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Tumour treatment vaccine CTP37CRM197 immunogen, preparation method and application thereof

A technology of CTP37CRM197 and CRM197, applied in the field of medicine, can solve the problems of not reaching the theoretical cross-linking rate and the difference in the cross-linking rate of sugars

Inactive Publication Date: 2008-12-03
QILU PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From the experimental results, although the chemical cross-linking rate is high, it has not reached the theoretical cross-linking rate, and the cross-linking rate of different sugars is also different, which shows that the cross-linking rate is also related to the cross-linking product, see: Francisco Betty, Paul Crestanti, Mike Fletcher, Croceo Ruchiraz. Water Solubility, Sedimentability and Kinetic Characteristics of Polysaccharide-Protein Crosslinked Vaccines

Method used

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  • Tumour treatment vaccine CTP37CRM197 immunogen, preparation method and application thereof
  • Tumour treatment vaccine CTP37CRM197 immunogen, preparation method and application thereof
  • Tumour treatment vaccine CTP37CRM197 immunogen, preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] 1. Materials used

[0104] 1.1 Main reagents

[0105] Monohydrate cysteine ​​hydrochloride (Cysteine ​​Hydrochloride Monohydrate), sulfhydryl group determination reagent Ellman reagent, cross-linking agent EMCS: product of Sigma company; Fluorescamine: product of Sigma company;

[0106] Others are domestic reagents.

[0107] 1.2 Main instruments and equipment

[0108] TGL-16 high-speed desktop centrifuge: product of Shanghai No. 6 Medical Device Factory.

[0109] Micropipettes: products of Eppendof, Germany.

[0110] HD95-1 protein detector and 3057 recorder: (Shanghai Kanghua Biochemical Instrument Factory). Peristaltic pump: Lange pump, YZ1515, Baoding Lange Constant Flow Pump Co., Ltd. BHW-IV electric heating constant temperature water temperature box: Beijing Medical Equipment Factory. J2-MC high-speed low-temperature centrifuge: Beckman company products. Ice machine (GRANT), 1-15 centrifuge (Sigma), Spectrumlad54 UV-Vis spectrophotometer (Shanghai Prism Tech...

Embodiment 2

[0137] 1. In vitro tumor inhibition experiment

[0138] 1.1 Anti-CTP37CRM197 antibody prepared by the present invention inhibits the growth of tumor cells

[0139] Add the antibody prepared in Example 1 to well-growing tumor cells, and measure the cell viability of different treatment groups (adding CTP37 group, adding antibody group, blank control group) by MTT method to determine the inhibitory effect of the antibody on tumor cells . The selected tumor cells are gastric cancer cell MKN45 and colorectal cancer cell SW480.

[0140] Tumor cell lines were cultured in vitro, and sterile CTP37 and CTP37 antibodies were added to 10% calf serum RPMI1640 culture medium, and divided into three groups: blank control group, CTP37 group (25ug / m1) and antibody group (30ug / m1).

[0141] Determination of cell viability: use thiazolium blue (MTT) colorimetric assay to measure cell viability, take 100ul of cell-containing culture fluid (cell concentration is 10 5 / m1), inoculated in 96-wel...

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Abstract

The invention relates to an immunogen and the preparation method thereof. The preparation method comprises the steps as follows: cross-linking peptide (hCG-CTP37) composed of amino acid molecules of Beta subunit C-terminal 37 of human chorionic gonadotrophin hormone in the presence of heterobifunctional cross-linking agent N-(Epsilon-maleimidocaproyloxy)succinimidyl (EMCS) using diphtheria toxin mutant CRM197 as carrier protein to obtain CTP37CRM197. The carrier protein has the advantages of high recovery rate, easy purification, and suitability for large-scale industrial production. The cross-linked product has the advantages of high cross-linking rate, short preparation time and reduced production cost. The invention also relates to an application of drug adopting appropriate immunoadjuvant as adjuvant in tumor therapy by employing the immunogen prepared by the method as effective component of therapeutic vaccine.

Description

(1) Technical field [0001] The invention relates to an immunogenic composition CTP37CRM197 using diphtheria toxin mutant CRM197 as an immune carrier and a preparation method thereof, as well as the application of the immunogen in the treatment of digestive tract tumors, belonging to the technical field of medicine. (2) Background technology [0002] CRM197 (cross-reacting materials 197) is a mutant that loses the toxicity of diphtheria toxin, see Wu Qida et al., Diphtheria toxin and related proteins I. Isolation and description of diphtheria toxin serotype-related mutant strains, Biochemistry, 1973.248 : 3838-3844. (Uchida, T., A.M.Pappenheimer, Jr.and R.Gregory.al., Diphtheria toxin and related proteins I.Isolation and properties of mutantproteins serologically related to diphtheria toxin.J.Biol.Chem.1973.248: 3838-3844.), it is the mutation of a base G to A in the base sequence of the wild-type diphtheria toxin, which leads to the mutation of the 52nd amino acid GLY to GLU...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K39/02A61P35/00
Inventor 王栋海李磊王晶翼
Owner QILU PHARMA
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