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CAR-T cells for cancer treatment: current design and future development
CAR-T cells for cancer treatment: current design and future development. Immunotherapy has been growing in the past decade as an alternative therapy for cancer treatment.
In this chapter, we studied CAR-T cells, which are genetically engineered autologous T cells that can express chimeric receptors specific to antigens expressed on the surface of tumor cells.
Although this type of personalized therapy is revolutionizing cancer treatment, especially B-cell malignancies, it still has some challenging limitations.
Here, we discussed the basic immunological and technical aspects of CAR-T cell therapy, the limitations that have compromised its efficacy and safety, and the current proposed strategies to overcome these limitations, so that CAR-T cells have greater Therapeutic application value.
Conventional cancer treatment methods, such as chemotherapy and radiotherapy, are effective in many cases, but they still have several limitations: (a) They are not specific and therefore affect two tumors the same as normal cells; (b) They cannot effectively eliminate tumor cells in all patients; (c) they weaken the body’s natural ability to defend against cancer cells, and (d) they may cause a variety of side effects that affect the treated patients.
In the past decade, immunotherapy has become an alternative treatment method for many types of tumors, which can be used alone or in combination with other conventional or innovative treatment methods . Immunotherapy consists of technical methods that use soluble or cellular components of the immune system for the treatment of cancer and immune-mediated diseases . Adoptive cellular immunotherapy is characterized by the use of cell subsets of the immune system for therapeutic purposes .
In recent years, the development of genetic engineering technology and cell-based biological processes has allowed the generation and expansion of genetically modified anti-tumor T cells, overcoming the actual obstacles that previously limited the use of tumor infiltrating lymphocytes (TIL) in immunotherapy programs  .
2. CAR design
CAR-T cells are T cells that have been genetically engineered to express chimeric receptors that target specific antigens. CAR (Chimeric Antigen Receptor) is a chimeric molecule of T cell receptor (TCR) fused with an antigen recognition domain, such as a single-chain fragment of a monoclonal antibody (scFv) . Therefore, despite having natural TCR, CAR-T cells can still recognize specific antigens on the surface of other cells through CAR receptors. In contrast to TCR-mediated recognition, CAR antigen recognition does not depend on the major histocompatibility complex (MHC).
CAR molecules can bind to antigens recognized by their scFv regions on the surface of tumor cells. Antigens can be proteins, carbohydrates or lipids, because antibodies/scFv can bind to such molecules. For all targeted cancer therapies, the target must be specific to cancer cells to prevent damage to healthy cells and tissues.
B-cell tumors are considered to be ideal drug candidates for CAR-T cell targeted therapy. B cells can be easily targeted by specific and selective markers (such as CD19). In addition, CD19 is not present in most normal tissues (except for normal B cells), which makes it a relatively safe target/antigen .
Currently, CARs are divided into three generations according to their intracellular signal transduction domains. The first generation consists only of the extracellular domain (scFv) and the intracellular CD3ζ domain (Figure 1a). The second-generation CAR has a unique CD28 or 4-1BB molecular costimulatory domain, which improves the clinical efficacy of the treatment by increasing the survival rate of modified T lymphocytes (Figure 1b) [5,6]. The third generation CAR contains molecules with three or more cytoplasmic costimulatory domains. In addition to CD28 and 4-1BB, CD27, ICOS or OX40 can also be coupled, theoretically improving the activation, survival and effectiveness of genetically engineered T lymphocytes (Figure 1c). However, so far, the second-generation CAR has shown the best clinical results and safety [7, 8].
3. Lymphocyte activation and CAR transduction
The production of CAR-T cells begins with the collection of peripheral blood mononuclear cells (PBMC) from patients through blood sampling or peripheral venous blood collection. Initially, unfractionated has been used as a raw material for its CAR-T cells to produce T cells. Since the composition of PBMC may vary greatly, this technique may produce inconsistent CAR-T cell products.
Immunomagnetic beads can be used to separate T cell subpopulations, thereby removing monocytes, thereby inhibiting the activation and expansion of T cells [9, 10], thereby producing CAR-T cell products rich in T cells (total CD3, CD3+ CD4+ Or CD3+CD8). However, the CD4:CD8 ratio varies greatly between patients. The number and frequency of CD4 and CD8 subgroups receiving various chemotherapy treatments for cancer vary greatly. Compared with healthy individuals, there are more CD8 than CD4 T cells. This may affect the preparation of anti-tumor activity CAR-T cell products .
The functions of CD4 and CD8 T cells, their ability to proliferate and persist in the body, and their ratio in peripheral blood (CD4:CD8 ratio) are different. In addition, the CD4 and CD8 subgroups (natural, memory stem cells, central memory, effectors and regulators) differ in their extracellular and intracellular markers and their metabolic and epigenetic signaling pathways [12,13]. In this case, the CD4 and CD8 subsets can be separated during the preparation of CAR-T cell products, and they can be added in a determined ratio at the end of the expansion.
