CAR T-cells profiling in carcinogenesis and tumorigenesis: An overview of CAR T-cells cancer therapy

Highlights

• CAR T-cells cancer therapy has a notable clinical potential in treatment of solid tumors.

• CAR-modified T cells have this ability to terminate and remove B cell malignancies.

• CAR T-cell therapy containing CD19+ B-cell malignancies targeting has confirmed as an impressive clinical aims, but there is a concern about treatment that eliminates and remove normal B cells during this process which lead to B cell aplasia.

• The anti-tumor immune response to this mechanism can durably identify the tumor antigens and comprehensively improve it.

Abstract

Immunotherapy of cancer by chimeric antigen receptors (CAR) modified T-cell has a remarkable clinical potential for malignancies. Meaningly, it is a suitable cancer therapy to treat different solid tumors. CAR is a special recombinant protein combination with an antibody targeting structure alongside with signaling domain capacity on order to activate T cells. It is confirmed that the CAR-modified T cells have this ability to terminate and remove B cell malignancies. So, methodologies for investigations the pro risks and also strategies for neutralizing possible off-tumor consequences of are great importance successful protocols and strategies of CAR T-cell therapy can improve the efficacy and safety of this type of cancers. In this review article, we try to classify and illustrate main optimized plans in cancer CAR T-cell therapy.

Introduction

The development and progress of chimeric antigen receptors (CARs) have revolutionized T-cell based immunotherapies for some cancers. Transformed cellular receptors (TCR) have broad applications in immune-based therapies. In this way, a short comprehensive look on T (CAR T) cell therapy indicates that CAR T-cell therapy is considered as a practical factor for relapsed or refractory tumors, especially for many serious cancers. Innovations and novelties in the combination, mechanism and manufacturing of CAR T-cell s have led to an impressive progressions in efficacy and consistency, considerably with the progression of fourth-generation CAR-T cells [1], [2]. However, their ability to identify target antigens is limited and needs the original Major Histocompatibility Complex (MHC) in the supply of antigens [3]. Also, CARs are capable of detecting a wide range of antigens without the need for MHCs so that it will lead to further developments in the clinical treatments of patients than TCR. CARs consist of an antigen identifier region called single-chain variable fragment (SCFV) integrated with signaling chains of the TCR complex. Specifically, it can be explained that the basic structure of CARs includes an extracellular region of SCFV connected to a hinged region resulting in its flexibility. This structure is connected to the cell membrane region, and more importantly, with portions of the intracellular signaling pathway, which leads to changes in the functionality, stability, and capability of CARs [4].

The ability of CARs to identify antigens without the need for MHC provides the advantage that it will no longer require the compatibility of Human Leukocyte Antigen (HLA) between donor and recipient. The first generation of CARs consisted of a single intracellular chain called CD3. Since the antigen-MHC combination results in T-lymphocyte stimulation in normal TCRs, the second and third intracellular signaling chains were also added in designing the next generation of CARs to improve activity, stability, and ability of the second and the third generations of CARs [5].

Researchers propagated T-cells in vitro, stimulated simultaneously for 56 days, and observed reductions in less differentiated cells with a high division potency. They finally achieved a functional and reliable generation of modified T-cells. All clinical evidence obtained from the study resulted in the second generation of CAR protein-expressing T-cells.

One of these remarkable successes was the use of the second generation of CD19 targeting CAR T-cells, the therapies based on which showed excellent efficiency against blood diseases. Given the successful activation of CAR T-cells, they can be utilized as one of the critical therapies against recurrent malignancies related to Β-cells and expansion of these methods in the treatment of other cancers, including solid tumors. Studies concerning new therapeutic molecules and therapies based on cell immunity pathways that have provided a good ground for future progress in therapeutic T-lymphocytes to promote the design of more efficient CAR proteins, optimization of specific T-cell production, and linking the patient's previous status and new therapies [6].

A variety of CARs have the targeting capability of diverse tumor specific antigens/tumor associated antigens (TSAs/TAAs). These CAR T-cells function considerably against tumor cells both in vitro and in vivo, including targeting the antigens on tumor cell surface in hematologic malignancies and solid tumors. In general, antigens identified by CAR require to be expressed on target cancer cell surfaces, an event considered as a great disadvantage of this method. However, recent research has shown that certain types of antigens expressed within tumor cells can also be identified with special variants of CARs called T-cell receptor-mimic antibody (TCRm) obtained from TCRm ESK1 monoclonal antibody (Fig. 1).

