Gene modification strategies for next-generation CAR T cells against solid cancers

CAR T cells are autologous or allogeneic T cells genetically engineered to express a synthetic chimeric antigen receptor (CAR). These cells are emerging as a promising cancer immunotherapy. A typical CAR has an extracellular single-chain variable fragment (scFv), fused through a transmembrane domain to intracellular costimulatory signaling domains. The recognition of tumor antigens by scFv activates the intracellular costimulatory signaling domain, subsequently stimulating T cell proliferation and eliminating tumor cells through cytolysis and cytokine release. Based on the structure of the intracellular region, CARs are divided into three generations. First-generation CARs contain only one intracellular signaling domain, CD3ζ, to provide signal 1, but this is insufficient to trigger CAR T cell expansion and engender continuous antitumor activity in vivo [1]. In contrast, second- and third-generation CARs utilize first-generation CARs as a backbone and incorporate one or two additional costimulatory signaling domains, such as CD28 or 4-1BB (CD137), respectively [2]. Unlike T cell receptors (TCRs), CARs recognize specific surface targets on tumor cells in a major histocompatibility complex (MHC)-unrestricted manner, effectively preventing immune escape by downregulating MHC expression in tumor cells [3] (Fig. 1a). In addition to peptide antigens, CARs also recognize carbohydrates and glycolipid antigens, thus expanding the range of tumor antigen targets [4].

To date, CAR T cell-based immunotherapy has yielded remarkable clinical success in patients with CD19-positive leukemia or lymphoma [5, 6]. Owning to these results, Kymriah, a CAR T therapy developed by Novartis, has been approved by the US Food and Drug Administration (FDA) and the European Commission for the treatment of relapsed or refractory acute lymphoblastic leukemia in children and young adults. In October 2017, the US FDA permitted Yescarta, another CD19-targeting CAR T cell product, to treat relapsed or difficult-to-treat large B cell lymphoma [7, 8].

However, there remain a series of challenges that need to be urgently tackled in CAR T cell therapy. In the manufacturing process of CAR T cells, most of the currently available CAR T cells are from autologous T cells. Generation of autologous CAR T cells require huge costs and a long production cycle, limiting their large-scale clinical application. Therefore, universal CAR T cells derived from allogeneic healthy donors are created as an “off-the-shelf” product. Graft-versus-host disease can be avoided by abating TCR and human leukocyte antigen class-1 (HLA-I) expression in universal CAR T cells with gene-editing technologies [9] (Fig. 1b). Apart from this, desirable efficacy of CAR T cells has not been achieved in solid tumors, due to multiple limiting factors. Five major challenges of using CAR T cells to treat solid tumors have been summarized [10]. These include the heterogeneity of tumor antigens, toxicity control, inadequate infiltration, poor proliferation and persistence, and the immunosuppressive tumor microenvironment (TME). Due to the heterogeneity of solid tumors, CAR T cells targeting a single antigen may result in tumor recurrence due to excessive growth of antigen-negative cells or cells expressing low antigen levels. The migration of CAR T cells to the tumor site is the primary step in achieving effector function. Nevertheless, insufficient expression of chemokine receptors in CAR T cells and physical barriers inhibit their homing to tumor sites. Costimulatory signals and cytokine production are also associated with CAR T cell function [11]. The absence of costimulatory molecules can lead to CAR T cell inactivation, while the incorporation of costimulatory molecules strengthens CAR T cell function [12]. The presence of cytokines, as signal 3, is a prerequisite for the full activation of CAR T cells, but exhausted CAR T cells have lower levels of cytokine receptor expression and reduced reactivity to inflammatory cytokines [13], causing the abatement of cytotoxic function. In addition, the TME weakens the function of T cells through a variety of factors, such as the upregulated and sustained expression of inhibitory receptors [14], which is the main characteristic of exhausted T cells [15]. These problems highlight the significance of developing new methods to optimize CAR T cell structure and overcome the treatment hurdles raised by solid tumors.

Herein, we outline and analyze several novel strategies for enhancing CAR T cell function, to improve therapeutic responses against solid tumors, including targeting multiple antigens, overexpressing cytokine or chemokine receptors, enhancing costimulatory signals, and eliminating negative T cell regulators. These next-generation CAR T cells have shown promise in preclinical models and they have great prospects for the treatment of solid tumors.

Antigen-specific recognition is a prerequisite for effective CAR T cell therapy. To avoid immune escape and improve safety, tumor-associated antigens (TAAs) that are specifically and highly expressed in tumor tissue are usually selected as targets for CAR T cells [16, 17]. However, these TAAs are also expressed in normal tissues and therefore, despite their low levels, off-target cross-reactions still occur with CAR T cell therapy. In addition, in contrast to hematological tumors, solid tumors are characterized by antigen heterogeneity. CAR T cells preferentially kill tumor cells with high expression levels of the targeted antigens, but they may not eliminate cells with lower antigen density [18]. Various studies have shown that CAR T cell treatment results in the down-regulation of targeted antigens, which is one of the reasons for tumor recurrence [19].

