Single-domain antibody for CD47

A single-domain antibody and expression vector technology, applied in the direction of antibodies, anti-tumor drugs, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problem of harsh storage conditions, low stability, and long development cycle and other issues, to achieve the effect of broad application prospects

Inactive Publication Date: 2015-07-29
JIANGSU CHUNSHENTANG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to their special structural properties, nanobodies have both the advantages of traditional antibodies and small molecule drugs, and almost perfectly overcome the shortcomings of traditional antibodies such as long development cycle, low stability, and harsh storage conditions.

Method used

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  • Single-domain antibody for CD47
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  • Single-domain antibody for CD47

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Construction of Nanobody Library against CD47

[0036] (1) First synthesize CD47 polypeptide, mix 1 mg CD47 with Freund's adjuvant in equal volume, immunize a Xinjiang dromedary, once a week, immunize 7 times in total, and stimulate B cells to express antigen-specific nanobodies;

[0037] (2) After the 7 times of immunization, 100 mL camel peripheral blood was drawn, lymphocytes were separated and total RNA was extracted;

[0038] (3) Synthesize cDNA and use nested PCR to amplify VHH;

[0039] (4) Digest 16 μg pCMECS3 phage display vector and 8 μg VHH with restriction enzymes PstI and NotI and connect the two fragments;

[0040] (5) The ligation product was transformed into electroporation-competent cells TG1, and the CD47 nanobody library was constructed and the library capacity was determined. The library size was 5.85×10 8 CFU; In addition, the insertion rate of the constructed library was detected, and 24 single clones were randomly selected for PCR dete...

Embodiment 2

[0041] Example 2 Screening process for Nanobodies against CD47

[0042] (1) Dissolve in 100mM NaHCO 3 , 20 μg of CD47 in pH 8.2 was coupled to a NUNC microtiter plate, and placed overnight at 4°C;

[0043] (2) Add 100 μL 0.1% BSA the next day, and block for 2 hours at room temperature;

[0044] (3) After 2h, add 100μL phage (2×10 11 1 immunized camel single-domain antibody phage) at room temperature for 1 h;

[0045] (4) Wash 5 times with PBS+0.05% Tween-20 to wash off unbound phages;

[0046] (5) Use 100mM TEA (triethylamine) to elute the phages that specifically bind to CD47, infect Escherichia coli TG1 cells in the logarithmic phase, and incubate at 37°C for 1 hour to produce and purify the phages for the next round of screening , the same screening process was repeated for 3 to 4 rounds, and the enrichment was gradually obtained.

Embodiment 3

[0047] Example 3 Using phage enzyme-linked immunoassay (ELISA) to screen specific single positive clones

[0048] (1) From the cell culture dish containing phage after the above 3-4 rounds of screening, select 96 single colonies and inoculate them in 1 mL of TB medium containing 100 ug / mL ampicillin (1L TB medium contains 2.3 g of KH 2 PO 4 , 12.52g K 2 HPO 4 , 12g peptone, 24g yeast extract, 4mL glycerol), after growing to the logarithmic phase, add IPTG at a final concentration of 1mM, and culture overnight at 28°C.

[0049] (2) The crude antibody was obtained by infiltration method, and the antibody was transferred to an antigen-coated ELISA plate, and left at room temperature for 1 hour.

[0050] (3) Unbound antibodies were washed away with PBST, and mouse anti-HA tag antibody (anti-mouse anti-HA antibody, purchased from Beijing Kangwei Century Biotechnology Co., Ltd.) was added, and left at room temperature for 1 hour.

[0051] (4) Unbound antibodies were washed away ...

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Abstract

The invention discloses a single-domain antibody, and particularly relates to a single-domain antibody for a CD47 extra-cellular fragment. An amino acid sequence is indicated as SEQ ID NO.9. The single-domain antibody consists of 4 framework areas and 3 complementary decision areas, the amino acid sequence of the four framework areas is indicated as SEQ ID NO. 1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, the amino acid sequence of the three complementary decision areas is respectively indicated as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7. The invention discloses nucleic acid for encoding the single-domain antibody, an expression carrier comprising the nucleic acid and a host cell comprising the expression carrier. The single-domain antibody for the CD47 can be specifically combined with CD47 and is wide in application prospect for researching the CD47 curative antibody medicine.

Description

technical field [0001] The invention relates to a single-domain antibody against CD47, specifically designs a camel-derived single-domain antibody against the extracellular segment of the CD47 molecule, and belongs to the technical field of nanobodies. Background technique [0002] CD47 is a membrane glycoprotein widely expressed in multiple species and various tissues. It interacts with inhibitory receptor signal regulatory protein a as a receptor and ligand, and can form a CD47-SIRPa signaling complex. The complex has the function of mediating bidirectional signal regulation and regulating the process of various immune responses. On normal hematopoietic stem cells (hematopoietic stem cells, HSCs), the significance of CD47 expression is to maintain its relative stability in the body. In the study of malignant diseases such as leukemia, non-Hodgkin's lymphoma, bladder cancer and breast cancer, it was found that tumor cells had elevated CD47, and high expression of CD47 indi...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N15/70C12N1/21G01N33/68A61K39/395A61P35/00A61P35/02
CPCA61K39/395C07K16/28C12N15/63G01N33/53
Inventor 万亚坤孟红卞忠华
Owner JIANGSU CHUNSHENTANG PHARMA
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