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Humanized anti-human-interleukin-1[belta] monoclonal antibody, preparation thereof and applications thereof

A monoclonal antibody, interleukin technology, applied in anti-cytokine/lymphokine/interferon immunoglobulin, application, antibody and other directions, can solve the problems of low activity, low yield, decreased antibody affinity, etc. Biological activity, high biological activity, high affinity effect

Inactive Publication Date: 2014-02-19
JIANGSU T MAB BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, such antibodies often encounter problems such as decreased affinity, low activity, and low yield.

Method used

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  • Humanized anti-human-interleukin-1[belta] monoclonal antibody, preparation thereof and applications thereof
  • Humanized anti-human-interleukin-1[belta] monoclonal antibody, preparation thereof and applications thereof
  • Humanized anti-human-interleukin-1[belta] monoclonal antibody, preparation thereof and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Preparation of hIL-1β monoclonal antibody

[0054]1) Preparation of hybridoma cells: Rabbits were immunized with recombinant hIL-1β (rhIL-1β). After the rabbits produced a specific immune response, the spleen cells of the immunized rabbits were immortalized with the rabbits according to the method described in US Patent No. 7,429,487. Chem cells 240E-W2 were fused together. After fusion, cells are cultured in a medium containing hypoxanthine, aminopterin, and thymidine (HAT), selected for hybridoma growth, and after 2 to 3 weeks, hybridoma cells begin to appear colony.

[0055] 2) Screening of hybridoma cells: Use enzyme-linked immunosorbent assay (ELISA) to analyze the culture medium growing hybridoma cells to detect whether monoclonal antibodies against antigens are produced. Coat the 96-well plate with 20 μg / ml hIL-1β, add the culture supernatant to be tested, use the culture medium as the negative control, and the corresponding immune rabbit serum as t...

Embodiment 2

[0060] Example 2 Purification of hIL-1β monoclonal antibody

[0061] 1. Mabselect (Protein A) affinity chromatography

[0062] 1) Solution preparation:

[0063] Equilibration buffer A: 20mM PB, 0.15M NaCl, pH7.0

[0064] Elution buffer B: 20mM sodium citrate-Na 2 HPO 4 , pH3.2

[0065] 2) Purification

[0066] After equilibrating with the equilibration buffer, the sample was loaded on the Protein A affinity chromatography column, after re-equilibration, eluted with 100% elution buffer, and the eluted peaks were collected.

[0067] 2 SP Fastflow Cation Exchange Chromatography

[0068] 1) Solution preparation

[0069] Equilibration buffer A: 15mM PB, pH6.0

[0070] Elution buffer B: 20mM PB, pH7.2

[0071] 2) Purification

[0072] Use 1M Tris-HCl pH 9.0 to adjust the pH to 6.0 for the eluted peak obtained in the first step of purification. Liquid A balances the filler and loads the sample. 100% B is eluted and the eluted peak is collected.

[0073] 3 Q Fastflow A...

Embodiment 3

[0083] Example 3 Affinity Determination of hIL-1β Monoclonal Antibody

[0084] The binding affinity of hIL-1β monoclonal antibody was determined by Fortebio Octet. The antigen rhIL-1β was first biotinylated and then desalted on AKTA with Coarse G25. The first peak was collected about 1.68ml, and the converted concentration was about 0.15mg / ml. The concentration of hIL-1β monoclonal antibody is 1.8mg / ml, diluted to 40nM with PBS buffer, then diluted sequentially by 2 times, so that the concentration is 20, 10, 5, 2.5, 1.25, 0nM, used according to Fortebio Octet QK The determination of the affinity constant was carried out according to the standard, and a total of 2 determinations were made (see the attached image 3 ). The results are shown in Table 1, the affinity constant of hIL-1β monoclonal antibody is between about 10 pM and about 25 pM.

[0085] Table 1 Dissociation equilibrium constant (KD) of hIL-1β monoclonal antibody

[0086] Testing frequency KD...

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Abstract

The invention relates to a humanized anti-human-interleukin-1[belta] monoclonal antibody, preparation thereof and applications thereof. The light chain of the monoclonal antibody has an amino acid sequence shown as SEQIDNO:1, and the heavy chain of the monoclonal antibody has an amino acid sequence shown as SEQIDNO:2. The antibody can be used for treating various diseases such as acute gout, rheumatoid arthritis, II-type diabetes, non-infectious uveitis, etc. which are related to interleukin 1[belta] pathological functions.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a humanized anti-human interleukin-1 beta monoclonal antibody and its preparation and application. Background technique [0002] Human interleukin-1β (hIL-1β) is a pro-inflammatory cytokine secreted by different types of cells such as monocytes and macrophages, which mainly stimulates such as adrenocorticotropic hormone, platelet factor-4, prostaglandin E2 , interleukin-6 and interleukin-8 and other inflammatory regulators to trigger a series of biological effects. hIL-1β can stimulate local or systemic inflammatory responses by activating the hIL-1 receptor on any type of cell. [0003] hIL-1 family cytokines are associated with many diseases (Dinarello CA, N Engl J Med (2009) 360:2467–70), family members include hIL-1α, hIL-1β and hIL-1Ra, among which hIL-1β is the main , is a cytokine that plays a key role in the inflammatory response. In the central nervous system, hIL-1 is re...

Claims

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Application Information

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IPC IPC(8): C07K16/24C12N15/13C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12Q1/68G01N33/577A61K39/395A61P19/02A61P29/00A61P27/02A61P19/06A61P3/10
Inventor 裘霁宛郭箭吴亦亮于东安田静黄岩山
Owner JIANGSU T MAB BIOPHARMA
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