All CAR-T cells, both autologous and allogeneic, are genetically-modified T cells to express the specific CAR molecule. Moreover, one of the main strategies to enable the allogeneic use of CAR-T cells manufactured from healthy donor T cells involves the addition of extra genetic modifications in the manufacturing process.
Genetic strategies to enable allogeneic use of CAR-T cells
As previously mentioned, the two main potential problems of the allogeneic use of T cells are GvHD and immune rejection. The former can be avoided by eliminating the TCR, usually through the knockout of the constant domain of one of its chains (α or β), or by replacing some TCR subunits that impedes its antigen recognition function [
45,
93]. However, although the absence of TCR controlled the tumour burden in vivo without alloreactivity, the long-term persistence was lower compared to CAR-T cells with endogenous TCR in patient-derived mouse xenografts [
94].
Regarding immune rejection, it is avoided by preventing the expression of HLA class I (HLA-I) molecules by knocking out their common subunit β2-microglobulin (encoded by
B2M gene), which prevents the recipient’s T cells from recognising the therapeutic cells as foreign through their TCR [
36]. However, HLA-I negative T cells are susceptible of being lysed by host natural killer (NK) cells; to avoid this, a study applied the strategy of inducing the constitutive expression of mutant B2M-HLA-E and B2M-HLA-G fusion proteins [
95]. Furthermore, activated T cells also express HLA class II (HLA-II) molecules, whose incompatibility can trigger the activation of receptor alloreactive CD4
+ T cells [
36]. Therefore, some authors propose for allogeneic CAR-T cell manufacturing knocking out either the
CIITA gene, which is an important transcriptional activator of HLA-II genes [
39], or the
HLA-DRA,
-DQA and -
DPA genes, which encode the α-chains of HLA-II molecules, which are less polymorphic than β chains, thus resulting in higher efficiency for eliminating HLA-II expression [
36].
Different gene editing technologies have been utilised for deleting the aforementioned genes, such as the CRISPR/Cas9 system [
35], zinc finger nucleases (ZFNs) [
58], or transcription activator-like effector nucleases (TALENs) [
57]. CRISPR/Cas9 technology is the most widely used because it has demostrated a remarkably low rate of off-target mutagenesis in T cells [
40,
48]. In addition, a specific high-fidelity Cas9 mutant, called eSpCas9, did not cause any detectable off-target effect, making it an even safer technology [
48]. To study the expected decreased alloreactivity, Ren et al. co-cultured TCR
−/HLA-I
− T cells and irradiated allogeneic peripheral blood mononuclear cells (PBMC) and observed that they triggered only a minimal response, which was hypothesised to be mediated by NK cells from the PBMC population [
48]. Moreover, this group developed an allogeneic CAR-T cell prototype by introducing the gRNAs directed to eliminate TCR and HLA-I expression in a lentiviral vector together with the CAR transgene, thus achieving constitutive expression of the gRNAs and the consequent increase in the knockout efficiency and population homogeneity [
35,
40].
Since multiplex editing with Cas9 nuclease may cause risk of gene rearrangements and chromosomal instability due to double-strand breaks, it has also been proposed the use of base-editing proteins, such as BE3 and BE4, aimed at inducing exon skipping by disrupting splice acceptor sites or creating premature stop codons, thus avoiding double-strand breaks and minimising the genetic risk [
56].
Another technique employed to produce allogeneic CAR-T cells is TALEN, which has achieved only double gene disruption, in contrast to the CRISPR/Cas9 system by which quadruple knockouts have been reported [
35]. This is owed to the complexity of the construct and the difficulty of targeting several genes with TALEN technology, which is also the case with ZFN [
48]. Some groups have developed TALEN-edited CAR-T cells with different modifications such as TCR
−/CD52
− [
25,
41,
43] or TCR
−/dCK
− [
34], which avoid GvHD due to elimination of TCR and are resistant to other concomitant treatments such as alemtuzumab, a chemotherapeutic agent which targets CD52, or to purine nucleoside analogues, antitumour prodrugs activated by the enzyme deoxycytidine kinase (dCK), respectively [
25,
34,
41,
43].
