July 7, 2022

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Promote innovative strategies for CAR-T cell treatment of solid tumors

Promote innovative strategies for CAR-T cell treatment of solid tumors


Promote innovative strategies for CAR-T cell treatment of solid tumors.  In the past few decades, genetically modified CAR T cells have made exciting achievements.

The addition of specific CARs enables T cells to identify specific tumor cells in an MHC-independent manner.

The fourth-generation CAR can release transgenic products, and has enhanced proliferation and anti-tumor efficacy [1].

Significant results have been achieved in the elimination of hematological malignancies by adoptive infusion of CAR T cells, especially in achieving regression of B-cell lymphoma and persistent leukocyte disease [2-4].

The FDA subsequently approved two CD19 CAR T cell products targeting B cell tumor antigens to combat relapsed or refractory B cell malignancies, setting off a climax in CAR T cell research [5, 6].

These achievements have stimulated the exploration of further application of CAR T cell therapy against solid tumors, and many preclinical or clinical studies have been initiated to evaluate the role of CAR T cells [7-10].

However, the current results of CAR T cell treatment of solid tumors are still unsatisfactory.

It is assumed that several factors have caused significant differences in the clinical impact of hematological malignancies and solid tumors. The lack of unique tumor antigens and heterogeneous antigen expression is the key factor leading to side effects and antigen escape [11].

Moreover, due to the disorder of the vascular system in solid tumors and the compactness of the matrix, it is difficult to transport CAR T cells from the blood to the tumor site after infusion [12]. In addition, the hostile tumor microenvironment (TME) is another obvious obstacle that severely hinders the function and persistence of CAR T cells [13].

In order to overcome the challenges associated with solid tumors, various strategies have been developed, including optimized CAR structures and innovative combination therapies, aimed at enhancing the specificity, permeability and efficacy of CAR T cells and reprogramming the inhibitory conditions. In this review, we will elaborate on the main challenges posed by solid tumors and the feasible strategies to support CAR T cell therapy.


Selection of CAR T cell targets

Choosing a suitable target on tumor cells is very important. It will not only affect the accurate identification and removal of tumor cells by CAR T cells, but also affect the safety of tumor treatment. Since the normal tissues of some targets may also express non-specific tumor antigens, they may be damaged by the target and extra-tumor toxicity [16]. In order to improve safety and reduce off-target toxicity, much attention has been focused on optimizing CAR constructs with improved tumor antigen selectivity and specificity.

One way to improve specificity is to design bispecific CAR T cells whose signaling pathways are connected to costimulatory signals and activation signals (Figure 1A). Only when CAR T cells encounter two antigens expressed on tumors at the same time, T cells can be activated to produce a powerful effect [17].

Similarly, it has also been studied to improve the ability to control CAR T cells to turn them on or off under specific conditions (Figure 1A). Switch CAR T cells are designed to be conditionally activated only under conditions that can induce foreign molecules to accurately control the activation of CAR T cells. This effect can be achieved by dividing the key recognition and activation signals of CAR into different modules, which can only be combined with the application of heterodimerized small molecules [18]. Using a bifunctional small molecule “switch” composed of folic acid and fluororesin isothiocyanate (folate-FITC), CAR T cells can specifically recognize tumor cells that overexpress folate receptors [19].

Promote innovative strategies for CAR-T cell treatment of solid tumors

Subsequently, some studies have proposed another method of inhibiting T cell function, if the toxic reaction occurs through engineering suicide genes or using antibody-mediated killing, also known as non-switching CAR T cells [20, 21].


Tumor antigen heterogeneity and tumor antigen escape

Solid tumors are composed of molecularly heterogeneous subgroups, with different antigen expressions in different tumors or individuals. The expression of heterologous target antigens limits the outcome of CAR T cell therapy and is related to subsequent clonal escape, tumor resistance, and recurrence after removal of most immunogenic epitopes [23]. It has been demonstrated that in patients with recurrent glioblastoma, the tumor resistance caused by the loss of tumor antigens, epidermal growth factor receptor (EGFR) targeting CAR T cell therapy [24]. Therefore, addressing tumor heterogeneity and overcoming tumor escape are essential to achieve long-term remission.

