June 20, 2021

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The emerging vision of immune cell therapy

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The emerging vision of immune cell therapy

The emerging vision of immune cell therapy



The emerging vision of immune cell therapy. Fundamental advances in immunology, molecular biology, and virology, as well as technological advances in cell manufacturing and genetic engineering, have led to exciting developments in immune cell therapy, and T cell therapy has become the most advanced therapy in this category.

Tumor Infiltration The adoptive transfer of T cells and T cells can express recombinant T cell receptors that recognize tumor antigens. It mediates an impressive response rate in some solid cancers. Chimeric antigen receptor-modified T cells are effective against all standards.

Drug-resistant B-cell malignancies showed impressive responses, and virus-specific cytotoxic T lymphocytes (CTL) effectively controlled some viral infections in immunocompromised hosts (Figure 1).

This success has enabled immune cell therapy to develop from small-scale research in research institutions to global commercial enterprises. The continuous advancement of immunobiology and synthetic biology, the rapid advancement of clinical-scale genetic engineering and gene editing technology, and the integration of private sector investment will enable immune cell therapy to have a greater impact on human health in the coming decades. From this perspective, we summarize the current status and future prospects of immune cell therapy for cancer, infectious diseases, autoimmune and other diseases.

The emerging vision of immune cell therapy

Figure 1. Immune cell therapy for the treatment of human diseases


Recent advances in synthetic biology and bioengineering have expanded the applicability of immune cell therapy, including cancer, infection, allogeneic transplantation, and autoimmunity. CAR-T or NK cells, engineered TCR and TIL therapies have been and will continue to be tested in hematology and solid cancer. Tregs, CAR-Tregs and CAAR-T cells are being developed to treat various autoimmune diseases and prevent rejection of transplanted tissues.



T cell therapy for cancer

Adoptive transfer of tumor-infiltrating lymphocyte TIL

Studies conducted in the late 1980s and early 1990s showed that 25%–50% of patients with metastatic melanoma received autologous tumor infiltrating T cells (tumor infiltrating lymphocytes [TILs]) plus recombinant human leukocytes expanded in vitro The infusion therapy of Interleukin-2 (rhIL-2) experienced a lasting complete remission. These impressive results are groundbreaking because they provide irrefutable evidence for human tumor-specific T cell-mediated immunity and lead to molecules that are the basis for T cells to recognize autoantigens and neoantigens. .

Other studies have demonstrated the key role of lymphopenia-induced steady-state cytokine elevations in supporting the expansion of adoptively transferred T cells, demonstrating that lymphocyte clearance therapy is a key component of effective adoptive T cell therapy for cancer.

Although the overall clinical effect of TIL therapy in oncology is limited, partly because non-melanoma patients cannot reliably produce sufficient amounts of bioactive TIL, receiving infusions in patients with metastatic colorectal cancer or breast cancer is patient-specific After TIL treatment with neoantigens, promising results were also observed. .


T cells expressing engineered TCR

Advances in genetic engineering have allowed researchers to clone tumor-reactive T cell receptors (TCRs) from the TIL of responding patients, and express TCRs in T cells amplified from the peripheral blood of other cancer patients, providing potential for therapeutic applications An unlimited number of cells (Figure 2). Although it is technically feasible to generate T cells expressing this “engineered TCR”, the early experience of applying this method to the clinic faces some challenges.

One problem is the pairing of transgenic, tumor-specific TCR alpha and beta chains with endogenous receptors, which leads to low levels of transgenic TCR expression and the risk of off-target toxicity. By optimizing the vector design and adding cysteine ​​and/or murine elements into the transgenic α and β chains to induce preferential pairing of the transgenic protein.