Also, the tumor angiogenesis process is considered a suitable target for CAR T-cells based therapies. Optimal utilization of each method can exert tremendous impacts on the treatment of all cancer types [7].

Among all clinical trials, CD19 has been one of the important tumor antigens in all types of leukemia with widespread interest. CD19 is an ideal antigen for targeting hematologic malignancies associated with B cells stemming from its high and integrated expression on B-lymphocyte cell surface. Ferry et al. (2014) reported successful treatment of a B-cell lymphoma called diffuse large B-cell lymphoma (DLBCL), which was of a high capability and validity in the treatment of B-cell related hematologic malignancies together with chemotherapy [8]. In the same year, another study with the same strategy reported the recovery of 27 children (out of 30 cases) with acute lymphoblastic leukemia with recurrent periods. Accordingly, treatment with CAR T-cells targeting CD19 antigen enabled researchers to overcome the limitations of traditional methods and stimulate recovery in patients with recurrent malignancies, thereby establishing this method as strong support for later developments [9].

CD20 is an activated glycosylated phosphoprotein expressed on the surface of B- lymphocytes. Liu et al. (2017) designed special clinical trials to examine the influence of the third generation of CAR T-cells that specifically target CD20 antigens in patients with a variety of B-lymphocyte related lymphomas [10]. The treatment process was well implemented on patients with promising clinical results. The noteworthy point is that a threshold exists for the concentration of antigen, which is necessary for the proper function of this treatment, though, it may have no effect on increased malignancy in patients [11].

CD30 is a member of the TNF (Tumor Necrosis Factor) receptor superfamily. A wide range of malignant T-cells in Hodgkin's lymphoma (HL) Non-Hodgkin's lymphoma (NHL) can specifically express CD30, which can be used as a target antigen. Lymphocytes, hematopoietic stem cells, and progenitor cells (HSPCs) express CD30 on their surfaces only after activation. Based on research in 2016, CAR T-cells based treatments that target CD30 antigens derived from HRS3scfv are very good therapies in the treatment of CD30+ malignancies, which do not target healthy lymphocytes and HSPCs [12].

CD33 is a myeloid cell surface antigen that is not expressed on blood stem cells or within the hematopoietic system, but it can be expressed on the surface of natural B-lymphocytes, activated T-lymphocytes, and natural killer (NK) cells; it can be utilized as a therapeutic target in patients with Acute Myeloid Leukemia (AML) [13].

Studies have demonstrated that leukemic cell lines and tumor progenitor cells are somehow efficiently used in vitro together with CAR T-cells that target CD33 antigen on the surface of these cells. A notable point is reduced tumor cell numbers in murine models with AML treated with CD33-targeting CAR T-cells compared to untreated murine models. This also indicates the effectiveness of this system under laboratory conditions. Accordingly, it can be concluded that this class of CAR T-cells plays a significant role in preventing the disease progression of AML patients [14].

CD123 is a remarkable target on leukemic stem cell surfaces, the expression of which has been greatly reduced in HSPCs in comparison to the two previous groups. Research has shown that CAR T-cells targeted by CD123 antigen receptor act vigorously not only in vitro but also in clinical conditions and in widespread AML-affected animal models [15].

Wilms tumor 1 (WT1) is expressed increasingly in many cancers such as hematologic malignancies, acute and chronic leukemia, and most of the solid tumors. In aa research, CAR T-cells were designed, called 28Z WT1, which target the WT1 intracellular protein in human CAR T-cells28z WT1s designed explicitly for the WT1-HLA-A complex. The results showed that these CAR T-cells not only target the intracellular antigens but also show no tendency to target T-cell surface-expressing proteins [16].

In addition to tumor-specific antigens/tumor-associated antigens (TSAs/TAAs), tumor angiogenesis is also considered as a target of CAR T-cells. In a series of cancer-combating methods, researchers target tumor vascular cells, instead of the cell itself, using CAR T-cells targeting VEGFR2 (Vascular Endothelial Growth Factor Receptor2). Since the angiogenesis process is associated with cancer progression and metastasis to other body parts, this method seems to be more potent in coping with cancer. Previous study presented evidence of increased growth and survival rate in inhibited tumor cells by the use of CAR T-cells against VEGFR2 tumors together with extracellular interleukin-12 (IL-12) in a cancerous murine model [17].

