EGFRvIII antibody, conjugate thereof, preparation method and application

An antibody, the technology of SEQ ID NO. 184, applied in the field of biomedicine, can solve the problem of lack of EGFRvIII antibody and other problems

Inactive Publication Date: 2019-06-21
KAIHUI SCI & TECH DEV SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The technical problem to be solved by the present invention is to provide a high-affinity and specific EGFRvIII antibody and its preparation method and application in order to overcome the current deficiency of EGFRvIII antibody

Method used

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  • EGFRvIII antibody, conjugate thereof, preparation method and application
  • EGFRvIII antibody, conjugate thereof, preparation method and application
  • EGFRvIII antibody, conjugate thereof, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1E

[0237] The preparation of embodiment 1 EGFRvIII antibody

[0238] (1), preparation of immunogen A

[0239] The nucleotide sequence containing the amino acid sequence encoding the extracellular region of human EGFRvIII protein (NCBI: NP_005219.2, missing 30-297 amino acids) was cloned into the pCpC vector with human IgG Fc fragment (hFc) (purchased from Invitrogen, V044 -50) and prepare the plasmid according to the established standard molecular biology method. For specific methods, refer to Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). HEK293 cells (purchased from Invitrogen) were transiently transfected (PEI, Polysciences) and used FreeStyle TM293 (Invitrogen) was expanded at 37°C. After 4 days, the cell culture fluid was collected, and the cell components were removed by centrifugation to obtain the culture supernatant containing the extracellular dom...

Embodiment 2

[0261] Example 2 Production and Purification of Lead Antibody

[0262] The concentration of antibodies produced by hybridoma cells is low, only about 1-10 μg / ml, and the concentration varies greatly. Moreover, various proteins produced by cell culture in the culture medium and fetal calf serum components contained in the culture medium have varying degrees of interference with many biological activity analysis methods, so small-scale (1-5 mg) antibody production and purification are required.

[0263] The hybridoma cells obtained in Example 1 were inoculated into T-75 cell culture flasks and acclimatized and passaged for 3 generations with a production medium (Hybridomaserum free medium, purchased from Invitrogen). When the growth state is good, inoculate the cell culture spinner bottle. Add 200 ml of production medium to each 2-liter culture spinner bottle, and inoculate the cell density at 1.0╳10 5 / ml. Close the bottle cap tightly, and place the spinner bottle on a spinne...

Embodiment 3

[0268] The assay of embodiment 3 leading antibody

[0269] A. Enzyme-linked immunosorbent assay (ELISA) to detect the binding of antibody to EGFRvIII protein

[0270] The purified EGFRvIII antibody obtained in Example 2 was reacted with human EGFRvIII-hFc protein.

[0271] Dilute the purified immunogen A (EGFRvIII-hFc) obtained in Example 1 (for its preparation method, see Step (1) of Example 1) with PBS to a final concentration of 1.0 μg / mL, and then add 100 μl per well to a 96-well ELISA plate .Seal with plastic film and incubate overnight at 4°C, wash the plate twice with plate washing solution [PBS+0.01% (v / v) Tween20] the next day, add blocking solution [PBS+0.01% (v / v) Tween20+ 1% (w / w) BSA] blocked at room temperature for 2 hours. Pour off the blocking solution, add the purified EGFRvIII antibody 100 μl per well obtained in Example 2. After incubation for 2 hours at 37° C., wash the plate with plate washing solution [PBS+0.01% ( v / v) Tween20] wash the plate 3 times. A...

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Abstract

The invention discloses an EGFRvIII antibody, a conjugate thereof, a preparation method and application. The antibody comprises complementarity determining regions (CDRs) of the EGFRvIII antibody: oneor more of a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and / or one or more of a light chain CDR1, a light chain CDR2 and a light chain CDR3, the amino acid sequences of antibody are described herein. The EGFRvIII antibody has high affinity with an EGFRvIII protein, can enter a cell after being coupled with a small molecule toxin such as MMAE, and performs cytotoxic killing effect on EGFRvIII positive cells. Therefore, the EGFRvIII antibody can be applied to preparation of medicines for treating tumors and the like.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an EGFRvIII antibody and its conjugate, preparation method and application. Background technique [0002] Human epidermal growth factor receptor (EGFR, human epidermal growth factor receptor, also known as her-1 or Erb-B1) is a 170kDa transmembrane protein receptor, encoded by the proto-oncogene c-erbB, and the inner region of the membrane has a casein Amino acid kinase activity (Modjtahedi et al., Br. J. Cancer 73:228-235, 1996; Herbst and Shin, Cancer 94:1593-1611, 2002). The full-length sequence of EGFR is numbered P00533 in the SwissProt database. EGFR regulates a variety of cellular physiological processes through signal transduction pathways mediated by tyrosine kinases, mainly including cell proliferation and differentiation, cell survival and apoptosis, angiogenesis, and cell mitosis and cell metastasis (Atalay et al., Ann .Oncology14:1346-1363, 2003; Tsao and Herbst, Signal ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K47/68A61K38/08A61K39/395A61P35/00G01N33/68
CPCA61P35/00G01N33/57407G01N2333/71A61K38/00A61K47/6851C07K16/2863C07K2317/24C07K2317/56C07K2317/565C07K2317/73C07K2317/34C07K2317/92A61K47/68031A61K47/6889A61K47/6803A61K47/6849A61K2039/505C07K2317/33C07K2317/51C07K2317/515G01N33/57492
Inventor 张莹杨翠青刘思琪张瑜王荔娜刘礼乐
Owner KAIHUI SCI & TECH DEV SHANGHAI
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