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CAR-T CELL-BASED IMMUNOTHERAPY DEVELOPMENT OF CAR-T CELLS

In the CAR-T cell constructing processes, the first step is to eukaryotically express the target antigen. Mice are vaccinated with the antigen, and the B cells are collected to generate hybridomas with myeloma cells using cell fusion technology. After several rounds of antigen-specific screening, hybridomas with the highest affinity with the target antigen are collected, and the total RNA is isolated and reverse transcribed for sequence analysis. Sequences of the antibody variable region are cloned into CAR vectors, including lentivirus, retrovirus, and transposome systems. Total white cells are collected from the patient’s blood by pheresis methods and separated with flow sorting to obtain the CD4 and CD8 T cells, which are then in vitro matured and expanded. After infection or transfection, the CARs are expressed in the T cells to finally generate the CAR-T cells.

Antigen gene

Creative Biolabs CAR/TCR-related Products & Services

VH & VL

CAR vectors

Immunize mouse

Tumor antigen

Expression vector

DNA

B cells

IL-2, IL-7, IL-15, IL-21

Transduction with CAR

Hybridomas

RNA

Screening CD4&CD8 T cells

+/–

Expansion

Beads or artificial APCs

Flow sorting column separation

Pheresis CAR-T cells

Quality control

Total white cells

Return to patient

Vector Systems for CAR Engineering

2

Lentivirus System Transfer plasmid of 1G and 2G

5’LTR U3

R

U5

Prom.

Ψ/RRE

Envelope plasmid of 1G and 2G

3’LTR

CAR

U3

R

gag

pol

vif

vpr

vpu

gag

5’LTR with no U3 Prom.

R

pol

Transfer plasmid of 3G

Ψ/RRE

U5

Prom.

rev tat RRE rev tat RRE

Packaging plasmid of 2G Prom.

Prom.

U5

Packaging plasmid of 1G Prom.

Lentivirus is HIV-1 based and the most mature system for T cell engineering. The first (1G) and second (2G) generation lentivirus systems both consist of three parts: transfer, envelope, and packaging plasmids. A regulator plasmid, which regulates viral transcription and nuclear export of transcripts, is supplemented to form the third generation. Gamma retrovirus system is quite similar to lentivirus except for few elements including promoters. Transposons are dual component systems composed of one plasmid carrying the CAR (transposon) and the other carrying the transposase.

Flow Chat of CAR-T cells generation

1

R

gag

pol

Prom.

U3

R

RRE

Ψ

Donor plasmid gag

pol

CAR

env

pA

Prom.

pA

rev

pA

Transposome System

Transfer plasmid U5

pA

Regulatory plasmid of 3G

Retrovirus System 5’LTR

nef

Envelope plasmid of 3G

U5

Packaging plasmid of 3G Prom.

pA

pA

ΔU3 3’LTR

CAR

VSV-G

Donor plasmid

3’LTR U3

R

TIR

U5

Prom.

CAR

TIR

Transposase expression plasmid

Envelope plasmid Enve.

Prom.

Transposase

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CAR T Cell Based Immunotherapy Development of CAR T Cells  

In the CAR-T cell constructing processes, the first step is to eukaryotically express the target antigen. Mice are vaccinated with the anti...

CAR T Cell Based Immunotherapy Development of CAR T Cells  

In the CAR-T cell constructing processes, the first step is to eukaryotically express the target antigen. Mice are vaccinated with the anti...

Profile for car-t
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