Modification of allogeneic CAR-T cells via gene editing technology is one of the major strategies to avoid GvHD and host-mediated allorejection. Gene editing technology is based on two mechanisms: double-strand breaks can be repaired to mediate gene knock-in through homologous recombination or gene knockout through non-homologous end joining when template strand is introduced or not, respectively (14). Currently, a series of powerful gene editing tools, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9), have played a role in preclinical and clinical studies of allogeneic CAR-T cells (15–17).

Genetically editing donor CAR-T cells to eliminate endogenous αβ TCR may effectively prevent TCR-mediated alloreactivity without compromising CAR-dependent function. Torikai et al. (15) first proposed that TCR-negative allogeneic anti-CD19 CAR-T cells, in which αβ TCR was deleted through ZFN-driven gene editing, showed the expected cytotoxicity of CD19 redirection without responding to TCR stimulation. This pioneering work introduced gene editing technology into allogeneic CAR-T cells, paving the way for its further use by utilizing different editing tools. More recently, a two-in-one strategy that inserting CAR gene into TCR α constant region (TRAC) through CRISPR system was designed ( Figure 2A ) (18). The produced allogeneic CAR-T cells lacked the unwanted graft-versus-host response, and were protected from the side effects of CAR transduction, such as virus usage and random integration. This two-in-one strategy further suggested that integrating CAR gene into targeted gene locus through gene editing may be a promising approach to both avoid the defects of CAR transduction and achieve the desired goal of targeted gene knockout. For example, CAR insertion in PDCD1 locus may resist exhaustion of CAR-T cells (19, 20).

Several strategies have also been developed to escape host rejection. One of the most commonly used in clinical settings is disrupting CD52 in donor CAR-T cells combined with lymphodepletion through Alemtuzumab, a humanized anti-CD52 monoclonal antibody ( Figure 2B ) (21, 22). CD52 is widely distributed on the surface of lymphocytes and many other hematopoietic cells, so pretreatment with Alemtuzumab can prevent host rejection and facilitate the expansion and survival of infused CD52-decifient CAR-T cells, which are resistant to Alemtuzumab. Since HLA-I molecules are expressed on the surface of most nucleated cells, abrogating their expression in CAR-T cells is another feasible strategy to evade the recognition by the recipient T cells. Disruption of β2-microglobulin (B2M) gene was reported to efficiently eliminate the expression of HLA-I molecules, as B2M is a highly-conserved component of HLA-I heterodimer. Nevertheless, the removal of HLA-I may lead to the activation and killing of NK cells, suggesting the limitations of direct deletion. Therefore, different modification methods were proposed to guarantee no ‘missing-self’ response by host NK cells. One is to express a single-chain HLA-E with minimal polymorphism fused to the B2M locus, which can effectively prevents killing of NKG2A⁺ NK cells ( Figure 2D ) (23, 24). Another is to construct an inventory containing HLA-C-retained cells that disrupts both HLA-A and HLA-B alleles. An induced pluripotent stem cell bank containing 12 lines of HLA-C-retained cells was found to be immunocompatible with >90% of the world population (25), demonstrating the construction of a cell inventory is potentially viable ( Figure 2E ). While not expressed in naive T cells, HLA class II (HLA-II) molecules are upregulated in activated T cells under the control of several trans-acting regulatory factors such as RFX5 and CIITA (26–28). Previous studies found that mutations in these regulatory genes led to lack of HLA-II antigen expression and CD4⁺ T cell-dependent immune response (29), indicating depletion of them would be a strategy to overcome host rejection caused by HLA-II-restricted recognition ( Figure 2F ). In the case of treating T cell malignancies, CD7-targeting allogeneic CAR-T cells with genetic modifications of CD7 depletion are also an encouraging approach to resist fratricide in addition to T cell-mediated rejection ( Figure 2C ) (30). As CD5 is also a pan-T marker acting as an inhibitory regulator of TCR signaling and regularly expresses on ~85% of T cell malignancies (31), it can also be an attractive target against T cell malignancies in allogeneic CAR-T therapy.