Recently, researchers have been working on designing multi-target CAR T cells, aiming to inhibit tumor antigen escape by recognizing multiple antigens. Tandem CAR T cells have two scFVs that can recognize different antigens linked to a CAR construct (Figure 1B). A group compared the effects of tandem CAR T cells co-expressing HER2 and IL13Rα with monospecific CAR T cells in a mouse model of glioblastoma, and found that the former inhibited the reduction of antigen escape and the increase of tumor clearance [25 ]. At the same time, CAR T cells expressing multiple CAR constructs have been developed. Similarly, another study found that in an autologous mouse model, trivalent CAR T cells targeting three different glioblastoma targets showed antigenic heterogeneity among different patients and promoted tumor clearance [26] . Targeting mutant antigens or non-tumor cell antigens in the tumor environment, such as fibroblasts or tumor vasculature, can also overcome antigen escape [27]. As shown in one group, EGFRvIII CAR T cells that recognize EGFRvIII inhibited tumor growth in a glioblastoma model [28].

Promote innovative strategies for CAR-T cell treatment of solid tumors


Limited CAR T cell delivery

Successful transport from the blood to the tumor site is a prerequisite for CAR T cells to attack tumor cells. However, limited delivery to solid tumors is another major obstacle. Different from the easy access to hematological malignancies, there are some factors in solid tumors that lead to low T cell homing efficiency and limited tumor site infiltration.

One factor is the mismatch of chemokines expressed in tumor tissues, which are usually incompatible with chemokine receptors (CCR) on CART cells [33]. In addition, the physical barrier composed of dense extracellular matrix (ECM) and the abnormal tumor blood vessels in solid tumors further inhibited infiltration [12]. Therefore, many strategies have been proposed to overcome the limited T cell trafficking.

Local infusion of CAR T cells to the tumor site or cranial cavity seems to be a potential strategy, and it can also avoid the toxicity and off-target effects of systemic injection. The intracranial administration and intraperitoneal CAR T cell delivery for breast cancer brain metastases and ovarian cancer were evaluated separately [34-36]. However, in the case of extensive tumor metastasis, these strategies are not feasible.

Promote innovative strategies for CAR-T cell treatment of solid tumors

It is necessary to explore other strategies to enhance T cell infiltration and achieve precise cell homing. By directly optimizing CAR T cells to express a well-matched CCR, CAR T cells can better interact with chemokine ligands expressed by tumor cells (Figure 1C). Moon et al. used lentiviral vectors to obtain CCR2b-expressing anti-mesothelin CAR T cells, which interact with chemokine ligand 2 (CCL2) secreted by cancer cells, and showed improved performance in xenogeneic mesothelioma transplantation models. T cell infiltration, accompanied by improved anti-tumor activity [37]. Recently, another study found that CAR T cells expressing CXCR2 can promote T cell homing and enhance anti-tumor response [38]. In addition, other CCRs, such as CCR4, have also been proven to be effective to varying degrees [39].

In order to enhance the ability of migration and infiltration, another possible method includes destroying the physical barrier in solid tumors by designing CAR T cells to recognize stromal cell-related antigens or secrete stromal degrading enzymes (Figure 1C). One of the attractive targets appears to be the protease fibroblast activation protein (FAP), which is expressed on fibroblasts in many solid tumors [40]. In addition, the evaluation of FAP CAR T cells in a variety of tumor models showed effective tumor suppression and a reduction in the number of FAP-positive stromal cells [41]. In another study, CAR T cells modified by heparinase (HPSE) were designed to supplement the HPSE dysfunction in the matrix, and also showed the effect of promoting matrix degradation and T cell infiltration [42].


Immunosuppression of tumor microenvironment

Even if CAR T cells are successfully transported, another major obstacle is that highly immunosuppressive TME can render them unresponsive [43]. Solid tumors are usually characterized by inhibitory TME composed of multiple immunosuppressive molecules and inhibitory immune cells. Inhibitory immune cells (such as regulatory T cells) can hinder the proliferation and cytotoxicity of CAR T cells through contact-dependent methods, while certain inhibitory molecules (such as transforming growth factor-β (TGF-β) and IL-10 ) Can promote T cell unresponsiveness caused by indirect contact [13]. Recently, various strategies have emerged to overcome immunosuppressive TME and make it beneficial to the survival and proliferation of T cells (Figure 1D).