In complementary studies with CAR T-cells targeting VEGFR2, tumor-specific TCR (T-cell Receptor)-expressing cells were presented for cancerous murine models and their synergetic antitumor activity induced a stronger response against cancer and additionally improved the survival rate of patients. Results of such studies can be promising in the treatment of a wide range of cancers through targeting tumor angiogenesis by CAR T-cells [18].

Other antigens have also been studied in blood malignancies and solid tumors both in vitro and in vivo clinical examinations, including CD138 in Multiple Myeloma (MM), Natural Killer Group 2 member D (NKG2D), leukemia, and Carcinoembryonic antigen (CEA) in colorectal cancer [19].

As mentioned earlier, CAR T-cells have drastically been successful in the treatment of hematologic malignancies. Consequently, CAR T-cell based therapeutic techniques have been developed against blood antigens such as CD22, CD20, and CD11. Nonetheless, the capability of CAR T-cells has been less evaluated in the treatment of solid tumors, which is assumed to be due to toxic side effects and lack of proper responses to the treatment by patients. Despite conducting 81 tests on hematologic malignancies using CAR T-cells, only 51 cases have been documented for solid tumors. Because the effect of CAR T-cell based therapies in solid tumors is far lower than hematologic malignancies, several factors have so far been found that contribute to the improvement of such inefficiencies (Fig. 2). There are a number of differences between hematologic malignancies and solid tumors, and examination of individual aspects revealed that all kinds of manipulations should be integrated to improve most CAR T-cell based therapies in solid tumors further [20].

Typically, hematologic malignancies develop in a widespread manner. Also, antigens available in such malignancies are homogenous and are expressed in most tumor cell populations. In solid tumors, on the other hand, targeted antigens are heterogeneous and differ not only in a single tumor, but also between primary and metastatic tumors. Hence, treatment with CAR T-cells in solid tumors is associated with multiple problems. The first is the delivery method of CAR T-cells as these cells need to face with a proper chemical signaling pathway to be released in sufficient numbers to tumor cells. Abnormal angiogenesis system in tumors decreases the ability of CAR T-cells to penetrate into the tumor. On the other hand, physical barriers such as the tumor-surrounding stroma prevent adequate penetration of CAR T-cells. Eventually, multiple tumor suppressors such as cellular checkpoint pathways, cytokines, and disrupted metabolic byproducts all pose a great challenge on the use of CAR T-cells [21].

PSMA is a membrane-bound glycoprotein with 750 amino acids, which is expressed in large quantities on the endothelia of many solid tumors, especially prostate cancer cells. Both animal models with prostate cancer and laboratory tests of PSMA antigen-targeting CAR T-cells have a clearly specific activity on the surface of cancer cells against the tumor. With continuous efforts of researchers, the second generation of these anti-PSMA CAR T-cells was developed to enhance the efficiency of the first generation. The second generation of these CAR T-cells releases more cytokines than the first generation and also multiplies at a higher rate in vitro. In addition, these second generation of CAR T-cells seems to suppress prostate tumors more strongly in animal models. According to these results, an optimum method can be achieved by improving these methods at the clinical phase [22].

EGFR is an antigen identified in 2017, which is expressed in approximately 30% of glioblastoma cases and is associated with poor prognosis. Miao et al. in 2014 investigated D-270 MG intracranial tumors and found that CAR T-cells targeting EGFR III molecules could suppress tumor growth and improve the vitality of murine models [23]. In another report, it was similarly shown that mice with glioma were cured well with the third generation of these CAR T-cells [21].

Surprisingly, the use of these CAR T-cells at the clinical stage in patients with brain cancers was associated with successful expression of EGFRvIII on the surface of their cells. Considering the high potential and effective therapeutic efficiency, results of clinical trials on CAR T-cells targeting EGFRvIII have received considerable interest in glioblastoma treatment [24].

GD2 (Disialoganglioside) protein is overexpressed among most of the solid tumors, such as neuroblastoma, retinoblastoma, glioma and Ewing family of tumors in children and adults. In previous studies, the anticancer property of GD2-targeting CAR T-cells in neuroblastoma cells has been demonstrated in vitro and animal models with neuroblastoma in clinical conditions. A clinical phase trial revealed tumor necrosis in four neuroblastoma cases, which were recovered entirely through treatments based on this class of CAR T-cells and no side effects of this treatment were observed in 11 patients during a 20-month follow up. As a result, the use of CAR T-cells against GD2 has provided the researchers with a targeted therapeutic modality [25].