In the near future, as part of the biological process of CAR-T cell therapy production, specific T cell subsets can be isolated before activation/transduction or after expansion to regulate the cellular composition of CAR-T cell products. Of course, controlling the cellular composition of CAR-T cell products will reduce their variability and improve their proliferation, persistence and efficacy in vivo.
4. The limitations of current autologous CAR-T therapy and the next development direction
Although CAR-T cell therapy has been clinically successful in recent years, this treatment method still has many inherent conceptual limitations.
The conventional CAR currently in use has a single target specificity, and due to the heterogeneity of most tumors, it may lead to tumor escape [17,18]. Another limitation of current therapy is that traditional CAR design cannot directly control the reactivity of CAR-T cells. After infusion, CAR-T cells can expand to 10,000 times in response to their antigens, making the degree of response unpredictable, which may cause serious side effects . In recent clinical trials, approximately one-third of patients experienced severe fever and inflammation, and all patients had developed chronic B-cell hypoplasia. B cell hypoplasia can be alleviated by the administration of gamma globulin.
Among the side effects, the most serious is the “cytokine release syndrome” (CRS) that is potentially life-threatening. In patients with increased tumor burden, CRS seems to be aggravated and is often accompanied by macrophage activation syndrome (i.e. uncontrolled activation and proliferation of macrophages) and tumor lysis syndrome (i.e. sudden intracellular material after tumor lysis) Released into the bloodstream). Fortunately, the effects of CRS can be reduced by reducing the number of injected T cells and/or by administering anti-IL-6 receptor monoclonal antibodies (Tocilizumab, Actemra®) and steroids .
Some strategies to improve the efficacy and safety of current CAR-T therapies are currently being discussed (Figure 2).
4.1 CAR transduction of allogeneic T cells
The current autologous CAR-T cell therapy is aimed at patients. Therefore, they require labor-intensive work and a long production time (usually 3-4 weeks to produce CAR-T cell products). Although it does not take long to consider personalized treatment, it is much longer than many non-personalized cancer treatments, which can be used almost immediately by patients.
Due to the low lymphocyte count and the invaded cells present in cancer patients, the expansion and production of self-modified T cells is not an easy task. Due to production failure, approximately 9% of patients receiving Kymriah (tisagenlecleucel) pilot treatment cannot obtain the product . Therefore, ready-to-use allogeneic genetically modified T cells made from healthy donors are attractive in many ways.
Allogeneic donor and recipient cells may have incompatibility in the human leukocyte antigen (HLA) complex, leading to severe graft-versus-host disease (GvHD) . On the other hand, rejection can lead to the removal of CAR-T cells, leading to treatment failure. To solve this problem, HLA knockout allogeneic CAR-T cells (UCART19) were developed to prevent allogeneic reactions . The early results of the UCART clinical study showed that 4 of the 6 patients who received the initial dose of treatment relapsed 4-6 months after treatment, and 1 of them had skin GvHD, which indicated that HLA molecules are part of CAR-T cells. The expression is allergic ].
In order to solve these problems, research is moving towards the next generation of CAR-T therapies, which are allogeneic therapies or “off the shelf” therapies, which can be mass-produced from healthy donor cells and used in multiple patients. For allogeneic products, many questions still need to be answered, such as whether T cells from one donor can be fully expanded, or must one donor make multiple donations, and whether comparable CARs from different donors will be obtained -Is it better to use batches of T cell products, or to use a large number of donor lymphocyte pools for large-scale production? Of course, for different donors, the individual variability at the cell level is usually high, but cells collected from the same donor at different times also show a certain degree of variability.
4.2 Management of CAR-T cell therapeutic toxicity
In some cases, CAR-T cells can expand uncontrollably in the body, which may be related to life-threatening toxicity and chronic B cell dysplasia. Therefore, there is a need to control the proliferation of engineered T cells in patients. To meet this challenge, a switchable CAR (sCAR) has been developed [30, 31]. sCAR-T cells can control the activity of CAR-T cells in dose titratable by using antibodies or small molecule-based switches. Another advantage of this system is that it can switch to different targets. In addition, CARs with suicide genes have been developed to avoid uncontrolled amplification in vivo .
On the other hand, the immunosuppressive tumor microenvironment may have a negative impact on the expansion and activity of CAR-T cells in the body. One strategy to overcome this limitation is to co-modify CAR-T cells with pro-inflammatory cytokines such as interleukin 12 (IL-12) and interleukin 18 (IL-18). IL-12 is a pro-inflammatory cytokine produced by DCs and macrophages, and has been shown to promote the maturation of DCs and increase the proliferation of T cells .
