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Frizzled7 targeted humanized antibody and preparation method and application thereof

A humanized antibody, targeting technology, applied in biochemical equipment and methods, chemical instruments and methods, botanical equipment and methods, etc., can solve the problems of reducing the affinity and loss of the original antibody

Active Publication Date: 2020-05-12
SHANGHAI UNIV OF MEDICINE & HEALTH SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, simple CDR transplantation tends to lose or reduce the affinity of the original antibody (Jones PT et al., 1986, Nature, 321:522-5; Al-Lazikani B et al., 1997, J MolBiol, 273:927-48 )

Method used

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  • Frizzled7 targeted humanized antibody and preparation method and application thereof
  • Frizzled7 targeted humanized antibody and preparation method and application thereof
  • Frizzled7 targeted humanized antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1: Preparation of anti-Fzd7 monoclonal antibody hybridoma cells

[0068] 1. Immune animals: the immunized animals used in the present invention are BALB / c mice, which are provided by Shanghai First People's Hospital (Songjiang) Animal Experiment Center, the immune adjuvant is purchased from Sigma, and the immunogen is recombinant human Fzd7 extracellular CRD domain protein (rhFzd7). BALB / c mice were immunized by conventional immunization methods. Immunization was performed every four weeks after primary immunization, and three times in total. The antibody titer of immunized mouse serum was detected by indirect ELISA method, and the mouse with the highest immune titer detection value was selected for booster immunization once, and the spleen of the mouse was taken three days later for cell fusion.

[0069] 2. Cell fusion:

[0070] 1) Preparation of myeloma cells (Sp2 / 0): resuscitate Sp2 / 0 two weeks before fusion and place in 5% CO 2 , Subculture in a const...

Embodiment 2

[0076] Example 2: Preparation and identification of mouse-derived anti-Fzd7 antibody

[0077] 1. Ascites collection: After the hybridoma cells were expanded and cultured, the mice were injected intraperitoneally. After 7-10 days, the abdominal cavity of the mice was obviously enlarged, and the ascites was collected at this time. Centrifuge at 5000g for 20min at 4°C to remove cell debris and oil in ascites (Western blot detection of the combination of four strains of ascites and antigen, figure 1 The results showed that 1#, 2#, 3# ascites were better combined with the antigen rhFzd7), and then an equal volume of glycerol was added and stored at -20°C.

[0078] 2. Purification of mouse anti-Fzd7 antibody: Purify the antibody with Protein G affinity chromatography column.

[0079] Specific steps are as follows:

[0080] 1) Wash the Protein G affinity chromatography column with 10 times the column volume of water.

[0081] 2) Wash the Protein G affinity chromatography column wi...

Embodiment 3

[0100] Example 3: Cloning and sequencing of heavy and light chain V region genes of 1# hybridoma cell line

[0101] 1. Extraction, amplification and preliminary identification of heavy and light chain V region genes of anti-Fzd7 monoclonal antibody SHH002

[0102] 1) Extraction of total RNA: Collect hybridoma cells in logarithmic growth phase, extract total RNA with RNA extraction kit (Shanghai Sangon Biotech), dissolve in 20-50 μL RNase-free water, and store at -70°C.

[0103]2) cDNA synthesis: the first strand of cDNA was synthesized by reverse transcription using total RNA as a template (the reverse transcription kit was purchased from Shanghai Sangon Biotech).

[0104] 3) PCR amplification of antibody heavy and light chain V region genes:

[0105] A. Polymerase: PrimerSTAR high-fidelity polymerase

[0106] B. Primers: degenerate primers designed according to the upstream and downstream gene sequences of the heavy and light chain V regions of the hybridoma cell strain ant...

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Abstract

The invention relates to a Frizzled7 targeted humanized antibody and a preparation method and application thereof. The Frizzled7 targeted humanized antibody has a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQID No.11, and the amino acid sequence of the light chain variable region is as shown in SEQID No.12. The Frizzled7 targeted humanized antibody SHH002-hu1 disclosed by the invention reserves high affinity (the KD values of a mouse-derived monoclonal antibody SHH002 and a recombinant humanFzd7 protein are both smaller than 1*10<-12>M) of the mouse-derived monoclonal antibody SHH002 and the recombinant human Fzd7 protein, is specifically combined with Fzd7, and does not have cross reactions with other receptors in a Fzd family, so that the target missing risk of the antibody in possible clinical application is reduced. The SHH002-hu1 can be combined with Fzd7 on the surfaces of tumor cells to block a Wnt signal channel, can be effectively combined with Fzd7 in tumor tissue of a patient, has potential tumor targeting properties, and is a new candidate medicine for anti-tumor treatment in the future.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a humanized antibody targeting Frizzled7 and its preparation method and application. Background technique [0002] The Wnt signaling pathway is a highly conserved signaling pathway in the evolution of species, which plays a vital role in the early development of animal embryos, organ formation and tumor development. The Wnt protein family binds to the N-terminal extracellular domain CRD (cysteine-rich domain) of the receptor Frizzled (Fzd), interacts with Dvl (disheveled) protein and activates downstream signaling pathways. Fzd protein is a kind of Wnt receptor family with seven times transmembrane molecular structure, there are 10 member proteins in this family, Frizzled-7 (Fzd7) is one of them. Studies have shown that Wnt receptor Fzd7 is abnormally expressed in different types of cancer (including triple-negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), liver can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/867C12N5/10G01N33/68A61K39/395A61K47/68A61P35/00
CPCA61K51/103A61K47/6849A61P35/00C07K16/2863C07K2317/24C07K2317/33C07K2317/56C07K2317/76C07K2317/92
Inventor 解伟黄钢王进张坤驰周兆丽王风仙张艳赵慧杰黄燕娟孙吉
Owner SHANGHAI UNIV OF MEDICINE & HEALTH SCI
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