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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