Fusion protein containing CD40 antibody and IL-15 and preparation method and application of fusion protein

A fusion protein and monoclonal antibody technology, which is applied in the preparation methods of peptides, chemical instruments and methods, and medical preparations containing active ingredients, etc. , to achieve the effect of enhancing tumor targeting, reducing lung metastases, and reducing average

Active Publication Date: 2021-10-08
北京华奥玄德生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there has been no report on the fusion protein of anti-human CD40 antibody and IL-15

Method used

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  • Fusion protein containing CD40 antibody and IL-15 and preparation method and application of fusion protein
  • Fusion protein containing CD40 antibody and IL-15 and preparation method and application of fusion protein
  • Fusion protein containing CD40 antibody and IL-15 and preparation method and application of fusion protein

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Experimental program
Comparison scheme
Effect test

Embodiment approach

[0031] According to a preferred embodiment of the present invention, the monoclonal antibody or its immunologically active fragment capable of binding to CD40 includes a monovalent antibody, a single-chain antibody, a double-chain antibody, a chimeric antibody or their derivatives or homologues, or

[0032] Fragments or Fv fragments of immunoglobulins, or

[0033] Antibody fragments or polypeptides containing antigen binding domains are included.

[0034] In the present invention, the derivatives and homologues refer to proteins that basically maintain the same biological function or activity as the monovalent antibody, single-chain antibody, double-chain antibody, and chimeric antibody. That is, the derivatives and homologues encompass changes in the amino acid sequence that are present in non-essential positions of the corresponding antibodies.

[0035] Wherein, the immunoglobulin fragment is at least an immunologically active segment of the immunoglobulin molecule, such a...

Embodiment 1

[0120] Prepare fusion protein:

[0121] (1) Obtaining the target gene:

[0122] The synthetic plasmid containing the target fragment synthesized by Zhongmei Taihe Biotechnology (Beijing) Co., Ltd. was used as a template, and PCR was carried out according to the primers and templates described in Table 1, the reaction system described in Table 2, and the reaction conditions described in Table 3 Amplify to obtain the corresponding target gene fragment.

[0123] Table 1

[0124]

[0125]

[0126] Table 2

[0127] reaction system volume 10×ExTaq buffer (purchased from Takara Company, product number is RR001A) 5μL dNTP mixture (each 2.5mM) (purchased from Takara Company, item number is RR001A) 4μL Upstream primer (10μM) 2.5μL Downstream primer (10μM) 2.5μL Template (0.1 μg / μL) 1μL ExTaq (purchased from Takara Company, catalog number RR001A, 5U / μL) 0.25 μL h 2 o

Make up to 50μL

[0128] table 3

[0129] ...

Embodiment 2

[0136] The preparation method of the fusion protein adopted in this embodiment is similar to that of Example 1, the only difference being:

[0137] The expression vector constructed in step (2) contains the light chain gene of the monoclonal antibody that can bind to CD40, the heavy chain gene of the monoclonal antibody that can bind to CD40, and the sushi region gene of the receptor alpha of cytokine IL-15 and the fusion gene of IL-15 gene (such as figure 2 Plasmids 1 and 4 shown in ).

[0138] The expressed fusion protein was designated as fusion protein 2.

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Abstract

The invention discloses a bispecific fusion protein containing a cytokine IL-15 and a CD40 antibody or an immunocompetence fragment and a preparation method and application of the bispecific fusion protein. The fusion protein comprises the human CD40 antibody and the cytokine IL-15 with different structural formulas, and is obtained by co-transfection and expression of different plasmids. The fusion protein disclosed by the invention can activate APC cells, T cells and NK cells at the same time, and the tumor targeting property of the cytokine is enhanced; DC cell maturation and IL-12 secretion can be stimulated, the growth of mouse melanoma can be significantly inhibited, and the volume and average pulmonary metastasis of the melanoma can be reduced.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a bispecific fusion protein comprising cytokine IL-15 and CD40 antibody or immunologically active fragments and a preparation method and application thereof. Background technique [0002] CD40 is a 40-50kDa type I transmembrane glycoprotein belonging to the tumor necrosis factor receptor (TNFR) family, which is widely expressed in various antigen-presenting cells (APCs) (such as dendritic cells DCs) and B lymphocytes And other non-immune cells (such as epithelial cells and platelets, etc.), and also expressed on the surface of some tumor cells. CD40 plays an important role in both cellular and humoral immunity. [0003] CD40 can activate APC cells, especially DC cells, after binding to its natural receptor CD40L or activating CD40 antibody. Activated DC cells increase the ability of antigen presentation by up-regulating the expression of other co-stimulat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/22C12N15/62C12N15/85A61K38/20A61K39/395A61P35/00
CPCC07K16/2878C07K14/5443C12N15/85A61K39/3955A61K38/20A61K35/00C07K2319/00
Inventor 段海峰解晶张晶孟文华杨继业
Owner 北京华奥玄德生物医药科技有限公司
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