The emerging vision of immune cell therapy

Figure 2. Continuity of immune cell therapy


Non-engineered immune cell therapy (left) has also shown clinical efficacy in various disease backgrounds, including peripheral Treg isolated from patients. These Tregs are expanded and reinjected into patients to treat autoimmune diseases, GVHD or Organ rejection. Tumor infiltrating lymphocytes (TIL) are separated from the resected tumor (usually from a patient with melanoma), expanded in vitro, and reinjected into the patient. The stored donor virus-specific CTLs are thawed, amplified, and reinjected into HLA-matched recipients to treat chronic infections.

Engineered immune cell therapy (right) is produced by first extracting or drawing blood from a patient, isolating T cells, and then using viral or non-viral methods to insert a transgene encoding a synthetic receptor. Examples of engineered T cells include:

(1) T cells expressing engineered TCR composed of TCR α and β subunits;

(2) T cells expressing CAR (CAR-Ts) or NK cells (CAR-NKs), consisting of The extracellular antigen-binding domain is fused with the intracellular domain involved in TCR signal transduction;

(3) CAAR T cells (CAAR-Ts), in which the chimeric receptor is composed of the antigen-binding domain targeting self-reactive B cells ;

(4) CAR-Tregs, in which Tregs are isolated from peripheral blood and designed to express a CAR, redirecting them to tissues affected by autoimmune diseases.

Before being reinjected into the patient, all engineered T cell types are further expanded in vitro. It is worth noting that T cells modified to express CAR or TCR mainly contain cytotoxic effectors, because current culture methods do not significantly enrich Tregs.

The second challenge is to identify safe and effective TCRs for genetic transfer included in the TIL population. A large proportion of TILs present in melanoma tumors can recognize self-antigens expressed at low levels on healthy tissues, highlighting the potential of engineered TCRs to induce significant targeted, non-tumor toxicity (see Table 1).

Compared with TCR that recognizes foreign antigens, TCR that recognizes self-antigens usually exhibits low efficacy, partly related to low affinity caused by thymus selection.

To improve the effectiveness of TCR in recognizing tumor-associated self-antigens, researchers used yeast or phage display or enhanced their affinity by immunizing mice with human antigens. Tumor-reactive TCR with enhanced affinity usually shows enhanced efficacy, but in clinical trials, because it recognizes low-level antigens on normal tissues, it increases the risk of intra-target and extra-tumor toxicity.

TCRs with enhanced affinity are also prone to cross-reactions, as shown in two deaths in a study, in which T cells were designed to express high-affinity TCR HLA-A targeting melanoma antigen gene (MAGE)-A3- *01 The derived peptide in the background cross-reacts with the titin expressed on the heart tissue.

In a related example, a high-affinity TCR designed to target MAGE-A3 induced fatal neurotoxicity, possibly due to cross-reaction with MAGE-12-derived peptides expressed in brain tissue.

The emerging vision of immune cell therapy

Methods have been developed to predict off-target toxicity of engineered TCRs, and some high-affinity TCRs have demonstrated safety and significant clinical activity. The most notable is the high-affinity TCR (c259), which recognizes peptides derived from NY-ESO-1/LAGE-1 expressed on HLA-A2.

Approximately 50% of patients received the lymphocyte clearance preparation program, followed by 1-10×109 T cells expressing c259, with or without rhIL-2, and experienced sustained anti-tumor effects. Despite the good response rate, complete tumor eradication has not been observed in most patients, and work is underway to better understand the basis for resistance to this therapy.

Good clinical results have also been observed in NY-ESO-1/LAGE-expressing multiple myeloma patients treated by adoptive transfer of c259-expressing T cells after autologous hematopoietic stem cell transplantation (HSCT).

Similarly, T cells expressing engineered TCR targeting the tumor-associated antigen WT-1 showed good safety and showed impressive results in preventing the recurrence of acute myeloid leukemia when administered after HSCT.

