Breakthrough! CAR-T Upgrade: iPSC can mass-produce general-purpose CAR-T and cure a variety of cancers!

The first patient treated with CAR-T has been rehabilitated for ten years

In 2012, Dr.

Grupp (Figure 2) said: "I'm not just a doctor, I'm also a scientist

CAR-T therapy is hindered by multiple factors and its popularity is limited!

To date, patients treated with CAR-T have used their own donors, while those who have received a large number of treatments sometimes lack a sufficient number of functional T cells available for autology; Moreover, the expansion and engineering of autologous T cells requires waiting, but some patients with severe disease do not have enough time; In addition, CAR-T is expensive, and the price of the two CAR-T therapies listed in China is more than one million (WuXi Juno 1.

Harvard Medical School: Large-scale production of CAR-T using IPSC can cure more patients Previous studies have shown the possibility of using human iPSCs to generate T cells for adoptive T cell therapy, but iPSC-CAR-T

Epigenetic regulators play a key role

The results show that

The researchers first induced human erythrocyte-derived IPSCs (cell line 1157) to form embryoid bodies to produce hematopoietic endothelial (HE) cells; CD34+ HE cells are collected and monolayered on tissue culture dishes coated with Notch Delta-like ligand 4 (DLL4) and VCAM-1; HE cells were cultured with multiple cytokines (SCF, Flt3, IL-7, IL-3, TPO) in SFEMII culture medium to initiate T cell differentiation; During this time, HE cells undergo a transition from endothelial to hematopoietic cells (EHTs), producing floating cells containing CD5+ CD7+ T cell precursors (Prots); On day 14, floating hematopoietic cells are collected, replicated on new DLL4-coated plates, and then cultured with Flt3 and IL-7 for 3 weeks; Prot cells continue to expand and go through a brief CD3+ CD4+ immature CD4 single-positive (Isp) phase, then activate the expression of CD8 to form CD4+ CD8+ double-positive (Dp) cells (Figure 6).

After 5 weeks of differentiation, CD3+ TCRαβ+ T cells were

To determine whether inhibition of EZH1 promotes IPSC in vitro T cell differentiation, the researchers performed shRNA-mediated EZH1 gene knockout during T cell differentiation of IPSC-derived CD34+ HE cells

Fig.

As an alternative to shRNA-mediated EZH1 gene knockout, the researchers constructed doxycycline-induced CRISPR interference (CRISPRi) into IPSC-derived CD34+ HE cells to transcriptionally inhibit the expression
of EZH1.
During T cell normalization (0-2 weeks), CRISPR-mediated EZH1 gene knockout led to a significant increase in the production of CD3+ T cells, while EZH1-CRISPRi induced after Prot cell formation (2-5 weeks) failed to promote T cell differentiation
.
In control cells, the expression of EZH1 increases significantly during HE entry into Prot cells and is down-regulated
after the Prot phase.
EZH2 is a homologous of EZH1, also as a catalytic sub-of the PRC2 complex, highly expressed
in both control and EZH1 shRNA-treated cells, and in late stages of T cell differentiation.
This observation is consistent with previous findings that EZH1 instead of EZH2 performs the function of inhibiting lymphatic system promise in HE cells, and explains why inhibition of EZH1 has no effect on
the later stages of T cell differentiation.
After 6 weeks of differentiation, IPSC-SFT cells in the control group contained a significant proportion of DP T cells, while EZ-T cells consisted mainly of
CD8 or CD4SP cells.


After EZ-T cell differentiation, CD3+ TCRαβ+ increased significantly, and CD3+ TCRγδ+ T cells decreased significantly, indicating that EZH1 gene knockout promoted differentiation to αβ T cells rather than γδ T cells
.
The EZH1 gene knockout also resulted in a decrease in CD1a, which is expressed in thymic cells but not in mature peripheral blood T cells, further suggesting that EZ-T cells exhibit a more mature T cell phenotype
.
The matrix-free differentiation protocol supports the production of CD8αβ T cells, and EZH1 gene knockout promotes higher yields of CD8αβ T cells, with almost no detection of the number of
congenital CD8ααα T cells.
Together, these analyses suggest that inhibition of EZH1 promotes the efficient differentiation of IPSC cells into mature SP T cells
in vitro.