Among primary problems in the use and delivery of CAR T-cells are challenges in their migrations, adequate penetration into tumors, and tissue toxicity. One of the factors leading to the increased ability of CAR T-cells against hematologic malignancies is the hematologic origin of tumors and CAR T-cells, which hence attain an increasing tendency to migrate to areas such as bone marrow and lymph nodes.

On the other hand, solid tumors release such cytokines as CXCL5 and CXCL12 to prevent the migration of T-lymphocytes to these regions, and usually, chemokine receptors on T-cells are not sufficiently compatible with the characteristics of tumor chemokines. All these results in the migration of a minimal number of T-lymphocytes to the tumor. The number of T-lymphocytes migrating to the tumor can be increased through the creation of chemokine profiles expressed with tumor cells and by the construction of engineered CAR T-cells capable of expressing optimal chemokine receptors [21]. For example, engineered cells with CXCR2 expression are capable of migrating to a wide range of tumors with CXCL1 expressing cells. CCR2b-expressing and CCR4 receptor- containing CAR T-cells can be used in mesothelioma and neuroblastoma cancers and in the Hodgkin’s lymphoma, respectively. In addition, the tumor surrounding stroma is able to secrete a variety of chemokines; therefore, tumor placement site and stroma cytokinesis have profound effects on the tumor chemokine profile. Besides, instead of manipulating the chemokine receptor on CAR T-cells, chemokines released by tumor cells can be modified to adapt to typical receptors on CAR T-cells [26].

Direct injection of CCL5 and IL5 expressing adenoviruses into neuroblastoma tumors leads to the penetration of CAR T-cells into the tumor and better control of the tumor. Likewise, an injection of Herpes simplex virus merged with EGFR-CAR-NK-92 cells in tumors is very effective before metastasis, leading to a wide penetration of T-cells deep into the tumor. This procedure can be performed in primary malignancies and metastasis using viral carriers such as Vaccinia virus or other delivery methods such as cell carriers. Additionally, changes can be made in the tumor microenvironment (TME) to be able to receive CAR T-cells better [27].

Another barrier facing CAR T-cells prior to entering the TME with immunosuppression properties is a physical obstacle that reduces sufficient penetration of these cells into the tumor. Myeloid cells suppressing the immune system are capable of absorbing into the TME and can prevent the penetration of CAR T-cells into the relevant tumor. In addition, these myeloid cells and tumor fibroblasts lead to the creation of fibrotic extracellular matrix, which is, in turn, a difficult barrier to the penetration of CAR T-cells. Heparanase (HPSE) is an enzyme that degrades heparin sulfate in peptidoglycans and is the most abundant constituent of the extracellular matrix. In laboratory tests, heparanase depletion was observed in culture media of CAR T-cells. Thus, increased expression of heparanase in CAR T-cells or direct targeting of non-malignant stroma with CAR T-cells against TAA protein, the fibroblast activating protein, eliminates this effect followed by increased penetration of CAR T-cells into the tumor and its TME [28].

An ideal target in immunotherapy is prostate-specific membrane antigen (PSMA). This antigen is indeed found only on the membrane of malignant prostate cells and the vasculature endothelium of some tumors and cannot be found on normal vessels. Delivery of PSMA-containing CAR T-cells to murine models with ovarian cancer results in tumor withdrawal, though this is not a complete response given the heterogeneous PSMA expression in the tumor. Accordingly, it can be suggested that the integration of tumor targeting and tumor-specific antigens with CAR T-cells results in their elevated capability against solid tumors with low and heterogeneous expression on antigens [22], [29].

Despite the high capacity of T-cells with modified CAR protein, their application in the treatment is accompanied by a high toxicity potential. The extent of this toxicity has been reported in various studies, including the most hazardous Cytokine Release Syndrome (CRS), Macrophage Activation Syndrome (MAS), neurotoxicity, and Tumor Lysis Syndrome (TLS). CRS and neurotoxicity seem to occur in B-cell associated malignancies and are fortunately curable in most cases. CRS is associated with high levels of various circulatory cytokines and includes IL-6 and γ-interferon being apparently associated with high tumor activity and disease progression. CRS has been frequently observed to occur together with MAS and involves a slight increase in IL-16. Tocilizumab monoclonal antibody can inhibit CRS and MAS, and also reduces inflammation through binding to and inhibition of IL-16 activity [30].