To overcome the challenges associated with targeting tumor-associated self-antigens, several groups attempted to identify TCR that mediates the recognition of tumor-specific neoantigens. Immune response to neoantigens seems to play an important role in immune checkpoint suppression of anti-tumor response after treatment, and TCR that recognizes neoantigens does not require affinity maturation and is expected to show safety characteristics. Autologous T cells expressing engineered patient-specific, neo-antigen-targeted T cells have been produced on a small scale, but the costs associated with this personalized approach may not be applicable on a large scale.

One strategy to overcome this challenge is to focus development on engineered TCR therapies for shared neoantigens. Several recurring “hot spot” mutations have been found in common oncogenes, such as phosphatidylinositol 3-kinase (PI3K), Ras and p53, and some peptides derived from hot spot mutations on certain HLA alleles Expression as observed in the KRAS G12D mutation in colorectal cancer and the H3K27M mutation in brain tumors. In addition, there is renewed interest in identifying TCRs that target the breakpoint regions of oncogenic fusion proteins.

It is possible to generate a TCR library, identify peptides expressed on a range of HLA alleles, and use next-generation sequencing and major histocompatibility complex (MHC) typing to identify patients suitable for such treatments. However, the relatively small proportion of patients who identify TCRs that exhibit potency and specificity and match HLA types and tumor hotspot mutations limits the broad applicability of this strategy.


The future development of engineered TCR therapy

The continued progress of T cell therapeutics that incorporate engineered TCR will require technology to improve efficacy, specificity, and safety. So far, the considerable effort required to develop effective and safe TCRs has limited the availability of such therapeutic drugs to a small set of alleles, such as HLA-A2, leading to treatment for patients with less common HLA alleles Major obstacles.

Progress in this field is ongoing, including the recent method of modifying the TCR variable domain framework region, which can increase expression levels and enhance efficacy, while limiting the risk of cross-reactivity, and new technologies to generate de novo TCR or identify tumor responsiveness Antigen targets of T cells.

In addition, many engineering improvements are under development to improve the efficacy of adoptively transferred T cells in the treatment of cancer (Figure 3). Integrating these improvements into cells expressing tumor-reactive TCR can improve the efficacy. Examples already in clinical trials include a test for NY-ESO-1/LAGE-1 specific TCR T cells using CRISPR/Cas9 (NCT03399448) and immune checkpoint inhibitors (NCT02775292 and NCT03709706) to manage the project T cell test.

Figure 3. Engineering strategies to enhance adoptive T cell therapy


Complex bioengineering methods are being developed to improve the efficacy, specificity and safety of T cell therapy. Suicide switches, gates, and adaptor CAR platforms are being developed to reduce CAR-mediated toxicity. The ectopic expression of c-Jun or the gene deletion of NR4a factor confers resistance to CAR T cell failure, which may improve the efficacy and durability of solid tumors.

CAR T cells can also be designed to secrete specific factors to enhance expansion or persistence (for example, IL-7, IL-12, IL-15 or IL-21), reduce the need for lymphatic clearance programs, and resist suppressive tumors Microenvironment (for example, secretion of IL-18, expression of truncated transforming growth factor β [TGF-β] receptor), or as a tumor-specific drug delivery vehicle (for example, local secretion of anti-PD-1).


Chimeric antigen receptor (CAR) T cells for B cell malignancies

CAR combines the extracellular antigen targeting domain, which usually contains single-chain variable fragments (scFv), with the intracellular signaling domain, which is usually derived from TCR. CAR-expressing T cells are not restricted by MHC. Regardless of the type of HLA, they can be used in patients and prevent drug resistance caused by down-regulation of MHC, which is common in cancer. Technology is now readily available to generate scFv and other binding agents for essentially any cell surface molecule (for example, modified proteins, lipids, sugars, and MHC restricted peptides), and to design binding agents with a wide range of biochemical properties.