Combination of these strategies to overcome both host-mediated allorejection and GvHD has been intensively studied in the field of allogeneic CAR-T therapy (16, 19, 32). For instance, a recent study successfully generated CAR-T cells with immune-evasive properties by inserting CAR and HLA-E gene into TCR and B2M loci, respectively (33). These engineered cells were proved to evade the attack of T cells and NK cells, as well as extend their antitumor activity and persistence both in vitro and in vivo. The success of combination strategy in preclinical models enables the use of allogeneic T cells for adoptive T cell therapy, supporting the translation of research into clinical practice. To date, a number of clinical trials using gene-edited allogeneic T cells as the source of effector cells for CAR-T cell therapy are underway worldwide, most of which target hematological malignancies (34–36). UCART19 is the first-in-class allogeneic CAR-T product developed for the treatment of CD19-positive hematological malignancies, which is engineered to eliminate the expression of TCR and CD52 through TALEN system. A clinical trial (NCT02746952) based on UCART19 showed great expansion and antitumor activity of CAR-T cells in patients with relapsed or refractory B-cell acute lymphoblastic leukemia, in which only 2 patients developed grade 1 acute cutaneous GvHD (37). Many other clinical trials have also shown no or minimal GvHD and host rejection in gene-edited allogeneic CAR-T products, indicating the safety in clinical applications.

Although gene editing technology is an excellent tool to modify allogeneic T cells, it actually carries various potential risks associated with carcinogenicity and chromosomal abnormalities. CRISPR-Cas9 editing was reported to potentially trigger p53-mediated programmed cell death, while downregulation of p53 made the cells more likely to be edited (38, 39). The findings suggest that p53-mutant cells are easier to be edited, therefore raising the risk of cell carcinogenesis. Moreover, cells with mutations in other cancer driver genes, such as the KRAS gene, have a competitive advantage after CRISPR-Cas9 editing (40). Numerical or structural chromosomal abnormalities are also hidden dangers of CRISPR-Cas9 editing (41, 42). CRISPR-Cas9 cleavage may cause chromosomal aberrations in human T cells, including chromosomal truncations, translocations and frequent aneuploidy (43, 44). All the reports point that global detection is needed to eliminate potential risks if CRISPR-Cas9 gene-edited cells are used for treatment. As an alternative, non-gene editing technologies have been explored to mitigate the risk of GvHD and host immune rejection.

Unlike gene editing technology that performs precise genetic manipulation in TCR locus to avoid alloreactivity of donor CAR-T cells, non-gene editing technology prefers to disrupt the downstream mechanisms of action. Since the formation of the TCR/CD3 complex is required for antigen recognition and downstream signal generation (45), inhibition of its assembly or cell surface expression may effectively prevent endogenous TCR-driven T cell activation. From the mRNA level, knocking down any subunit of TCR/CD3 complex through specific short hairpin RNA (shRNA) is one of the effective strategies to inhibit this complex formation ( Figure 3A ). On this basis, CYAD-211, a non-gene edited allogeneic CAR-T product that co-expresses CD3ζ shRNA with BCMA CAR via a single viral vector, was subsequently explored. A phase 1 trial (NCT04613557) evaluating CYAD-211 in adult patients with relapsed or refractory multiple myeloma showed an encouraging antitumor effect with a favorable safety profile (46), suggesting that knockdown of components in the complex is feasible. Several means of affecting the formation of TCR/CD3 complex on cell surface at the protein level have also been proposed. As the complex is assembled in the endoplasmic reticulum (ER), a specific protein expression blocker (PEBL) that consists of anti-CD3ϵ scFv and peptide predicted to anchor to ER was designed, thereby retaining the complex intracellularly ( Figure 3B ) (47, 48). Besides, expression of a TCR inhibitory molecule (TIM) that consists of a truncated CD3ζ peptide can result in dominant suppressive effects in TCR downstream signal through competitively form TCR/TIM complex ( Figure 3C ). By co-expressing a NKG2D CAR and a TIM, the CAR-T product CYAD-101 was proved to have antitumor activity without GvHD in mouse models and clinical trials (NCT03692429) (49, 50).