Another type of toxicity caused by CAR T-cell based treatments results from targeting of antigen-expressing normal cells even at low levels. This kind of toxicity was initially reported in the first clinical phase of renal carcinoma treatment. Patients underwent treatment with T-cells, whose CAR protein targeted carbonic anhydrase CA1X antigen. Meanwhile, a great number of treated patients developed significant toxicity in their liver tissues, and it seemed that this toxicity rooted in CA1X expression in the biliary epithelial cells. Hence, the treatment process was stopped immediately in these patients. Unfortunately, the first report of lethal toxicity resulting from targeting normal cells by CAR T-cells was observed in a number of patients with colorectal cancer undergoing treatment [31].

Patients suffered from severe respiratory disorders and cardiac arrest and lost their lives after 5 days due to multiple disorders in several vital organs. Researchers speculated that the used CAR T-cells targeted ERBB2 antigen expressed at low levels in the lung epithelial cells leading to toxicity in the respiratory system tissues and triggering a cascade of secreted cytokines, eventually causing the deaths of patients [32]. In fact, upon depletion of B cells in the recipient’s body immune system, an antigen targeting event can somewhat be predicted to occur on normal cells. This process occurred in most patients undergoing treatment with CAR T-cells targeting CD19 antigen, and B-lymphocyte aplasia varies from few months to several years depending on the conformation (spatial structure) of the identifier CAR protein. To prevent this toxicity, immunoglobulin replacements can be prescribed monthly for patients and perform a long-term follow up to avoid late side effects of B-lymphocyte aplasia [33].

To inhibit severe and chronic toxicity, suicide genes can be introduced by carriers to the cells of recipient patient, which are used for the delivery of CARs. Also, the developed toxicity can be reduced through the simultaneous expression of antibody-binding epitopes (e.g., CD20 and EGFR) on the cell surface [34].

Other approaches are based on CARs with unstable and self-restrictive expression and the use of suppressing antibodies and steroids. Finally, CAR genes can be easily incorporated into T-cells using targeted carriers and improve the level of clinical care. On the other hand, this process might theoretically increase the risk of insertion mutations such as stem cell therapies in primary immunity defects. On the contrary, numerous studies involving more than 500 cancer patients and a one-year post-treatment follow-up suggest the safety of retrovirus gene transduction into T-lymphocytes. However, it is very early to judge that this insert viral is safe, and consequently, it should be examined in larger populations [35].

Besides the CAR T-cells induced toxicity in an organism, reduced risk of detrimental mutations decreased autoimmune disorders, and providing better and more efficient generations of CAR T-cells are also among the challenges facing such treatments. To improve the safety of patients undergoing CAR T-cell treatments, it is necessary to consider further solutions with better efficiencies [36].

Retroviruses can be used as T-cell gene carriers with modified CARs. Since the gene of interest is randomly introduced to the genome in the conventional use of these viruses, the virus may settle in the proto-oncogene region, and the retrovirus entry to that site may activate the oncogene and lead to malignancy. In other words, there is a high and strong risk of gene mutations and tumorigenesis upon the integration of related T-cells. According to previous investigations, TAL effector nucleases (TALEN) and CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) system are considered robust tools for targeted delivery. Accordingly, therapeutic transgenes such as retroviruses carrying CAR T-cells are securely inserted to areas of the chromosome that do not interfere with internal gene activity, and there is no increased risk of cancer development [37], [38], [39].

Furthermore, a form of T-lymphocyte that could persistently express CD40L and was able to increase T-cell proliferation and the influence of the immune system on the tumor. This method improved the CAR/CD40 T-cell specificity in detecting tumor CD19 antigen, with a considerable effect on TME as well. As a result, researchers strive to achieve the best combination state and introduce a new generation of efficient CAR T-cells in all types of cancer treatment in the near future [40] (Table 1).