Promote innovative strategies for CAR-T cell treatment of solid tumors

Some research has been devoted to modifying CAR T cells to overexpress cytokines that promote inflammation, such as IL-12, IL-15 and IL-18. These cells are called armored CAR T cells [44-46]. Contrary to the toxicity caused by systemic injection of stimulating molecules, this method can safely regulate the local microenvironment. It is confirmed that the secretion of IL-12 by engineered T cells can induce the proliferation, viability and cytotoxicity of ovarian cancer, as well as resistance to apoptosis and PD-L1-induced functional inhibition [47]. In the leukemia model, it was also found that IL-15-expressing anti-CD19 CAR T cells achieve persistence and induce sustained remission, the mechanism may be related to the formation of memory cell subpopulations [48]. Similarly, in a neuroblastoma metastasis model, optimizing GD2 CAR T cells with IL-15 can improve anti-tumor ability [49]. In addition, related studies on CAR T cells that secrete IL-18 have shown that these cells have enhanced proliferation and infiltration capabilities, and can recruit endogenous immune cells to regulate TME [50, 51].

Other strategies to isolate CAR T cells from inhibitory molecules also need to be considered, such as blocking the immunosuppressive signals in CAR T cells and TME (Figure 1D). Since TGF-β signaling is an important inhibitory pathway, a viable alternative to promote anti-tumor response is to block TGF-β signaling. One group found that in preclinical prostate cancer models, TGF-β dominant negative receptor (DNR) can effectively protect CAR T cells from the interference of immunosuppressive cytokines, thereby inhibiting signal transduction [52]. Alternatively, CAR T cells engineered with receptors convert TGF-β signals into 4-1BB or IL-12 stimulation signals, exert a powerful anti-tumor effect, and develop resistance to immunosuppressive signals [53]. Another group also found that TGF-βCAR T cells have enhanced cytotoxicity and enhanced anti-tumor immune function, which can protect neighboring immune cells from TGF-β-mediated immune suppression [54].

Similarly, studies on pancreatic cancer models have shown that CAR T cells expressing inverted cytokine receptors can promote T cell survival. The extracellular part of IL-4 fuses the receptor and the intracellular fragment of the IL-7 receptor to convert the immunosuppressive cytokine signal into an activation signal [55]. Methods to induce endogenous anti-tumor immune responses and enhance costimulatory signals have also been explored [56]. In addition, metabolic disorders such as nutritional deficiencies, low pH and hypoxia are another characteristic of TME, and metabolic competition further limits the activity of CAR T cells [57]. Reforming the metabolic pathways of CART cells may be another strategy. For example, under hypoxic conditions, the transcription factor hypoxia inducible factor 1-α (HIF-1α) can be stably expressed. By modifying CAR T cells to express HIF-1α, these cells have the ability to resist hypoxia, leading to increased CAR expression and oncolytic effect to improve anti-tumor effect [58].

Recent research has focused on the use of gene silencing technologies, such as CRISPR/Cas9 or short hairpin RNA (shRNA), to directly knock out the gene encoding the inhibitory receptor PD-1 in CAR T cells. For example, a group of anti-CD19 CAR T cells silenced PD-1 through CRISPR/Cas9, and showed salvage T cell activity and improved anti-tumor efficacy in a PD-L1 positive tumor model [61]. The silencing of PD-1 gene by MSLN CAR T cells leads to down-regulation of endogenous PD-1 levels, enhanced T cell expansion and tumor cell lysis [62].

In addition, modifying CAR T cells to express switch receptors seems to be an attractive strategy to overcome immune checkpoints or inhibitory molecules, which can convert inhibitory signals into stimulus signals [63]. The PD-1 switch receptor is created by fusing the extracellular region of the immunosuppressive signal with the activated intracellular domain (Figure 1E), and converts the PD-L1 signal into a stimulus signal, thereby resisting T cell dysfunction and promoting tumors Faded [64]. Attempts to modify CAR T cells with dominant negative PD-1 receptors have also been explored (Figure 1E). Due to the lack of transmembrane segments and intracellular signal transduction domains, T cells with DNR have the ability to compete with endogenous cell receptors for binding to PD-1, but cannot transduce inhibitory signals [65]. In addition, it has been shown that engineered CAR T cells that can release PD-1 bloking antibodies can enhance the anti-tumor effect of CAR T cells [66].