It has also been reported a strategy that combines TCR deletion with CAR transgene integration by using adeno-associated virus (AAV)-based vectors, in which the CAR is integrated in a targeted manner in the
TRAC gene locus, thus avoiding the random integration of CAR gene that occurs with lentiviral vectors. Integration is achieved by homology-directed recombination after disrupting the
TRAC gene by a nuclease such as Cas9 [
24],
TRAC megaTAL [
52], or TRC1-2 (a single-chain variant of I-CreI) [
53]. Jo et al. went a step further by targeting with TALEN the
TRAC and
B2M genes where they inserted the CAR and HLA-E encoding-genes, respectively, using AAVs. In this study, they observed an increase of persistence and antitumour effect of the allogeneic CAR-T cells in presence of NK cells in vivo and in vitro, respectively, due to HLA-E expression [
60]. On the other hand, to avoid the use of viral vectors, Yang et al. used the same CAR targeted integration strategy using a TALEN nuclease and naked double-stranded DNA that included the CAR sequence [
54].
In relation to immune rejection, K3 and K5, protein ubiquitin-ligases of human herpes virus-8, have been demonstrated to down-regulate together HLA class I (HLA-A, -B, -C) and HLA class II (HLA-DR), as well as the NK cell activating ligands MICA and MICB, thus avoiding both T cell and NK cell citotoxicity. This strategy would contribute to the production of allogeneic CAR-T cell therapies less prone to elimination by the recipient’s immune system [
51]. Another challenge to be met, especially in the treatment of solid tumours, is the difficulty of trafficking and infiltration of the tumour and the immunosuppressive tumour microenvironment, together with the appearance of T cell exhaustion phenotype upon repeated activation [
96]. In this regard, the inhibition of immune checkpoints using monoclonal antibodies is a widespread strategy in the field of oncology, given its good results in enhancing antitumour immunity. However, this approach entails the potential risk of breaking peripheral tolerance, which might trigger autoimmune responses [
38]. Therefore, the silencing of genes encoding molecules involved in this signalling axis, including programmed cell death 1 (PD1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4), which are inhibitory receptors related to T cell exhaustion and the immune escape of tumour cells, are being studied with the aim of increasing cytotoxic activity [
24,
35,
38,
48]. Also, Ren et al. performed the knockout of Fas receptor to prevent the attenuation of CAR-T cell activity due to Fas-FasL cell signalling that triggers activation-induced cell death [
35].
To increase the safety of these therapies, suicide switches can be inserted, such as inducible Caspase 9, which produces apoptosis upon administration of a small molecule drug [
55], or rituximab-binding domains, which allow CAR-T cells to be depleted by administering this monoclonal antibody [
25,
37,
42,
43].
In case of treating T cell malignancies, a major obstacle is that the CAR target antigen is shared by CAR-T cells and malignant T cells, which leads to therapeutic T cell fratricide. To avoid this, the target molecule should be eliminated in the therapeutic cells. Cooper et al. developed an “off-the-shelf” CAR-T cell therapy by eliminating TCR and CD7 expression in CAR-T cells directed towards CD7 antigen [
47]. In this regard, GC027, a CAR-T cell product that shares these characteristics, has been studied in a phase I clinical trial with promising preliminary results [
46].
Moreover, in order to avoid the residual TCR
+ T cells that might cause GvHD, Juillerat et al. induced the transient expression of an additional CAR that targets CD3, which eliminate the TCR
+ CAR-T cells, leading to a very high proportion of TCRαβ
− population in the final CAR-T cell product [
44].
The strategies involving additional genetic modifications (besides introducing the CAR transgene) are very versatile and offer unlimited options not only to allow the allogeneic use of CAR-T cell products, but also to improve different characteristics of the therapeutic cells to increase persistence, infiltration, antitumour efficacy, safety profile, etc. Although high levels of efficiency can be achieved, for example, in gene knockout in human T cells (e.g. around 70–90%
TRAC gene disruption obtained by using different procedures based on CRISPR/Cas9 technology [
35]) and gene modification technologies are rapidly improving, these procedures need an important previous work to be optimised, and usually additional selection steps are needed during the manufacturing process to obtain the purified CAR-T cells with the desired phenotype.