Another new discovery in the modern world is the use of stem cells for the treatment of chronic diseases. They are non-specific cells with the ability to transform into specific cell types having a specific function. The unique ability of these cells is in their multipotential (pluripotency) and long-term self-regenerative ability, which enable them to repair and replace damaged tissue at the site of injury and thereby use them as suitable sources for wound healing [41], [42]. These cells are able to release a variety of active factors with therapeutic potential and local and systematic effects. Additionally, these cells are inherently capable of migrating to injured sites, which results in the chemical absorption of stem cells at the site of injury mediated by cell surface receptors such as chemokine receptors [43]. Stem cells are undifferentiated cells that are able to proliferate in suitable in vitro culture conditions for unlimited time and transform into specific cell types. In the body, these cells differentiate to produce a variety of tissues. Stem cells are generally used from two embryonic and adult origins [44]. Researchers isolate these cells from the eyes, muscles, skin, adipose tissue, spleen pulp, umbilical cord, and the gastrointestinal tract epithelium of adults. Depending on the tissue of residence, adult stem cells transform into various cell types in that tissue. Stem cells derived from bone marrow, peripheral blood, and umbilical cord blood are sources of transplantation, of which peripheral blood is the best source of stem cells for transplantation [45].

Stem cells are divided into two groups of pluripotent and multipotent stem cells (PPSCs and MPSCs, respectively), with the former having two sources of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) (Fig. 3) [46]. Mesenchymal stem cells (MSCs) are types of adult stem cells originating from MPSCs, which are non-hematopoietic, have the high proliferative capacity, and are able to differentiate into different T-cells being completely dependent on existing media conditions and factors. These cells have the ability to transform into mesodermal cell lines (e.g., various osteocytes, chondrocytes, and adipocytes), ectodermal cell lines (e.g., neurocytes), and endodermal cell lines (e.g., hepatocytes). It is worth noting that the potential for proliferation and differentiation depends on the source of their isolation. For example, the genetic stability of MSCs derived from adult stem cells is influenced by age and environmental stresses [47], [48].

MSCs play their role in modulating the immune system by secreting a variety of cytokines and immune receptors, thereby regulating the microenvironment of their host tissue. Given the potential to transform into multiple cell lines, the ability to regulate the immune system, and secretion of various anti-inflammatory cytokines, these cells are considered a powerful tool in the treatment of chronic diseases and cancer cells, tissue engineering studies, and in vitro pharmaceutical applications [49]. Exposure of MSCs to pathogens influences their secretions, migration, proliferation, and differentiation, during which these cells increase secretions of their cytokines, chemokines, and other signaling molecules. According to the literature, MSCs are capable of orientation and migration in response to chemotactic signals released by pathogenic or cells infected with these agents, as well as abnormal cells [50].

MSCs have the ability to migrate toward primary tumors, which renders these cells to serve as a suitable carrier for transferring anticancer factors to the tumor site [14]. In fact, inflammatory factors recall MSCs to the site of inflammation. In addition, the therapeutic effect of these cells is not solely related to their differentiating ability, and rather, it is their secretory factors that adjust the innate and acquired immunity arms. The collection of these secretory factors is called secretome. Immunity regulations, angiogenesis, anti-apoptotic effect, in situ damage limiting, and recalling tissue progenitor cells are some of the secretome effects [51], [52].

MSCs do not activate the recipient’s immune system because they have very low MHC expression and lack MHC II, therefore do not activate NK cells. On the other hand, MSCs cause the transformation of TH2 responses to TH1 through stimulating the secretion of various cytokines from T-cells. The homing ability, pluripotency, the ability to produce regulatory effects on the immune system, and secretion of anti-inflammatory factors can raise MSCs as therapeutic sources of autoimmune, inflammatory, and degenerative diseases. The homing property of these cells, which means migration of these cells to the injury site, has been suggested as a cell-based therapy. The presence of inflammatory factors (e.g., IL1 and TNFβ) increases their migration [49].

MSCs can modulate immune responses through the secretion of their paracrine mediators and are able to inhibit both innate and acquired immunity. Activation of resident stem cells through chemotaxis signals decreases inflammation and improves tissue repair [53], [54]. By the production of apoptotic inhibitor proteins, MSCs decrease the expression of pro-apoptotic factors such as BAX, break down caspase 3 and, on the other hand, increase anti-apoptotic factors such as BCL2. This function of MSCs in healthy cells is very different from that of cancer cells; as mentioned above, they increase apoptosis in cancer cells both in vitro and in vivo [55].