03 EZ-T cells exhibit molecular characteristics similar to peripheral blood TCRαβ T cells

CellNet's analysis of RNA-seq gene expression profiles showed that IPSC-derived T cells had a high degree of similarity with donor-derived T cells isolated from peripheral blood mononuclear cells (PBMCs) and were significantly different from low-differentiated pluripotent HSPCs; Detection of the expression of T cell characteristic genes found that IPSC-T cells differentiated by matrix-free method without EZH1 gene knockout (PSC-SF-T) were more similar to CB-HSPC-derived T cells than IPSC-T cells differentiated by OP9-DL1 matrix; The expression levels of these genes were assayed for all cell types, and the results again showed that EZ-T cells exhibited the gene expression profile most similar to αβT cells (Figure 8
).

Figure 8 EZ-T cells show molecular characteristics of mature TCRab T cells (Source: [1])

To explore the molecular mechanism of the mature phenotype of EZ-T cells, genoset concentration analysis (GSEA) was performed on the most significantly upregulated genes in EZ-T cells, and it was observed that the processes directly related to the differentiation or function of these genes were highly biologically abundant
.
There are relatively small amounts of differential expression of genes between control IPSC-SFαβT cells and cells with EZH1 gene knockout
.
TCRαβ T cells with EZH1 gene knockout expressed TRAC, TRBC2, CD2, and CD7 more abundantly and showed greater degree of downregulated residues (TRDC, KLRB1
).
Overall, inhibition of EZH1 during in vitro IPSC differentiation promotes the production
of αβT cells that exhibit a more mature phenotype.
During IPSC differentiation, EZH1 gene knockout promotes the maturation of T cells, and the resulting EZ-T cells exhibit the most similar molecular characteristics
to peripheral blood TCRαβ T cells.


04 EZ-T cells can produce T cells similar to memories after activation

iPSC-derived EZ-T cells reproduce the differentiation of primitive T cells, resulting in effector cells and T cell subsets
that exhibit memory-like phenotypes.


05 CAR EZ-T cells show enhanced antitumor activity in vitro and in vivo

When engineered with anti-CD19 CARs, EZ-T cells produced better antitumor effects in vitro and in vivo than control iPSC-SF-T cells lacking EZH1 knockout (Figure 9
).

Fig.
9 CD19 CAR EZ-T cells mediate more powerful tumor clearance in vivo (Source: [1])

When iPSC cell-derived T cells are further converted into CAR-T cells, the anti-tumor activity displayed is comparable to the anti-tumor activity of CAR-T cells produced by current methods used in clinical therapy, and when compared with the T cells manufactured by the previous iPSC cell method, these new cells have enhanced
the ability to kill cancer cells and remove cancer cells in living mice in the laboratory.


Study author Daley said: "After years of research, we now find that iPSC cells may eventually help develop novel therapies
for treating a variety of human diseases, such as cancer.
Universal iPSC cells can not only be effectively converted into CAR-T cells, but also can effectively create an enhanced type of CAR-T cells, which can be more similar to the gold standard clinical grade cells currently used, this new strategy may be able to achieve off-the-shelf CAR-T cell therapy and help more patients to be treated
in a timely and effective manner 。 While iPS cells are theoretically an infinite source of different cell types, researchers must overcome the challenge of spawning mature and fully functional T cells that can help make CAR-T cells.
In the past, because iPS cells were more likely to produce immature cells in a dish, researchers have been working to solve this problem
.
”

References:
[1]Jing R, Scarfo I, Najia MA, et al.
EZH1 repression generates mature iPSC-derived CAR T cells with enhanced antitumor activity.
Cell Stem Cell.
2022 Aug 4; 29(8):1181-1196.
e6.
doi: 10.
1016/j.
stem.
2022.
06.
014.
PMID: 35931029.