Innovative combination therapy

Although the remission rate of hematological malignancies is very high, patients still lack continuous remission after CAR T cell therapy, and eventually relapse of the disease. The function of CAR T cells is closely related to the clinical outcome of patients, and the activation, killing function and persistence of these cells in the body are dynamic processes affected by many factors (such as tumor factors, T cell factors and individuals). Factor [67]. Every aspect may affect the application of CAR T cells, so monotonous methods do not seem to be enough to solve these complex factors. In addition to direct genetic modification of T cells, alternative strategies to overcome insufficient expansion of CAR T cells and enhance persistence in vivo should also be explored [68-71]. By directly enhancing the function of T cells or by recruiting endogenous immune cells and reshaping TME, the latest developments in combination vaccines, biomaterials and oncolytic viruses have good application prospects for achieving the expected results.

CAR T cell therapy combination vaccine

By specifically inducing T cells to attack tumors, therapeutic cancer vaccines have become a key breakthrough in cancer treatment. Some studies have found that bispecific T cells express CARs for tumors and endogenous T cell receptors (TCRs) for strong immunogens (such as influenza virus), and they show strong expansion after immunization with immunogen vaccines Ability and anti-tumor activity [72-75].

Vaccination provides another strategy to further promote the expansion, activation and cytotoxicity of these CAR T cells in the body (Figure 1F). A common vaccine is a virus-based vaccine: for example, a cytomegalovirus (CMV)-based vaccine combined with adoptive infusion of T cells can synergistically promote tumor clearance [75]. In a variety of mouse models, vaccination with gp100-expressing virus vaccine can enhance T cell expansion and tumor regression [72]. A recent study on patients with B-cell acute lymphoblastic leukemia (B-ALL) also showed that even without lymph node dissection, the use of viral vaccines to stimulate natural TCR will allow CD19-modified virus-specific T cells to successfully expand and increase Increase sustainability [77]]. However, there is a risk of virus reactivation and viremia, and the safety of virus vaccines needs to be further evaluated.

In preclinical and clinical trials, other cancer vaccines containing soluble tumor-associated antigen (TAA) and dendritic cell (DC) adjuvants have been shown to activate TAA-specific effector cells and stimulate antibody production [78]. DC is a full-time APC, which can regulate natural and acquired immunity and is essential in the course of immunotherapy. A study has shown that DC vaccine can enhance T cell activation, expansion and anti-tumor effects of ACT in vivo [79]. In a clinical trial about Mel’s abnormality, after vaccinating a patient with a tumor antigen-carrying DC vaccine, tumor-infiltrating T cells were injected, resulting in complete remission of one person and stable disease in two people [80].

There are parallel results in the study of DC vaccine-mediated CAR T cell stimulation. One group found that GD2 CAR T cells prepared from virus-specific CTL killed the target cells when they were first cultured with neuroblastoma in vitro, but these T cells could not control the tumor after the second exposure. However, the virus-specific TCR can be reactivated by using DC or DC supernatant alone, and CAR T cell function can be rescued [73]. DC cell vaccination shocked with tumor-specific antigen peptides can enhance the expansion and function of intracellular oncoprotein WT1 CAR T cells in xenograft mouse models, and promote tumor clearance [81]. Although the DC vaccine may be a safe method to enhance CART cells, patients treated with cancer vaccines have shown moderate anti-tumor immune responses, and clinical treatment effects are very limited. These methods need to be improved in the future.


CAR T cell therapy combined with biological materials

There are some disadvantages when directly modifying CAR T cells to express supporting factors, such as complicated operations and uncontrollable systemic toxicity. Therefore, there is an urgent need to develop new strategies, and biological materials provide a potential way to supplement.More and more researchers are constantly exploring some methods based on nanomedicine to improve the function of adoptively injected T cells.

Synthetic nanoparticles can be designed to direct larger genetic cargo or drugs to target cell subpopulations in vivo through chemically coupled antibodies or ligands that bind to target cell surface receptors (Figure 1F) [82]. Some studies have explored the potential of liposomes to deliver stimulating molecules in ACT. Zhang et al. used PEGylated liposomes modified with cytokines or cell-specific antibodies to transfer cargo and simulate T cell expansion, and observed successful labeling and significant expansion of T cells in vivo [83].

Recently, backpack nanoparticles have been developed to connect supporting drugs to immune cells in an autocrine manner and enhance cell function [84]. Protein nanogels are another biological material that can carry therapeutic substances and release cargo based on antigen recognition. Li Tang et al. used protein nanogels to transport IL-15 super agonists to TME and observed that the efficacy of EGFR CAR T cells was significantly increased and the survival rate of tumor-bearing mice was improved [85].