MSCs can regulate the cell cycle to increase the amount of materials needed for the synthesis phase of the cell cycle, thereby increasing the proliferation of cancer cells such as colorectal cancer. These cells can elevate tumor growth and inhibit apoptosis, angiogenesis, and its metastasis in situ by activating Kb-NF pathway signals mediated by the AMPK/mTOR pathway, which activates the kb-NF pathway. Excessive mTOR pathway activity promotes colorectal cancer and stimulates the growth of MSCs [56]. In this account, if we take a short look on mTOR pathway we can say, the mammalian target of rapamycin (mTOR) creates two main structures including mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). These structures have many key roles in physiological activities, like metabolism, angiogenesis, cytoskeleton remodeling homeostasis, autophagy, biosynthesis of macromolecules, survival and finally protein synthesis. Remarkably, this molecular signaling pathway plays an important responsibility in differentiation, cell growth and interestingly, its deregulation status is suggested in pathological situations containing progression and neoplastic transformation in many different malignancies including gastrointestinal, prostate, breast and also liver cancer [57]. Furthermore, this pathway is included in the metabolically and functional regulatory processes, differentiation and regulating in adaptive or innate immune cells. Conspicuously, it can be used in clinical categories like organ transplantation. Importantly, mTOR may has an impressive activity in the regulation of immune cells, including T cells and macrophages and also by regulating the expression of the inflammatory parameters, comprising cytokines or chemokines, transforming growth factor (TGF)-β) and membrane receptors and ultimately Programmed Death 1. In MSC- derived secretome, there are high expression levels of ILs 6 and 8, both of which are associated with the development of colorectal cancer through activating the kb-NF pathway, thereby leading to tumor growth. Murine BALB/c in an in vivo tumor model and an MSC-derived secretome added to the medium led to increased invasion and proliferation of colorectal cancer cells. On the other hand, MSCs residing in different organs are able to help in damaged tissue repair [58].

Similar to all stem cells, one of the most important features of MSCs is their tendency towards tumors. Existing evidence indicates that adipose tissue-derived MSCs stimulate tumor growth and metastasis in cancer [59]. In contrast, there is other evidence on the potential of these cells to destroy the tumor by inhibiting tumor cell proliferation and inducing apoptosis therein. Obesity, which accumulates adipose tissue and increases the number of MSCs in this tissue, is associated with a variety of cancers such as breast and colon cancers [60].

All the above discussion demonstrates that the use of MSCs in treatment resembles using a double-edged sword [58].

To date, several markers have been introduced for a tumor antigen that is suitable for targeting. One of the most important issues is their specific expression only on tumor cells and not on normal cells. In addition, the target antigen protein should be involved in the growth and survival of tumor cells [61]. Some of these targets include epitopes derived from BCR/ABL displacement in CML, mutations known in the p21/ras protein of multiple malignancies and viral oncogenic proteins such as HPV (Human Papilloma Virus) protein in cervical cancer. In fact, most candidate antigens do not have all these attributes. Most of these antigens are non-mutated proteins expressed at high levels by ectopic cancer cells. It is worth emphasizing that high expression of these antigens in tumor cells compared to normal cells provide suitable T-cells with high affinity, which, in addition to identification and destruction of malignant T-cells, ignore very low expression of these antigens on the surface of normal cells. Tumor antigenic proteins with increased expression include WT1 gene in a variety of leukemia and solid tumors, such as Wilms and HER2/neu tumors in the breast and ovarian cancers [62], [63].

A rapidly emerging immunotherapy approach, called adoptive cell transfer (ACT), includes collecting patient's immune cells and using them for cancer treatment. Chimeric Antigen Receptor T-cell (CAR T-cell) is an example of Adoptive Cellular Immunotherapies (ACI), which themselves are a subset of complex biological therapies (CBTs). Unlike many conventional drugs, ACIs, including CAR-T-cells, are considered therapeutic - and not soothing - drugs so that they should be used only once or several times. It should be emphasized that the characteristic of ACIs means that only a small subset of patients with any cancer may be appropriate for treatment, and each ACI can separately be used for each patient [64].

Although there are different types of ACTs, only one called CAR T-cells treatment, has been approved so far by the Food and Drug Administration (FDA). Also, despite significant differences between these treatments, all share similar components. CAR is formed by fragments or domains of synthetic antibodies on the cell surface. Domains that are used can affect the detection and receptor binding to tumor cell antigen [23].