4.4 Development of CAR-T cells that recognize multiple antigens
The conventional CAR currently in use has a single target specificity. Due to the heterogeneity of most tumors, this method can lead to tumor escape. A new CAR design, tandem CAR, has been designed to express two more specific and safer antigen binding domains. Tandem CAR T cells are only activated when they recognize two different antigens at the same time. Hegede et al.  developed a tandem CAR with anti-human epidermal growth factor receptor 2 (HER2) and IL-13 mutant binding to IL-13 receptor α2 (IL-13Rα2), in which CD28 acts as Co-stimulatory molecules, and CD3ζ chain as the signal transduction domain. Therefore, it may bind to HER2 or IL-13Rα2 to reduce tumor antigen escape .
Recently, scientists from Boston University and the Massachusetts Institute of Technology (MIT) developed a new CAR technology called “Split, Universal and Programmable” (SUPRA) CAR, which simultaneously addresses tumor resistance and immune system The problem of over-activation and specificity . SUPRA-CARs are composed of two parts: one is a universal receptor expressed in T cells, called zipCAR, and the other is a tumor-targeting scFv adaptor, called zipFv. The zipCAR universal receptor is produced by the fusion of the intracellular signaling domain and the leucine zipper as the extracellular domain. The zipFv adaptor molecule is produced by fusion of an appropriate leucine zipper and scFv. The scFv of zipFv binds to tumor antigens, and leucine zipper binds and activates zipCAR in T cells . This SUPRA-CAR has unique characteristics that can adjust multiple variables, for example, (1) affinity between two pairs, (2) affinity between tumor antigen and scFv, (3) concentration of zipFv, and (4) zipCAR The expression level can be used to regulate the response of T cells. In vitro experiments show that SUPRA-CAR has the potential to increase tumor specificity and reduce toxicity. In vivo preclinical tests have shown that SUPRA-CAR can reduce the tumor burden in breast cancer xenograft mouse models and hematological tumor models . In addition, in vivo studies have confirmed that a variety of methods can be used to control the SUPRA-CAR system, such as CRS, to precisely regulate the SUPRA-CAR system .
Fig. Design and characteristics of SUPRA CAR system (A) Comparison between conventional CAR and SUPRA CAR design. The SUPRA CAR system consists of zipCAR and zipFv. zipCAR has a leucine zipper because of the extracellular part of CAR, while zipFv has scFv fused to an associated leucine zipper, and scFv can bind to the leucine zipper on zipCAR. (B) The SUPRA CAR system that uses different zipFvs to target multiple tumor antigens. K562 cells expressing Her2, mesothelin or Axl and CD8+ human primary T cells expressing RR zipCAR (n=3, mean±SD) were co-cultured in vitro. (C) Variables explored for characterizing the SUPRA CAR system: (1) Affinity between leucine zipper pairs; (2) Affinity between tumor antigen and scFv; (3) Concentration of zipFv; (4) SVF Expression level zipCAR. (D) The effect of the concentration of leucine zippers (SYN 3, SYN5 and EE) with different affinities between three zipFv and RR zipCAR on IFN g produced by primary CD4+ T cells (n = 3, mean ± SD). (E) The effect of zipper affinity, scFv tumor affinity and zipCAR expression level on the production of IFN-g by primary CD4+ T cells expressing RR zipCAR (n=3, average).
4.5 Improve the activity of CAR-T cells in the immunosuppressive microenvironment
Normally, CAR-T cells cannot migrate and/or penetrate to the tumor site. The incorporation of chemokine receptors into CAR molecules has been shown to improve the transport of CAR-T cells to solid tumors .
Another strategy to increase CAR-T cell infiltration in stromal stromal tumors is to incorporate heparanase into CAR molecules, which will produce and degrade the extracellular matrix components of tumor tissues in situ .
CAR-T cells are a revolutionary therapy for cancer patients. So far, satisfactory clinical results have been obtained for B-cell malignancies. However, for the treatment of solid tumors, CAR-T cells are not so effective. Therefore, many improvements are still needed. As described in this chapter, several promising methods are improving the therapeutic efficacy and safety of CAR-T cells.
At present, the greatest anti-tumor activity of CAR-T cells coincides with the highest side effects. Therefore, several safety adaptation measures have recently been proposed, such as suicide genetically modified CARs or split CARs (such as SUPRA-CAR).
In addition, the persistence of CAR-T cells in vivo needs other optimizations to provide longer-lasting protection against tumor recurrence. The development of allogeneic CAR-T cells and new design adjustments of CAR molecules will promote the development of this therapy. Synergistic therapy with other conventional or immunotherapies (such as immune checkpoint inhibitors) may expand the clinical application of CAR-T cells in the near future.
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