With a relatively small bank of donors, we can provide an "off-the-shelf" CAR or TCR-T cell solution for a large proportion of the population.Ĭonclusions. Importantly, these allogeneic T cells had comparable functional activity to their autologous T cell counterparts in in vitro assays (tumor cell killing and cytokine release) as well as in vivo tumor models. Using our proprietary T cell engineering process, we successfully generated allogeneic T cells with sequential KOs and insertion of a tumor-specific TCR or CAR with high yield. TRAC edited donor T cells did not induce GvHD in an immune compromised mouse model over the 90-day evaluation period. Edited T cells were protected from host NK cells, both in vitro and in an in vivo model engrafted with functional human NK cells. By knocking out select HLA class I and class II proteins, we were able to avoid host CD4- and CD8-T cell-mediated recognition. Alloreactivity by CD4 and CD8 T cells, NK killing, GvHD induction and T cell function was assessed in vitro and/or in vivo. An AAV-mediated insertion of a CAR or TCR into the TRAC locus is used in parallel with the TRAC KO step to redirect the T cells to tumor targets of interest. Additionally, we utilize potent TCR-α and -β constant chain ( TRAC, TRBC) gRNAs that achieve >99% KO of the endogenous TCR, addressing the risk of GvHD. To solve the issue of rejection by alloreactive CD4 and CD8 T cells, we knocked out (KO) select HLA class I and class II expression with a sequential editing process. Here, we developed an immunologically stealth "off-the-shelf" T cell strategy by leveraging our CRISPR/Cas9 platform and proprietary sequential editing process. Because activated T cells upregulate HLA class II, rejection by alloreactive CD4 T cells should also be addressed. To overcome this, researchers are exploring insertion of the non-polymorphic HLA-E gene, which can provide partial but not full protection from NK cell-mediated lysis. However, loss of HLA class I sends a "missing-self" signal to natural killer (NK) cells, which readily eliminate B2M null T cells. Other strategies include deletion of the B2M gene to remove HLA class I molecules and avoid recognition by host CD8 T cells. Although showing responses in the clinic, this approach carries the risk of infections and the durability of the adoptive T cells is uncertain. Some approaches utilize prolonged immune suppression to avoid immune rejection and increase persistence. The recognition of allogeneic cells by the host is a complex issue that has not been fully solved to date. Alloreactive T cells from unrelated donors can cause graft versus host disease (GvHD) for which researchers have successfully used nucleases to reduce expression of the endogenous T cell receptor (TCR) in the allogeneic product. There is a strong desire for an immediately available cell therapy option however, development of "off-the-shelf" T cells is challenging. Despite the success of autologous chimeric antigen receptor (CAR)-T cells, barriers to a more widespread use of this potentially curative therapy include manufacturing failures and the high cost of individualized production.
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