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Anti-human interleukin-4 receptor alpha monoclonal antibody, preparation method and applications thereof

A monoclonal antibody, humanized technology, applied in the field of its preparation, anti-human interleukin-4 receptor α monoclonal antibody, can solve the problem that patients have no cure

Active Publication Date: 2018-08-17
SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite this, 5%-10% of patients still have no current treatment

Method used

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  • Anti-human interleukin-4 receptor alpha monoclonal antibody, preparation method and applications thereof
  • Anti-human interleukin-4 receptor alpha monoclonal antibody, preparation method and applications thereof
  • Anti-human interleukin-4 receptor alpha monoclonal antibody, preparation method and applications thereof

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preparation example Construction

[0040]The preparation method of the anti-human IL-4Rα monoclonal antibody disclosed in the present invention comprises: cultivating the above-mentioned host cells under expression conditions, thereby expressing the anti-human IL-4Rα monoclonal antibody; isolating and purifying the anti-human IL-4Rα 4Rα monoclonal antibody. Using the above method, the recombinant protein can be purified into a substantially uniform substance, such as a single band on SDS-PAGE electrophoresis.

[0041] The method of affinity chromatography can be used to separate and purify the recombinant protein disclosed in the present invention. According to the characteristics of the used affinity column, conventional methods such as high-salt buffer, changing pH, etc. can be used to elute the protein bound to the affinity column. The fusion protein polypeptide on.

[0042] The humanized anti-human IL-4Rα monoclonal antibody disclosed in the present invention is obtained by the following method: using the ...

Embodiment 1

[0051] Example 1 Preparation of soluble IL-4Rα extracellular segment Fc tag and Flag tag antigen, positive antibody Dupilumab, IL-4, IL-13

[0052] The human IL-4Rα ectoantigen sequence was obtained from UniProt (UniProtKB-P24394). Sangon Biotech Co., Ltd. carried out codon optimization according to the codon usage preference of Cricetulus griseus and synthesized the N-terminal 26-232 amino acid fragment. Cloned into the pUC57 vector (from Sangon Biotechnology Co., Ltd.) to obtain pUC57-hIL4Rα-ECD. The constant region sequence of human IgG1 was synthesized according to the sequence of Secukinumab (see WHO Drug Information Vol. 23, No. 4, 2009. P342). The sequence was subcloned into pUC57 vector (from Sangon Biotechnology Co., Ltd.) to obtain pUC57-IgG1-CH. hIL4Rα-ECD and hFc (human Fc fragment) were spliced ​​by PCR method to obtain hIL4Rα-ECD-hFc fragment. Using pUC57-hIL4Rα-ECD as a template, the Flag tag (DYKDDDDK) was inserted into the C-terminus of the hIL4Rα-ECD fragme...

Embodiment 2

[0059] Example 2 Immunization of hIL-4RαECD-Fc

[0060] 100 μg / mouse hIL-4RαECD-Fc antigen was diluted to 75 μl with normal saline, mixed with an equal volume of Freund’s complete adjuvant, and after complete phacoemulsification, the 4-5 week-old Balb / c mice (purchased Subcutaneous multi-point injection was performed from Shanghai Lingchang Biotechnology Co., Ltd., animal production license number: SCXK (Shanghai) 2013-0018). Three weeks later, 50 μg / mouse protein was also diluted to 75 μl and mixed with an equal volume of Freund’s incomplete adjuvant. After phacoemulsification was complete, the mice were subcutaneously immunized at multiple points, and the immunization was repeated two weeks later. One week after the third immunization, the tails of all mice were cut to collect blood to separate serum, and the serum titer was detected by ELISA coated with hIL-4Rα-Fc antigen. For mice with serum antibody titer > 10,000, pulse immunization was performed one week after blood co...

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Abstract

The present invention belongs to the field of antibodies, and more particularly discloses a recombinant anti-human hIL-4R[alpha] monoclonal antibody, a preparation method and applications thereof. According to the present invention, the anti-human hIL-4R[alpha] monoclonal antibody can specifically bind to human hIL-4R[alpha], can well inhibit hIL-4 and hIL-13 induced proliferation of various celllines and the like, and can be used for preparing drugs for treatment of immune mediated Inflammatory reaction diseases.

Description

technical field [0001] The invention relates to the field of antibodies, and more specifically, the invention discloses an anti-human interleukin-4 receptor alpha monoclonal antibody, its preparation method and application. Background technique [0002] When the body is stimulated by an antigen, the antigen-specific lymphocytes in the body will recognize the antigen and undergo activation, proliferation, differentiation and other responses, and finally clear the invading antigen. T cells and B cells are the main effector cells. For different types of antigens, T cells can exert immune effects by 1) directly killing target cells and 2) secreting different types of cytokines to expand and enhance the immune response. Studies have shown that in allergic diseases such as allergic asthma, Th2-type cytokines such as interlukin (IL)-4, IL-5, IL-9 and IL-13 mediate the main pathological development. [0003] Asthma is the most common respiratory disease, often manifested by airway...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P37/02A61P29/00A61P11/06A61P37/08A61P17/00A61P11/02A61P1/00A61P17/06A61P19/02A61P19/08A61P27/02
CPCC07K16/2866A61K2039/505C07K2317/92C07K2317/565C07K2317/24C07K2317/76
Inventor 张成海党尉朱玲巧
Owner SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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