Resistance of allogeneic CAR-T cells to the recipient’s immune system requires eliminating alloreactive response mediated by T cells, NK cells and DSAs ( Figure 3D ). Activated lymphocytes temporarily upregulate the expression of several surface markers to provide additional costimulatory signals (51, 52), which can be targeted to eliminate host T cells and NK cells activated by allogeneic T cells, thereby preventing allogeneic T cells from being rejected. Based on the proof of principles, an alloimmune defense receptor (ADR) that consisted of an extracellular 4-1BB ligand-derived recognizing fragment fused to an intracellular CD3ζ chain was proposed to apply to allogeneic CAR-T cell therapy (53). The expression of ADR was shown to effectively resist host rejection and fratricide as the expression of ADR could downregulate or mask their own 4-1BB. As NK cells can be activated by ‘missing self’ recognition, Hu et al. (30) generated an NK cell inhibitory receptor (NKi), which was composed of the EC1-EC2 extracellular domain of E-cadherin and the CD28 intracellular domain. Due to the fact that E-cadherin negatively regulates NK cell function through KLRG1 binding and that CD28 intracellular domain enhances the inhibitory effect of NKi (54), the incorporation of NKi into CAR-T cells resulted in a reduction in NK cell-mediated cytotoxicity. Besides, previous studies showed that upregulation of CD47 expression specifically prevented NK cells from killing allogeneic stem cells and cells with HLA-I deletion, illustrating that ectopic expression of CD47 in CAR-T cells may be a novel strategy to solve NK cell-mediated rejection (55, 56). However, it still needs to be further evaluated in preclinical models. More recently, overexpression of CD64 was found to protect CAR-T cells from HLA and non-HLA antibody killing (13). Since CD64 is a high-affinity receptor for the fragment crystallizable (F_c) region of IgG, CAR-T cells that overexpress CD64 can capture IgG F_c and make DSAs inaccessible to effector cells and complement, thus allowing CAR-T cells to evade ADCC and CDC.

Invariant NKT (iNKT) cells are a CD1d-restricted, TCR semi-invariant T cell subpopulation with TCR containing an invariant Vα24-Jα18 chain. The monomorphism of CD1d gives iNKT cells the ability to overcome HLA-restricted GvHD in the allogeneic settings without inactivating their endogenous TCR, thus providing a safer source of effector cells for the ‘off-the-shelf’ CAR-T platform (63). Preclinical models showed that CAR-NKT cells had better localization ability to tumor tissues than conventional CAR-T cells and played a dual killing effect by targeting CAR-positive tumor cells and CD1d-positive M2 or tumor cells to reduce the immune escape of tumor cells (64–66). Moreover, they were able to facilitate maturation of DC through CD40L/CD40 and TCR/CD1d signaling and thus indirectly activate CD8⁺ T cells (67). The first clinical trial based on autologous CAR-NKT cells was conducted previously (NCT03294954) (68). NKT cells were engineered to co-express a CAR that could specifically recognize GD2-expressing neuroblastoma and IL-15 that could support the survival of NKT cells. The GD2.CAR.15 NKT cells showed enhanced persistence, better tumor localization and improved tumor control without significant toxicity, which suggests the great potential of CAR-NKT cells over conventional CAR-T cells. In addition, donor-derived iNKT cells were proved to exert antitumor function without exacerbating GvHD after allogeneic hematopoietic cell transplantation, indicating the possibility for ‘off-the-shelf’ production and use (69).