CAR T cells containing CD28 or 4-1BB inner domains show significant clinical activity; however, these products exhibit significant clinical activity in expansion speed (CD28 expansion rate is faster), peak expansion level (CD28 expansion degree higher), and persistence tendency ( 4-1BB shows greater durability). The differences in expansion kinetics and durability are related to clinical results, because CD19.28.z-CAR shows the highest response rate in lymphoma, CAR-mediated toxicity is higher, and CAR in CD19.BB relapses after CAR It seems lower.

The z-CAR used for B-ALL may be because CAR lasts at least 3-6 months for long-term control of this disease. These observations underscore the potential benefits of tailoring CAR T cell constructs to the unique characteristics of the target tumor. Although unproven, due to the rapid expansion kinetics, more aggressive tumors may require CD28 costimulation, while those that progress more slowly can be more safely controlled by integrating 4-1BB costimulated CARs T cells. In addition, scFv affinity and/or CAR density can be optimized to target tumors expressing heterogeneous antigen levels or to limit CAR-mediated toxicity (discussed in more detail below).


Significant toxicities were observed in patients treated with CD19 CAR-expressing T cells, including cytokine release syndrome (CRS), whereby the anti-tumor activity of CAR T cells resulted in high levels of secreted IL-6, IL-1 and Sepsis-like symptoms, as well as immune effector cell-associated neurotoxic syndrome (ICANS) related to blood-brain barrier endothelial cell dysfunction, are induced by a high inflammatory environment. Although these toxicities lead to serious and even fatal complications, treatment with reduced CAR T cell doses, steroid therapy, or IL-6R-blocking antibodies (ie tocilizumab) has been very effective.

It is worth noting that the treatment-related mortality rate in large multicenter trials is currently less than 5%, which is no different from other standard treatment options for these refractory diseases. Other pharmacological strategies, such as anti-IL1 receptor blockade or tyrosine kinase inhibitor dasatinib, which can effectively and reversibly inhibit CAR T cell function, have been tested in preclinical models and can be managed by standard CRS For patients who are refractory to therapy, there are ready-made alternatives approved by the U.S. Food and Drug Administration (FDA).

Engineered safety switches, such as inducible caspase 9, can deplete CAR T cells in severely toxic situations, but are irreversible in nature and have not been clinically tested. Combined with replacement of thymidine kinase derived from herpes simplex virus The suicide switch triggers an immunogenic response, leading to a decrease in the survival rate of adoptively transferred T cells, which indicates the potential risk of increased immunogenicity after the engineered expression of foreign proteins.


The clinical success of CD19-targeted CAR has been approved by the FDA and the European Medicines Agency, and has triggered extensive investment in CAR T cell research by academia and the private sector, including the development of CAR T cells for other targets in B-cell malignancies. CD22-targeted CAR has also shown significant success in ALL patients, with more than 80% of patients achieving complete remission after receiving the highest dose level of treatment, while B-cell maturation antigen (BCMA) targeting CAR induces a high proportion of multiple bone marrow The tumor patients were in remission. The observed high response rate of CAR T cells against B-cell malignancies is unprecedented, especially considering that most patients treated with these drugs are ineffective against all other therapies.

CAR T cells for non-hematological solid tumors In stark contrast to the success observed in B-cell malignancies, CART cells show no convincing evidence of activity in patients with solid tumors. The current concept believes that this may represent a fusion of several obstacles. A major challenge is the lack of identified cell membrane targets with high levels and uniform expression on solid tumors but limited expression on normal tissues. However, as researchers focus more on cataloging cancer surface groups, molecules with significant differential expression have been identified, including the Tn glycoform of MUC1 on adenocarcinoma, and GD2 ganglia on diffuse internal pontine glioma. GPC2 on neuroblastoma and PAPP-A on Ewing’s sarcoma. These results, coupled with the new understanding that CAR requires a high level of antigen density to achieve optimal activation, and the first project, optimistically believe that the therapeutic window for CAR T cells to target overexpressed cell surface molecules on solid tumors can be determined.