To improve CAR T cell antigen recognition, new CAR T cell strategies have been developed to simultaneously target two or more different TAAs or to mix different CAR T cells targeting a single antigen. CAR T cells co-targeting CD19 and HER2 display greater antitumor activity in vivo than CAR T cells with a single target [20]. IL13Rα2- and HER2-specific scFvs together form a bi-specific CAR (TanCAR) [21]. TanCAR can recognize each antigen individually to trigger activation, while its functionality is synergistically enhanced upon encountering both antigens [22]. Similarly, trivalent CAR T cells co-targeting HER2, IL13Rα2, and EphA2 effectively eliminate tumor cells [23]. Numerous studies have demonstrated that bi-specific CAR T cells co-targeting CD19 and either CD20 [24], CD22 [25], or CD133 [26] have the potential to reduce the risk of CD19-negative relapse.

Another multi-target strategy is to connect the scFvs of CD3 and the TAA using a flexible linker, to form bi-specific T cell engagers (BiTEs). BiTE-secreting CAR T cells can target multiple antigens, and also have the ability to activate and recruit bystander T cells [27]. Anti-EGFR BiTEs enhance CAR T cell efficacy in vivo and eliminate glioblastoma [28]. CAR T cells can kill tumor cells in a lower antigen density than BiTEs. And costimulatory molecules such as CD28 and 4-1BB can enhance the effect and killing function of BiTEs-activated T cells. Therefore, compared with BiTE or CAR T cells alone, BiTE-secreting CAR T cells display stronger antitumor ability through direct killing and induced bystander killing. Similarly, the construction of universal CAR T cells using adapters can target multiple TAAs without re-engineering T cells. Recently universal CAR structures, such as biotin-binding immune receptor, binding fluorescein isothiocyanate (anti-FITC CAR) and antibody Fc receptor (CD16 CAR), have been developed [29‐31]. And a SUPRA (split, universal, and programmable) CAR system has been developed, which is composed of a leucine zipper CAR (zipCAR) and a scFv binding to the leucine zipper (zipFv). By adjusting zipFv, this approach makes CAR T cells to target multiple antigens, and reduces potential immunogenicity [32]. Compared with traditional CAR T cells, these adapter systems (BiTEs and universal CARs) establish a bridge between CAR T cells and tumor cells, improving the safety and effectiveness of CAR T cells. In addition, the recognition ability of CAR T cells can also be improved by increasing antigen density. The exogenous addition of IL-22 increases the expression of MUC1. CAR-MUC1-IL22 T cells secreting IL-22 enhance recognition and cytotoxic ability against head and neck squamous cell carcinoma [33].

Targeting multiple antigens allows these CAR T cells to maintain their cytotoxicity, expand the range of recognized TAAs, and mitigate antigen escape. The modification of a multi-target CAR to recognize diverse tumor antigens results in more effective elimination of an established tumor than a unispecific CAR. Nevertheless, the combination of multiple targets increases the potential for off-target effects of CAR T cells. Multi-target CAR constructs greatly increase the genetic payload delivered to cells, which leads to reduced transduction efficiency [3]. As transposon-based gene transfer systems for stable genetic modification, Sleeping Beauty and piggyBac transposon systems have attracted much attention. After electroporation, this non-viral approach confers superior CAR expression and is less toxic compared to conventional plasmids [34, 35]. Moreover, optimizing T cell culture conditions and CAR structure have shown to improve transfection efficiency [36]. Therefore, multi-target CAR T cells require further study to improve their structural design and safety.

After homing to the tumor site, CAR T cells must undergo abundant expansion in order to eliminate the tumor in vivo. The major predictors of the clinical efficacy of CAR T cells are the proliferation and persistence of the T cells. Vast clinical trials of CAR T cell therapy have demonstrated poor persistence of T cells transfused in vivo, especially in solid tumors [16]. Therefore, improving the expansion and persistence of CAR T cells is the focus of current research.

The unremarkable efficacy of CAR T cells against solid tumors is associated with insufficient intratumoral T cell penetration and accumulation [92]. Improving the homing of CAR T cells to the TME is a prerequisite for them to exert therapeutic activity. To successfully infiltrate and accumulate in tumor sites, CAR T cells undergo a series of processes, involving the adhesion of endothelial cells and interactions between chemokines and chemokine receptors [93] (Fig. 3). Although the local infusion of CAR T cells results in the significant regression of glioblastoma, this approach may not be effective for all tumor types, especially metastatic disease [94].

