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Recombinant anti-hgf/dll4 bispecific antibody, its preparation method and application

A bispecific antibody and heavy chain technology, applied in the biological field, can solve the problems of regulatory barriers and high cost in the combined application of monoclonal antibodies

Active Publication Date: 2019-06-14
SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the combined application of monoclonal antibodies has the defects of adjustment disorder and high cost, which limit its clinical application.

Method used

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  • Recombinant anti-hgf/dll4 bispecific antibody, its preparation method and application
  • Recombinant anti-hgf/dll4 bispecific antibody, its preparation method and application
  • Recombinant anti-hgf/dll4 bispecific antibody, its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Gene Construction and Expression of Anti-DLL4 / HGF Bispecific Antibody

[0047] According to the anti-HGF monoclonal antibody Ficlatuzumab (IgG1, κ) sequence published by who.int (see WHO DrugInformation Vol.25, No.2, 2011.p171), compare the constant region sequence analysis of human IgG1 to obtain its VH and VL fragments . At the same time, according to the amino acid sequence of the anti-DLL4 monoclonal antibody Enoticumab (IgG1, κ) published by who.int (see WHO Drug Information Vol.27, No.1, 2013.p55), the nucleotide sequence was synthesized from the whole gene after codon optimization . The VH fragment of Ficlatuzumab was fused to the N-terminal of the Enoticumab heavy chain through the (ASTKGP) linker by Overlapping PCR; the VL fragment was fused to the N-terminal of the Enoticumab light chain through the (TVAAP) linker. Linker fragments were derived from N-terminal sequences of human IgG1 CH1 or CK, respectively. The primers for heavy chain Overlapping...

experiment example 2

[0049] Experimental example 2 Detection of anti-DLL4 / HGF bispecific antibody by SDS-PAGE and Western-blot

[0050] The purified anti-DLL4 / HGF bispecific antibody, Ficlatuzumab, and Enoticumab were tested for their purity and molecular weight by polyacrylamide gel electrophoresis under non-reducing (6%) and reducing (12%) conditions, respectively, and at the same time by Western-blot Further identify its properties and molecular weight. The Western-blot method is as follows: transfer the gel after electrophoresis to PVDF membrane by electrotransfer method, add HRP-labeled goat anti-human IgG (H+L) after blocking, wash twice with PBST, and finally develop color with DAB method. The results of polyacrylamide gel electrophoresis and Western-blot showed that under reducing conditions, both Ficlatuzumab and Enoticumab presented heavy chains and light chains with molecular weights of about 55KDa and 25KDa, while anti-DLL4 / HGF bispecific antibodies presented as The molecular weight i...

Embodiment 3

[0051] Example 3 Identification of the Binding Ability of Anti-DLL4 / HGF Bispecific Antibody to HGF

[0052] The binding of the anti-DLL4 / HGF bispecific antibody to HGF was detected by Biacore3000 (purchased from GE). HGF (purchased from R&D Company) was coated on the Biacore3000 chip at different concentrations, and its affinity was detected, and the parent antibody Ficlatuzumab was used as a control. The results are shown in Table 1. It can be seen that the affinity of the anti-DLL4 / HGF bispecific antibody to HGF is similar to that of the parent antibody Ficlatuzumab.

[0053] Table 1, Affinity of anti-DLL4 / HGF bispecific antibodies to HGF

[0054]

[0055] The binding activity of the anti-DLL4 / HGF bispecific antibody to HGF was detected by the human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cells, HUVEC) proliferation assay. Passage HUVECs with good growth status (purchased from: ATCC company) were inoculated into 96-well plates. After 2 days, 5...

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Abstract

The invention belongs to the biotechnical field and particularly discloses a recombinant anti-HGF / DLL4 bispecific antibody and a preparation method and application thereof. The anti-human HGF / DLL4 bispecific antibody can be combined with both HGF and DLL4. The antibody disclosed by the invention has amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4. The recombinant anti-human HGF / DLL4 bispecific antibody has an excellent anti-tumor effect.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically, the invention discloses a recombinant bispecific antibody, its preparation method and its effect on solid tumors. Background technique [0002] Malignant tumors are a major cause of death in human diseases. In recent years, due to the deterioration of the environment and the high pressure of life, the incidence of malignant tumors in my country has been increasing year by year. The treatment methods for malignant tumors are still mainly radiotherapy, chemotherapy and surgery, and the treatment effect is not satisfactory. With the rise of monoclonal antibody technology, the use of antibodies for biological targeted therapy has shown great room for development due to its incomparable advantages such as less toxic and side effects, and the possibility of specific and complete removal of cancer cells. However, due to the large heterogeneity of tumors, especially the tumor surface ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/46C12N15/13C12N15/85C12N5/10C12P21/02A61K39/395A61P35/00
Inventor 朱玲巧赵杰高宏海洪雪娟邓庭亭
Owner SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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