Because of the relative rarity that iNKT cells account for only 0.01%-1% of peripheral blood T cells (70), expansion of them from donor peripheral blood mononuclear cells (PBMC) to therapeutic level is crucial for clinical use. Rotolo et al. (66) built a system in which TCRVα24Jα18⁺ lymphocytes were activated by CD3/CD28 beads and cultured with an aliquot of irradiated autologous mononuclear cells at a 1:1 ratio in the presence of IL-15, resulting in an average 750-fold expansion of 3rd CAR19-iNKT cells after 3 weeks. Ngai et al. (71) also found that co-culture of iNKT cells with an aliquot of αGalCer-pulsed irradiated autologous PBMC in the presence of IL2 and IL-21 led to an average 600-fold expansion and increased CD62L⁺ frequency in 10-12 days. Currently, a clinical study of allogeneic CD19 CAR-NKT cells for relapsed or refractory B-cell malignancies is underway (NCT00840853) (57). The NKT cells were modified to express a CD19-targeting CAR, IL-15, and shRNA targeting B2M and CD74 to downregulate HLA-I and HLA-II molecules, respectively. The initial results indicated that these allogeneic cells were well-tolerated and could mediate antitumor responses in patients.

γδ T cells, whose TCR consists of γ- and δ-chain, are a subpopulation of T cells appearing in peripheral blood and barriers like intestine (72). Different from αβ T cells that recognize the MHC-peptide complex, γδ T cells play an important role in both innate and adaptive immune responses through multiple receptor-ligand interactions. In antitumor immunity, γδ T cells function in an MHC-independent manner of antigen recognition and tumor killing, as well as play an indirect role by activating B cells, αβ T cells and NK cells (73, 74). Multiple preclinical trials have found that the presence of γδ T cells after allogeneic stem cell transplantation do not induce GvHD (75, 76), a feature that makes them an attractive pool for allogeneic CAR-T therapy. A previous γδ T-based GD2 CAR-T study showed that CAR-γδ T cells could better migrate and infiltrate into tumor site, exert antitumor effects and cross-present antigens to tumor-infiltrating αβ T cells through uptake of released tumor-associated antigens (77). Other studies of CAR-γδ T cells also described several established production process of high-purity CAR-γδ T from PBMC (58, 59, 78). These harvested CAR-γδ T cells were shown to kill antigen-negative tumor cells without evidence of GvHD, indicating the ability to eliminate tumors even after antigen loss. All these characteristics mark their potential advantages over conventional CAR-αβ T cells. Currently, several allogeneic CAR-T cell therapies based on γδ T cells (NCT04735471, NCT05302037) are in phase 1 clinical trials for the treatment of hematological malignancies or solid tumors (60).

Double negative T (DNT) cells are a small subset of mature T cells that comprise approximately 1%-3% of T lymphocytes in human peripheral blood (79). Distinguished from conventional T cells and NKT cells, DNT cells lack both CD4 and CD8 in addition to not binding CD1d tetramers. DNT cells can recognize tumor cells through NKG2D or DNAM-1 and exert antitumor functions through cytokines or Fas/FasL signaling pathway (80). This MHC-independent and multi-targeted mechanism of action allows DNT cells to not cause GvHD and to kill tumor cells that do not express MHC molecules. Various preclinical models demonstrate that allogeneic DNT cells can target a range of tumor types and mediate significant cytotoxicity without triggering GvHD and host rejection (81–84). For example, healthy donor-derived DNT cells significantly inhibited the growth of late-stage lung cancer xenografts in mouse models (85). A Phase 1 clinical trial (NCT03027102) using healthy donor-derived DNT cells also validated objective responses without observed toxicity to normal tissues in patients with acute myeloid leukemia (86). These studies all suggest that DNT cells, which do not induce alloreactivity without modification, have the properties to be used in allogeneic settings.