As indicated by the name of CAR T-cell, T-cells are the backbone of treatment that plays a vital role in organizing immune response and killing cells infected with pathogens (any virus, bacteria, or another disease-causing agent). Treatment involves taking blood samples from patients and isolating their T-cells, followed by viral disarmament. Then, T-cells are genetically engineered to produce receptors on their surface, called chimeric antigen receptors (CARs). These specific receptors allow T-cells to identify and bind to a specific protein or antigen on a tumor cell. Once collected T-cells were engineered to express CAR-specific antigen, they are replicated to hundreds of millions of copies in the laboratory. The final stage is the injection of CAR T-cells to patients. If everything is arranged in a programmed manner, engineered cells are multiplied in the patient's body and identify and destroy cancer cells carrying antigens on their surface, which is steered by their engineered receptors [65].

The above-mentioned chimeric receptors are first made as chimeric cDNAs and manipulated properly so that they are capable of detecting specific molecules on the surface of tumor cells and lead to the activation of related T-cells by specific signaling. An advantage of this technology is that, unlike the αβ T-cell receptors (TCR), CAR receptors can identify antigens directly and without restriction through antigen providing polymorphic factors such as HLA [66].

For their function, receptors are dependent on intracellular stimulating signals. Thus, any CAR T-cell has an intracellular messenger and 'auxiliary-stimulus' domains that receive messages from surface receptors. Different applied domains can affect general cell function.

The advantages of cognitive immunotherapy strategies can be summarized in the following three items [18]:

• Independence of CAR T-cells from HLA molecules. Therefore, one type of CAR with an expression of a specific antigen can be used for all patients.

• The ability of malignant T-cells to interfere with the expression of antigens at their cell surfaces and escaping from immune system response. As such, CAR T-cells are still able to identify tumor cells irrespective of this process.

• Possibility of targeting a wide range of macromolecules such as proteins, carbohydrates, and glycolipids by CAR T-cells.

Creation of multiple genome modifications is one of the most appealing applications of the CAR T-cell system being considered an efficient technique for upgrading T-cell based immunotherapies. Even so, the low efficiency of DNA transfection process in gene targeting has limited the application of technologies generating multiple changes in primary T-cells [67].

Based on clinical trials in 2017, magnetic balls can easily be used for a 99% purification of genes encoding CAR T-cells. Genetically engineered T-cells never express GVHD. Hence they can be used as allogeneic CAR T-cells. A noteworthy point is the similar ability of modified T lymphocytes to normal T-cells both in vitro and in affected murine models. Previous studies have reported that T lymphocytes can be genetically engineered by genome changing technologies such as CRISPR/Cas9. These techniques actually influence the expression of internal T lymphocyte receptors through targeting their β and α chains, thereby inhibiting GVHD expression [68].

Because disturbing T-cell genes by lentivirus and adenovirus vectors is not associated with sufficient efficiency, researchers have increased the induced alteration efficiency using nucleus contamination methods by CRISPR components in CD4 T-cells. Although the toxicity created by this process has limited its application, research has shown that the downregulation of the HLA-1 gene dramatically reduces the immune system sensitivity to allogeneic T-cells thereby inhibiting rapid rejection of CAR T-cells carried by the host. However, evaluation of GVHD and host immune response by murine models with defective immunity in T lymphocytes cannot be generalized to the allogeneic transplantation events in humans. Accordingly, more accurate tests can be performed on nonhuman primates in the future to be able to confirm the safety of using these cells to inhibit the immune system of the recipient’s body. Before the clinical use of CAR T-cells modified by CRISPR/Cas9 for the treatment of patients, extensive sequencing techniques can be applied to ensure the modifications and the absence of unwanted mutations. In other words, it is necessary to examine and confirm the integration of CAR T-cell genome before their use in human clinical therapies. Universal CAR T-cell (U CAR T-cell), for example, CAR T-cell, has been invented by the TALEN technique through chromosomal translocation in this type of CAR T-cell-derived from three different donors, which shows no chromosomal rearrangement [69].

The therapeutic value of suppressing the immunosuppressive system, called PD-1, via its inhibition in CAR T-cells penetrating solid tumors and suppression of the triple genes (TCR, B2M, and PD-1) has been demonstrated in leukemic tumors. One of the potential limitations regarding inactivation of these three genes in CAR T-cells is possible stimulation of normal and passive killer cells and rejection of modified T-cells. The use of chemotherapy prior to this process can help reduce the rejection level of CAR T-cells carried to the host body by this factor through lymphocyte evacuation from the host body or using antibodies against natural killers to kill these cells [70].