A related issue is the heterogeneity of antigen expression in cancers in general, and solid tumors in particular, which has stimulated interest in the development of multispecific CARs, as described below (Figure 3). The effective treatment of solid tumors with cell therapy may also be hindered by limited trafficking. Recent work has shown that regional delivery can alleviate this challenge for CNS tumors and other cancers (such as mesothelioma), which mainly exhibit regional spread. Finally, it is well known that solid cancer has an inhibitory microenvironment, which inhibits CAR T cell function through a variety of ways, including the expression of checkpoint receptor ligands (such as PD-L1), hypoxia and nutrient consumption, and suppressive immune cells ( For example, regulatory immune cells). T cells [Tregs], myeloid-derived suppressor cells [MDSCs]). As described below, many engineering methods are being developed to address these challenges and enhance the effectiveness of CAR T cells against solid tumors (Figure 3).




T cell therapy for infectious diseases


Virus-specific T cell therapy

Clinical researchers in the early 1990s observed that some patients whose leukemia recurred after allogeneic hematopoietic stem cell transplantation might be relieved after infusion of T cells from a donor. These data ultimately provide convincing evidence for T cell-mediated anti-leukemia effects and the potential of such cells to treat uncontrolled viral infections, which is not uncommon in this case. In severely immunosuppressed patients, the allogeneic virus-specific CTL has a very high response rate to EBV infection and EBV-associated lymphoma, cytomegalovirus, adenovirus, BK virus and human herpes virus 6 infection, although the evidence for transplantation is limited. Minimal MHC matching products are used.

Due to the immunological insufficiency of the recipient, the therapeutic effect depends on the limited rejection of allogeneic cells, and the lack of GVHD may be due to the limited alloreactivity of CTL products lacking naive T cells. The “off-the-shelf” virus-specific CTL library can now be used to treat multiple viruses covering the vast majority of HLA alleles, so it may provide suitable products for most patients (Figure 2). Private investment is seeking to commercialize this therapy.

The success of these products has also raised the prospect of using virus-specific CTLs to target virus-related cancers, including cancers driven by EBV. However, since virus-associated cancer patients retain sufficient immunity to reject allogeneic cell products, these efforts are mainly focused on the adoptive transfer of autologous virus-specific CTL or genetically engineered virus-responsive TCR. Therefore, it is similar to the previously discussed methods. It is no different to use non-viral targeted T cells for cancer treatment.


Virus-specific CAR T cells for HIV infection

Since 1998, CAR T cell clinical trials for HIV infection have been launched. T cells are designed to express the first-generation CAR, using the CD4 outer domain to recognize the gp120 subunit of HIV Env protein as its antigen-binding domain, thereby being able to recognize HIV-infected cells. These trials have not proven effective in controlling HIV infection, but due to the simultaneous development of antiretroviral drugs, many patients have shown long-term survival. It is worth noting that 98% of the samples tested more than 10 years after the infusion showed evidence of persistent CAR T cells. There is no evidence that the vector integrated in the cells near the oncogene continues to clone expansion or enrichment, and there is no evidence. Indicates that there is continuous clonal expansion or cell enrichment. Evidence of the emergence of any replication-competent retrovirus/lentivirus. After more than 500 years of follow-up, this experience is the strongest evidence to date, proving that adoptive transfer of retroviral engineered T cells is safe and that such cells or their descendants can continue to exist for more than ten years.

Many efforts are underway to improve the efficacy of HIV-specific CAR T cells, many of which overlap with efforts to enhance CAR’s cancer function, including enhancing the persistence of CAR T cells and engineering multi-specificity to overcome viral heterogeneity. However, some challenges are uniquely related to CAR for HIV infection, including the need to design resistance to viral infection in the engineered T cells themselves. Researchers are trying to use CCR5 gene editing and overexpression of proteins that interfere with viral mechanisms. Since the toxicity associated with lymphocyte clearance protocols is unlikely to be accepted in this clinical setting, and because CAR biological activity must last for many years, engineering cells to ensure long-term persistence is a major focus. In a primate model, simian immunodeficiency virus (SIV)-reactive CAR T cells produced by HSC showed greater persistence after stopping antiretroviral therapy than those produced from blood And protection against viremia rebound, which improves the prospects of HSC-derived CAR-expressing T cells. In this case, cells may be the first choice. By co-expressing CXCR5, HIV-specific CAR T cells are co-expressed with CD4+ T follicular helper (Tfh) cells in lymphoid tissue B cell follicles, and work to target the HIV latent pool is also in progress (Figure 3).