Although the frequency of DNT cells in peripheral blood is relatively low, large-scale expansion to therapeutic levels can be reached under GMP conditions. Briefly, CD4- and CD8-negative PBMC are sorted and cultured in the presence of IL-2 and CD3 mAbs for 17 days, which allows the harvested DNT cells to reach an average fold and purity of 1558 and 91.9%, respectively (82, 83). As IL-33 has recently been reported to promote the proliferation and survival of DNT cells in vitro (87), adding IL-33 may be an optimized strategy to improve the current clinical expansion system for DNT cells. However, an optimal dosage-frequency regimen for IL-33 requires further exploration. More recently, Vasic et al. (88) generated CAR19-DNT cells by transducing CD19-specific CAR into DNT cells. These modified cells efficiently inhibited tumor growth with no off-tumor toxicity in mouse models, while they were able to evade the alloreactivity of mouse-derived T cells. Therefore, DNT cells have met several requirements for clinical application of allogeneic CAR-T products, including the efficacy, lack of alloreactivity and large-scale production.

Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved, unconventional T cell subpopulation, of which approximately 90% are phenotypically CD161⁺CD8⁺. MAIT cells have the unique semi-constant TCR that consists of TRAV1 combined with three kinds of TRAJ (TRAJ33, TRAJ12, TRAJ20) and a limited repertoire of β chains in human (89). The TCR can recognize modified derivatives from vitamin B2 synthesis pathway presented by MHC class I-related molecule MR1 on APCs, promoting the proliferation and secretion of cytokines, cytotoxic molecules and chemokines of MAIT in the face of most microorganisms. Moreover, they express several NK activating receptors, which act as a second pathway to initiate response. In antitumor immunity, MAIT cells can kill tumor cells via TCR or NK activating receptors, meanwhile boosting DC through upregulating CD40L and inhibiting TAM and MDSC through binding MR1 and NK ligand (90). MAIT cells are distributed in peripheral blood, liver, lung and intestinal lamina propria (91). Previous studies have showed that CD161⁺CD8⁺ MAIT cells upregulate tissue-specific chemokine receptors like CXCL6, CCR6 and CCR9 (92), the reason that they are highly enriched in non-lymphoid organs through tissue homing. Besides, MAIT cells present an effector memory-like phenotype and can produce IFN-γ and granzyme B upon stimulation. Since MR1 is a conserved molecule, MAIT cells are devoid of alloreactive potential, as confirmed by their failure to proliferate in response to alloantigen stimulation in vitro and to expand or participate in tissue damage during GvHD in vivo (93). This potential has also been proved by clinical data showing that the number of engrafted allogeneic MAIT cells is positively correlated with improved survival and less allogeneic adverse events (94). All the intrinsic characteristics of MAIT cells, including multiple targeting, memory phenotype and lack of alloreactivity, make them candidates for allogeneic cell therapy against cancer.

As engineering immune cells with CAR is an effective measure to enhance antitumor function, Dogan et al. (95) constructed anti-CD19 and anti-HER2 CAR-MAIT cells to evaluate their efficacy while compared with conventional CAR-T cells. They found CAR-MAIT cells showed similar or even higher cytotoxicity but lower proinflammatory cytokine secretion compared with conventional CAR-T cells in vitro assays, indicating MAIT cells may represent safer and more effective effector cells for CAR-T cell therapy. However, the efficacy and safety evaluation of CAR-MAIT cells in vivo and in allogeneic settings have not been studied yet, which means more efforts are needed to explore the allogeneic CAR-MAIT therapy. In order to apply allogeneic CAR-MAIT cells in clinical conditions, a key issue is to achieve efficient expansion of MAIT cells. There are two expansion protocols for MAIT cells. The first is co-culture MAIT cells with artificial APCs generated by immobilizing antigen-loaded MR1 tetramers and CD28 mAbs on cell-size latex beads (96). The protocol leads to a 60-fold expansion of pure MAIT cells in 3 weeks. Another is co-culture MAIT cells with autologous PBMC at a ratio of 1:10 in a serum replacement-supplemented medium in the presence of IL-2, which results in a 200-fold expansion of high-purity MAIT cells in 3 weeks (97). For better clinical application, it is worthwhile to optimize the amplification system of MAIT cells.