Cd47-car-t cells

A host cell and fusion protein technology, applied in the field of adoptive immune gene therapy of tumors, can solve the problems of high recurrence rate and low safety

Active Publication Date: 2018-08-21
GRACELL BIOTECH SHANGHAI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there are still many shortcomings in the current research on chimeric antigen receptors, and there are still problems such as high recurrence rate and low safety.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0148] Example 1 Production of CAR lentivirus

[0149] Lentiviruses were prepared by the following steps:

[0150] Day 1:

[0151] 1. Place 5×10 6 HEK293FT cells were seeded into 100 mm diameter petri dishes;

[0152] Day 2:

[0153] 2. Check to make sure the cells are 70%-90% confluent;

[0154] 3. Prepare the transfection complex for each 100 mm diameter Petri dish as follows:

[0155] a. In 1.5ml tube A: dilute 2.5μg CAR (chimeric antigen receptor) DNA plasmid (plasmid) and 20μL lentiviral packaging mix (ALSTEM, catalog number VP100; see Appendix B3) to 0.5ml DMEM or Opti -MEM serum-free medium, mix gently;

[0156] b. In 1.5ml tube B: Dilute 30μL Nanofect Transfection Reagent (ALSTEM, catalog number NF100) into 0.5ml DMEM or Opti-MEM serum-free medium, mix gently;

[0157] c. Add the NanoFect / DMEM in tube B to the DNA / DMEM solution (tube A), vortex for 5-10 seconds, and incubate the DMEM-plasmid-NanoFect mixture at room temperature for 15 minutes;

[0158] 4. Add a...

Embodiment 2

[0169] Example 2 lentiviral packaging system

[0170] Product Description

[0171] Product name: SuperLenti TM Lentivirus Packaging System

[0172] manual:

[0173] For the production of lentiviral particles, three components are generally required: 1) a lentiviral vector containing the foreign gene of interest, 2) a packaging vector containing all the necessary viral structural proteins, 3) expressing vesicular stomatitis virus (VSV) sugars Envelope carrier for protein (G). The third-generation lentiviral packaging system provides maximum biosafety because the lentiviral Rev gene is provided as a separate vector from other structural genes, further eliminating the possibility of reverse recombination of the vector into replication-competent virus particles. Third-generation lentiviral packaging mixes only support lentiviral expression vectors with chimeric 5’LTRs, where the HIV promoter is replaced by CMV or RSV, thus making it independent of TAT.

[0174] The SuperLent...

Embodiment 3

[0187] Example 3 Isolation of Peripheral Blood Mononuclear Cells (PBMC) in Whole Blood

[0188] Whole blood (Stanford University Hospital Blood Center, Stanford, CA) was collected from a single individual or multiple individuals (depending on the volume of blood required) and placed in 10 mL Heparin vacutainers (Becton Dickinson Company).

[0189] NOTE: Blood should be processed within two hours of blood collection to ensure maximum cell yield. Blood can be stored overnight at room temperature (indoors) for processing the next day; however, there will be some loss in cell yield. Blood should not be stored in empty tubes at 4°C. In a 50ml conical centrifuge tube, mix approximately 10ml of anticoagulated whole blood with sterile phosphate buffered saline (PBS) for a total volume of 20ml (PBS, pH 7 / 4, Ca-free 2+ / Mg 2+ ).

[0190] In a separate, sterile 50 mL conical centrifuge tube, pipette into 15 mL of Ficoll-Paque PLUS (GE Healthcare, 17-1440-03). Very gently layer 20ml ...

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PUM

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Abstract

The present invention provides a chimeric antigen receptor (CAR) fusion protein comprising from N-terminus to C-terminus: (i) a single-chain variable fragment (scFv) comprising VH and VL, wherein scFvhas an activity against CD47, (ii) a transmembrane domain, (iii) at least one co-stimulatory domains, and (iv) an activating domain. In one embodiment, the scFv is derived from a humanized anti-CD47antibody. The present invention also provides T cells modified to express the CAR of the present invention.

Description

technical field [0001] The present invention relates to the field of adoptive immune gene therapy for tumors, and more specifically relates to a CD47CAR-T cell, which overexpresses CD47 tumor antigen and effectively attacks tumor cells. Background technique [0002] Immunotherapy is an emerging and very promising approach to treating cancer. T cells, or T lymphocytes, are the immune system's potent armaments that continually search for foreign antigens and distinguish abnormal cells, such as cancer or infected cells, from normal cells. Genetically modified chimeric antigen receptor T cells (CAR-T) are a common approach to engineer tumor-specific T cells. The infusion of CAR-T cells targeting tumor-associated antigens (TAAs) into patients (termed adoptive cell transfer or ACT) represents an effective immunotherapy approach. The advantage of CAR-T technology over chemotherapy or antibody technology is that the reprogrammed engineered T cells can proliferate and persist in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17C12N15/86C07K14/7051C07K14/70521C07K16/2803C07K2317/24C07K2317/622C07K2319/03C07K2319/33C12N2740/15043
Inventor 曹卫
Owner GRACELL BIOTECH SHANGHAI CO LTD
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