T cell therapy for autoimmune and other diseases

Although improved therapeutics for autoimmunity have been introduced in the past few decades, more progress still needs to be made. Small molecule tyrosine kinase inhibitors and cytokine targeting antibodies show significant clinical efficacy, but they have extensive immunosuppressive effects and are not suitable for the full spectrum of autoimmune diseases. These challenges have prompted efforts to develop more targeted methods, such as adoptive transfer of Treg (Figure 2), which plays an important role in maintaining self-tolerance, maintaining immune homeostasis, and preventing autoimmunity.

The adoptive transfer of unengineered Tregs has produced impressive results in various autoimmune and GVHD mouse models, and has been proven to be safe and feasible in GVHD, organ transplantation, and type I diabetes (T1D), and It has been shown to last up to 1 year after infusion (Figure 1). However, the short-term and long-term effects of these therapies have not been proven.

CAR is being expressed in Treg as a strategy to improve the efficacy and specificity of Treg therapy. In a landmark study, researchers designed a CAR Treg specific to 2,4,6-trinitrobenzenesulfonic acid (TNBS) in a mouse model of colitis. CAR Tregs secrete inhibitory factors, proliferate and improve disease symptoms in an antigen-specific manner. Similar results were observed in mouse models of multiple sclerosis and transplant rejection. CAR Tregs showed therapeutic effects at suboptimal doses of non-engineered Tregs, providing evidence that CAR expression, in addition to enhancing specificity, also improves the efficacy of Treg therapy. In general, these studies provide a strong theoretical basis for the clinical testing of CAR Treg therapy.

One potential advantage of CAR Treg therapy over effect CAR T cell therapy is the maintenance of the target population, which may lead to continuous expansion, persistence, and lasting immunosuppression of Treg in the target tissue. In addition, although the off-target toxicity of effector CAR T cell therapy may cause severe tissue damage, the off-target effects of CAR Tregs are expected to be less severe, and may include long-term immunosuppression of non-diseased tissues, opportunistic infections, or suppression of local tumor immunity. If Treg expresses a CAR that exhibits an antigen-independent tonic signal, similar side effects may also occur, leading to constitutive and non-specific immunosuppression.

One obvious safety risk associated with CAR Treg is related to the potential plasticity of the Treg lineage. In the mouse model of T1D, Tregs exposed to inflammatory conditions in situ lost FOXP3 expression and transformed into effector-like T cells. In addition, CAR Tregs containing 4-1BB costimulatory domains are transformed into cytotoxic CAR T cells lacking immunosuppressive ability, which improves the prospect that transforming engineered Tregs into effector cells can enhance autoimmunity rather than suppress autoimmunity. If CAR A similar phenomenon may occur if there is effector CAR T cell contamination in products manufactured by Treg. Given that effector T cells expand faster and more robustly when activated compared to Tregs, a small group of effector CAR T cells can quickly outperform CAR Tregs and mediate destructive autoimmunity.