Chemokines and chemokine receptors play a pivotal role in mediating the directed migration of CAR T cells. Thus, equipping T cells with a chemokine receptor to better match and respond to tumor-derived chemokines may facilitate T cell migration ability. Integrin αvβ6-CAR T cells modified to express CXCR2 migrate more efficiently towards tumor-produced IL-8 [95]. Similarly, CAR T cells overexpressing CXCR1 or CXCR2 can significantly reduce tumor burden, without obvious toxicity, in various solid tumor xenografts [96, 97]. Further, forced expression of CCR4 by CD30 CAR T cells in a Hodgkin lymphoma model, enhances their homing to Reed-Stemberg cells secreting CCL17 and CCL22, the ligand of CCR4 [98]. Meso-targeted CAR T cells with CCR2b transduction result in increased migration and tumoricidal effect [99]. Furthermore, CXCR3 has been shown to be essential for the intravascular adhesion and penetration of adoptively transferred CD8⁺ T cells [100]. The expression of CXCL9, CXCL10, and CXCL11 (ligands of CXCR3), increased by chemotherapy drugs, enhances T cell trafficking to the TME [101], suggesting that CXCR3 is also a candidate for chemokine receptor modification of CAR T cells. Identifying an appropriate chemokine-chemokine receptor axis is of cardinal importance, but it requires numerous studies to confirm a specific chemokine with high expression levels in the targeted tumor and to overexpress the corresponding receptors on CAR T cells. The incorporation of a chemokine receptor does not affect the cytotoxicity of CAR T cells but exert superior antitumor activity. Of note, chemokines recruit not only effector T cells but also suppressor cells like regulatory T cells [102]. Moreover, factors like tumor types, stroma, and ambient cytokines may affect the tumor’s chemokine signature. Therefore, these aspects need to be taken into account when choosing the suitable chemokine receptor for CAR T cell constructs.

Engineering CAR T cells to deliver chemokines locally is another feasible strategy to recruit more immune cells into the tumor lesion. For instance, forced expression of CCL19 substantially enhances the chemotactic capability of CD20 CAR T cells, by inducing T cell and DC cell migration [79]. Co-expression of CCL5 and CXCL9 is key to CD8⁺ T cell recruitment, creating a loop to magnify lymphocyte engraftment [103]. However, it should be noted that the production of endogenous chemokines in CAR T cells may block chemokine receptors on their surface, resulting in attenuated migratory ability.

Other obstacles, such as the tumor blood vessels and extracellular matrix (ECM), also hinder the infiltration of CAR T cells [104]. Vascular endothelial growth factor receptor 2 (VEGFR2) plays a vital role in VEGF-mediated tumor angiogenesis and growth. CAR T cells targeting VEGFR2 display increased penetration and antitumor responses, by destroying tumor vascular endothelial cells [105]. Enhancing the trafficking of CAR T cells can also be achieved by targeting fibroblast activation protein (FAP), which is a surface marker of cancer-associated stromal cells (CASCs) [106]. FAP-CAR T cells can inhibit stromagenesis and angiogenesis by targeting FAP⁺ CASCs [107]. However, FAP-CAR T cells cause serious cachexia and bone toxicities by affecting FAP⁺ bone marrow stromal cells [108]. And the affinity and specificity of the FAP scFv deserve more attention to decrease the toxicity profile of this modification. Analogous to CASCs, heparan sulfate proteoglycan is an indispensable component of the ECM that can be degraded by heparanase. GD2 CAR T cells expressing heparanase enhance infiltration capacity and antitumor activity in a neuroblastoma xenograft model [109]. It is worth noting that intravenous injection of ECM-degrading enzymes in patients leads to increased thromboembolic events, so precaution like the addition of low molecular weight heparin need to be adopted.

There is increasing evidence that CAR T cells administered to patients gradually lose their effector function and fail to eliminate tumors, due to the TME and internal T cell factors [16, 110, 111]. T cell effector function is regulated by various factors, such as immune checkpoint molecules, transcription factors, metabolic molecules, and apoptotic genes [112]. Therefore, regulating the factors that impact T cell function may enhance the efficacy of CAR T cells (Table 2).

T cells engineered with CARs are ushering in a new era in cancer immunotherapy. Despite their spectacular success against hematological malignancies, CAR T cells are not effective for the treatment of solid tumors. CAR T cells play a role as combatants in the battlefront of tumor immunity, and their function directly determines therapeutic efficacy. In this review, we discussed external and internal factors that primarily impact CAR T cell function. Moreover, we summarized novel strategies developed to modify CAR T cells to improve their function. Targeting multiple TAAs improves the recognition ability of CAR T cells and reduces immune escape. CSSD optimization has been employed to strengthen costimulatory signaling (signal 2). Additional signal 3 can be provided by transgenic expression of cytokine, cytokine receptor or downstream factors for the optimal activation, proliferation, and persistence of CAR T cells. The overexpression of chemokine receptors on CAR T cells overcomes the obstacles of poor trafficking to tumor sites. Manipulating negative regulators in CAR T cells, such as the depletion of immune checkpoint molecules, helps to regain the effector response. Collectively, these approaches all contribute to exploiting the therapeutic potential of CAR T cells (Fig. 4).

However, adverse reactions, such as CRS and neurological toxicities, some of which can be fatal, have been reported in almost all clinical trials of CAR T cell therapy. The modification of CAR T cells can increase the effects of these adverse events. Therefore, the toxicity of CAR T cells must be taken into consideration. Furthermore, the implementation of additional clinical trials is greatly needed to determine whether the optimal CAR T cell modification can serve as a standard treatment option for patients with solid tumors. Thus, tremendous research efforts are required in the near future. Despite the current obstacles, optimized next-generation CAR T cells have the potential to eventually evolve into an efficient immunotherapy for solid tumors.