PD-L1 targeting immunotoxin as well as preparation method and application thereof

A PD-L1, immunotoxin technology, applied in the field of immunotoxin targeting PD-L1 and its preparation, can solve problems such as disease progression or recurrence

Pending Publication Date: 2022-01-11
FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, after an initial response to PD-1 / PD-L1 blockade, most patients eventually develop acquired resistance over time, leading to disease progression or relapse

Method used

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  • PD-L1 targeting immunotoxin as well as preparation method and application thereof
  • PD-L1 targeting immunotoxin as well as preparation method and application thereof
  • PD-L1 targeting immunotoxin as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Fluorescence-labeled Durvalumab tracks binding to PD-L1 and its internalization process

[0016] Method: The human lung cancer cell line PD-L1 / SPC-A-1 transfected with PD-L1 and the parental cell line SPC-A-1 cell line (named NC / SPC-A-1 in this specification) in logarithmic growth phase cell line). After digestion and counting, dilute to 5×10 with RPMI1640 complete medium containing 10% FBS 4 cells / ml, add to confocal special glass culture dish, add 1ml of cells per well, place at 37°C, 5% CO 2 Cultivate in an incubator for 24 hours, suck away the cell culture medium in the culture dish, and use RPMI 1640 to put the CUS 245C -FITC and Durvalumab-FITC were diluted to 5ng / ml to treat the cells. After the cells were treated for 1h, 2h, 6h, 12h and 24h, the liquid in the well was discarded, and the internalization was terminated with pre-cooled PBS. Discard the PBS in the well, and wash with pre-cooled PBS for 3 times, dilute Hoechst into 1× working solution a...

Embodiment 2

[0019] Example 2: Durvalumab and pumpkin protein mutant CUS 245C Coupling to prepare immunotoxin D-CUS 245C

[0020] Method: Take 2ml of Durvalumab (5mg / ml), dialyze with a dialysis tube with a molecular weight cut-off of 8-10kDa, and change the PBS dialysate every 3 hours (the amount of PBS is 1L each time), a total of 5 times; after dialysis, 16.45μl 20mmol / L SPDP solution (dissolved in DMSO), stirred at room temperature for 30 min, and the reaction product (D-PDP) was dialyzed with the above buffer solution overnight to remove excess SPDP. Take another 3ml CUS 245C (a total of 7.45mg), add an appropriate amount of dithiothreitol (DTT) dissolved in 0.01mol / L NaAc, so that the final concentration of DTT is 0.3mol / L, stir at room temperature for 30min, and perform dialysis with a 8-10kDa dialysis column. The method is the same as above, remove excess DTT; mix the above-mentioned Durvalumab after dialysis with CUS 245CMix in a small beaker, stir and react at 23°C for 18 hou...

Embodiment 3

[0023] Example 3: Immunotoxin D-CUS 245C In vitro antitumor activity assay

[0024] Methods: The PD-L1 overexpressing human breast cancer cell line MDA-MB-231 cells in the logarithmic growth phase and the PD-L1 transfected human lung cancer cell lines PD-L1 / SPC-A-1 and PD-L1 low The expressed lung cancer cell line NC / SPC-A-1 was inoculated on a 96-well culture plate, and the inoculation volume in each well was 4000 / 100 μL. After culturing for 24 h, the experimental group was added different concentrations of immunotoxin (D-CUS 245C ), squash mutant (CUS 245C ), Durvalumab (D) and the mixture of pumpkin protein and Durvalumab of corresponding concentration (D+CUS 245C ) 100 μL, and three replicate wells were made for each concentration; 100 μL of nutrient solution was added to the negative control group, and 200 μL of nutrient solution was added to the blank control wells for zero adjustment of the instrument. After 72 h or 120 h of action, discard the supernatant, add 100 ...

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Abstract

The invention relates to PD-L1 targeting immunotoxin as well as a preparation method and application thereof. The immunotoxin comprises a carrier and toxic molecules. The carrier is an antibody or other polypeptides capable of being combined with PD-L1 molecules; the toxic molecules are bacterial toxins, plant toxins and human-derived toxic molecules, and mutants of these toxins. The immunotoxin is formed by coupling the toxic molecule with a carrier capable of being combined with a tumor cell PD-L1 through a chemical cross-linking method, or the immunotoxin is expressed by fusing genes of the toxic molecule and the carrier through a gene engineering method, and the immunotoxin can be used for treating tumors. According to the invention, the chemical coupling immunotoxin D-CUS245C based on the cucurmosin mutant CUS245C and the PD-L1 monoclonal antibody Durvamab is successfully prepared; the immunotoxin has a remarkable targeted killing effect on PD-L1 overexpressed tumor cells, and the maximum targeted therapeutic index can reach 1,100,000 or above. Therefore, the PD-L1 targeting immunotoxin has a wide anti-tumor application prospect.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an immunotoxin targeting PD-L1 and its preparation method and application. Background technique [0002] Immunotoxins are specific cell-specific molecules that are coupled with carrier molecules (such as antibodies, cytokines, etc.) The hybrid molecule with killing ability, through the guiding effect of the carrier, the toxic molecule carried by it reaches the target cell, and then inhibits protein synthesis in the cell to cause cell death, and achieves the effect of specifically killing the target cell without damaging normal tissues. [0003] Programmed cell death-ligand 1 (PD-L1) is a type I transmembrane protein of the B7 family, expressed on the surface of various cancer cells and some immune cells. After PD-L1 on cancer cells binds to programmed cell death-1 (PD-1) on T cells, it can lead to T cell dysfunction, exhaustion and apoptosis, thereby inhibiting T cell mediation. Indu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/22C07K1/34C07K1/36A61K47/68A61K38/16A61P35/00
CPCC07K14/415C07K16/2827A61P35/00C07K2319/00A61K38/00
Inventor 谢捷明张彩云
Owner FUJIAN MEDICAL UNIV
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