invention includes a determinant complementary region and a frame region; the determinant complementary region of the nano antibody consists of CDR1, CDR2 and CDR3; an amino acid sequence of the CDR1 comprises 26th-32nd amino acids of SEQ ID NO.7 in a sequence table; an amino acid sequence of the CDR2 comprises 48th-55th amino acids of SEQ ID NO.7 in the sequence table; an amino acid sequence of the CDR3 comprises 94th-106th amino acids of SEQ ID NO.7 in the sequence table. The nano antibody Nb91 can be combined with T cells and CTLA-4, and can be applied to research and development of CTLA-4 molecular detection reagents, preparation of tumor inhibitors or tumor cell inhibitors and preparation of drugs for inhibition of CTLA-4 activity and promotion of T cell proliferation.

Description

technical field [0001] The invention relates to the anti-CTLA-4 nanobody Nb91 in the field of biomedicine, its preparation method and application. Background technique [0002] Cytotoxic T lymphocyte-associated antigen 4 (cytotoxic T lymphocytic associated antigen, CTLA-4 / CTLA4), also known as CD152, is a leukocyte differentiation antigen, a transmembrane glycoprotein molecule expressed on the surface of activated T cells, and belongs to immunoglobulin Members of the superfamily negatively regulate the proliferation of T cells and play an important role in the regulation of immune responses. 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, with extremely low toxic and side effects. New immunosuppressive drugs. [0003] There are many problems in the application of antibody drugs, such as long research and deve...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00B82Y30/00
CPCB82Y30/00C07K16/2818C07K2317/55C07K2317/565C07K2317/567C07K2317/569C07K2317/74C07K2317/76
Inventor 赵永祥卢小玲
Owner GUANGXI MEDICAL UNIVERSITY
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