In addition, nanoparticles have broken through the long-term barriers to the preparation of CAR T cells in vitro. A group of researchers who used DNA nanocarriers to transfer CAR genes that recognize leukemia cells found that circulating T cells can be quickly and accurately reprogrammed into in situ antigen-specific T cells and achieve the same effect [86].


The use of nanoparticles loaded with immunosuppressive inhibitors is another promising method to reverse inhibitory TME. Zhang et al. It was found that tumor-targeted liposomes encapsulating immunomodulators PI-3065 and 7DW8-5 inhibited monocyte-derived suppressor cells, while increasing endogenous anti-tumor cells, and provided two weeks for CAR T cell therapy Window.

During this infusion, CAR T cells can effectively infiltrate, expand robustly and promote tumor clearance in the mouse model [87]. Similarly, pegylated immunoliposomes transport small molecule inhibitors of TGF to ACT cells and maintain T cell proliferation in vitro [88].Nanoparticles can also transport antigens to lymphatic tissues and stimulate endogenous APCs to present antigens, thereby assisting CAR T cell therapy.

Ma Leyuan et al. realized the phospholipid polymer that binds albumin by connecting the small molecule or peptide ligand of CAR to the lipid tail to achieve lymph node targeting. As a result, the CAR ligand is effectively delivered to the lymph nodes by albumin and inserted into the APC membrane through the lipid tail to modify macrophages and DC. The subsequent endogenous cell activation further promotes the proliferation of CAR T cells and enhances the resistance. Tumor effect [89].

Recently, another study showed that nano-RNA vaccines can directly enhance the cytotoxicity of CAR T cells and overcome the problems of insufficient stimulation and low sustainability. This nanoparticle vaccine can transport CAR antigens to APCs in lymphoid tissues, and at the same time initiate a Toll-like receptor-dependent type I interferon-driven immune stimulation program [90].


Combination of CAR T cell therapy and oncolytic virus

As a virus that selectively infects tumor cells, oncolytic viruses not only directly lyse cells, but also induce endogenous anti-tumor immunity by recruiting immune cells and releasing pro-inflammatory molecules. In addition, the oncolytic virus can also be modified into a platform form to transport goods [91]. Therefore, the combination therapy of oncolytic viruses is a promising combination strategy to restore tumor immunosuppression and enhance CAR T cell function, especially the use of oncolytic viruses to transfer therapeutic drugs or genes to TME [91].

On the one hand, oncolytic viruses can present multiple tumor antigens to CAR T cells and help overcome antigen escape. Recently, CAR T cells combined with oncolytic viruses have been studied in vivo to express BiTEs specific to EGFR and have shown a synergistic effect in overcoming tumor heterogeneity [92]. On the other hand, transgenic oncolytic viruses encoding chemokines can attract CAR T cells to tumor sites and promote infiltration. For example, in a mouse model, the combination of CAR T cells and an oncolytic virus expressing the chemokine RANTES and the cytokine IL15 has been shown to improve infiltration and promote significant tumor regression [93].

Similar encouraging results were found in another oncolytic virus that expresses the chemokine CXCL11 [94]. Research also tried to overcome immunosuppression by modifying the oncolytic virus to express immunosuppressive agents and promote inflammatory factors. Oncolytic adenoviruses expressing cytokines IL-15, IL-2 and TNF have been shown to reverse immunosuppressive TME, promote CAR T cell infiltration and enhance persistence [95]. Another study showed that intracranial injection of oncolytic adenovirus combined with PD-L1 blocking small antibodies has the potential to improve the anti-tumor efficacy of HER2 CAR T cells [96].

CAR T cell therapy is still a potential strategy for cancer treatment. However, so far there has been no success in the treatment of solid tumors. The challenges of solid tumors, including lack of specific tumor targets, heterogeneous antigen expression, limited infiltration and inhibitory TME, are the main factors hindering the success of CAR T cell therapy. Various precise and controllable solutions are currently being modified to the CAR structure. Moreover, the combination therapy strategy in the pre-clinical model has been successful, indicating the potential to be transformed into clinical applications, and more clinical studies are needed in the future to evaluate the effect of combination therapy.


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