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Anti-human Delta like4 monoclonal antibody

A monoclonal antibody, antibody technology, applied in the direction of antibodies, anti-animal/human immunoglobulins, anti-tumor drugs, etc., can solve the problems of non-functional new blood vessels, tumor growth inhibition, etc.

Inactive Publication Date: 2014-05-21
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that D114 / Notch plays an important regulatory role in tumor angiogenesis, and inhibiting D114 / Notch signal transduction in tumor tissue will lead to the formation of a large number of non-functional new blood vessels in tumor tissue, which cannot effectively provide oxygen and oxygen for tumor tissue. Nutrients that ultimately lead to inhibition of tumor growth

Method used

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  • Anti-human Delta like4 monoclonal antibody
  • Anti-human Delta like4 monoclonal antibody
  • Anti-human Delta like4 monoclonal antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1, preparation of anti-hD114 monoclonal antibody:

[0026] (1) Immunized mice

[0027] Recombinant human Delta like4 (rhD114) was used as the immunogen, mixed with an equal volume of quick antibody adjuvant, shaken and mixed, and intramuscularly injected to immunize mice, each mouse was injected with 100 μl. On the 21st day, a dose of booster immunization was given in the same way. On the 35th day, the antigen pulse immunization was carried out according to the conventional method. After 3 days, the mice were sacrificed and the spleens were removed.

[0028] (2) cell fusion

[0029] For the removed spleen, a spleen single cell suspension was prepared by a conventional method, and the cell viability was detected by the trypan blue method, and the cell viability was determined to be >90%. Splenocytes and SP2 / 0 mouse myeloma cells were mixed at a ratio of 5:1, centrifuged and co-sedimented, and PEG (1450) was used for cell fusion at 37°C in a water bath. Fu...

Embodiment 2

[0032] Example 2, Amplification of Anti-hD114 Monoclonal Antibody Variable Region Gene

[0033] According to the conservation of the mouse antibody gene, the following primers were used to clone the variable region sequence of the anti-hD114 monoclonal antibody:

[0034] Primers for amplifying the 5′ end of the heavy chain variable region:

[0035] 1.ctt ccg gaattc SAR GTN MAG CTG SAG SAG TC

[0036] 2.ctt ccg gaattc SAR GTN MAG CTG SAG SAG TCW GG

[0037] Primers for amplifying the 3′ end of the heavy chain variable region:

[0038] gga agatct CTT GAC CAG GCA TCC TAGAGT CA

[0039] Primers for amplifying the 5′ end of the Kappa light chain variable region:

[0040] gg gag ctc GAYATT GTG MTS ACM CAR WCT MCA

[0041] Primers for amplifying the 3′ end of the Kappa light chain variable region:

[0042] ggtgcatgc GGATACAGTTGG TGCAGCATC

[0043] Among the above primers: R=A, G; Y=C, T; M=A, C; K=G, T; S=C, G; W=A, T; V=A, C, G; N= A, C, G.

[0044] Trizol Total RNA Extract...

Embodiment 3

[0051] Implementation example 3, preparation of anti-hD114 monoclonal antibody

[0052] 7 days after sensitization of 8-week-old BALB / c mice with 500 μl paraffin oil, intraperitoneal injection of 10 6 Hybridoma cells in logarithmic growth phase were injected into the peritoneal cavity of mice. One week later, the ascites of the mice was extracted. And measure ascites titer with indirect ELISA method, its titer reaches about 3,000,000, see attached figure 2 .

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Abstract

The invention relates to an anti-human Delta like4 (hD114) monoclonal antibody. The monoclonal antibody or a segment thereof is characterized by being capable of specifically combining with hD114, blocking off the inhibition of D114 on the proliferation of human umbilical vascular endothelial cells (HUVEC), and promoting the proliferation of the blood capillary in a chick embryo allantois membrane model. The invention specifically discloses a screening method and preparation method of the anti-human Delta like4 monoclonal antibody. The invention further discloses nucleotide and amino acid sequences, including nucleotide and amino acid sequences corresponding to complementary determining regions CDR1, CDR2 and CDR3, of heavy chain variable regions and light chain variable regions of the anti-human Delta like4 monoclonal antibody.

Description

technical field [0001] The invention specifically relates to the field of biotechnology, in particular, the invention relates to a novel anti-human Delta like4 monoclonal antibody. Background technique [0002] Since Folkman [1] Since the "tumor angiogenesis-dependent theory" was put forward in 1971, anti-tumor angiogenesis has become one of the important strategies for tumor treatment. At present, the purpose of inhibiting tumor angiogenesis is achieved by blocking vascular endothelial growth factor (VEGF) and its main receptor VEGFR2. [2] . However, clinical studies have found that a variety of tumors have antagonistic effects on VEGF and VEGFR2 inhibitors, and cannot effectively inhibit tumor growth. [3] . [0003] Delta like ligand4 (D114) widely exists in invertebrates and vertebrates. By interacting with its receptor Notch, it activates Notch signal transmission to cells and regulates the expression of downstream target genes, thus participating in the growth, deve...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13A61K39/395A61K48/00A61P35/00
Inventor 王旻王泽根涂孝洁金海珍厉道娟许卓斌张娟吴旻
Owner CHINA PHARM UNIV
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