Cell engineering and synthetic biology provide potential opportunities to mitigate this type of toxicity (Figure 3). In addition to the drug-induced suicide switch used in effector CAR T cells, an intrinsic suicide switch that activates autonomously when FOXP3 expression is absent or in response to inflammatory cytokine transcription can help protect CARTreg switching. Alternatively, ectopic expression of FOXP3 in CARTregs can reduce transformation by maintaining FOXP3 expression and promoting inhibitory functions. In order to prevent long-term immunosuppression of tumor immunity or opportunistic infection of target tissues, an adjustable platform can be used, in which CAR activity depends on the protein therapeutic agent injected into the patient. In autoimmune diseases that exhibit sudden and severe symptoms (ie, sudden) periods, such as rheumatoid arthritis or relapsing-remitting multiple sclerosis, such platforms can induce CAR Treg activity only during these periods, while at the same time Keep CAR Treg dormant to a minimum when disease symptoms appear.

Recently, researchers have developed a chimeric autoantibody receptor (CAAR) by using a creative strategy to use effector CAR T cells to treat autoimmune diseases (Figure 1). CAARs are similar to typical CARs, except that the extracellular antigen binding domain targets the B cell receptor (BCR) of self-reactive B cells. The researchers used a murine model of pemphigus vulgaris, in which autoreactive B cells targeting desmokerin mediate skin damage. T cells expressing CAAR with desmosonin 3 extracellular domain specifically target pathogenic B cells, thereby curing. This study provides an important proof of concept that CAAR T cells can be used for B cell-mediated autoimmune diseases, where specific autoantigens are clearly defined, such as rheumatoid arthritis and lupus erythematosus.

A recent challenging report used CAR T cells targeting fibroblast activating protein (FAP) to prevent fibrosis in a mouse model of fibrosis-induced cardiomyopathy. Fibrosis was induced within 4 weeks, and CAR T cells were given 1 week after the induction stimulus. CAR T cells infiltrate the heart, induce active fibroblasts to be killed, reduce fibrosis, and improve heart function. No significant toxicity was observed within 12 weeks, which may reflect the therapeutic window between high levels of FAP expressed on fibroblasts in inflamed heart tissue and low levels of FAP expressed on fibroblasts in normal tissues. It remains to be seen whether this time-dependent and antigen-dependent treatment window can be determined in the context of human fibrotic diseases, but this study proves the versatility of CAR T cells to treat a variety of human diseases and will definitely become a field Future research.




Next-generation engineering to solve major obstacles

Advances in genetic engineering, gene editing, cell reprogramming, and synthetic biology provide an increasingly powerful toolbox that can be used to design solutions to the current drug resistance and toxicity problems that limit the field. Many of these solutions are modular and have the potential to be integrated into individual cells and cell products in a variety of ways, thereby significantly increasing the complexity of immune cell therapeutics.


Antigen negative and low antigen escape after treatment with monospecific CAR T cells

Similar to other targeted therapies in oncology or infectious diseases (such as tuberculosis or HIV), selective pressure on any one target often leads to escape variants. Not surprisingly, antigen loss represents the main form of resistance to CAR T cell therapy. A related issue is the increasing recognition that optimal CAR T cell activation requires high levels of antigen compared to TCR therapeutics that can recognize very low levels of antigen. Although this feature can provide a therapeutic window for targeting antigens that are low expressed in normal tissues (such as GD2 ganglioside or mesothelin), as observed in clinical trials, by selecting targets with sub-threshold levels Antigenic variants may also develop resistance to CAR therapy CD22-CAR.

In order to solve these problems, efforts are being made to design effective multispecific CAR T cells (discussed below; Figure 3). By changing the affinity of scFv to adjust the antigen density threshold of CAR T cell activation, up-regulate the antigen density on target cells, and target tissues. Matrix to prevent the escape of mutant tumor cells, or engineering methods to enhance the induction of natural immunity, thereby expanding the CAR-induced immune response to include bystanders, antigen-losing variants.


Give multiple specificities to improve curative effect (OR gate)

Engineering combinatorial antigen recognition can improve the efficacy of CAR T cells by overcoming antigen escape and/or increasing the pool of targetable antigens (Figure 3). The administration of multiple CAR T cell products is a strategy for multiple antigen targeting. However, this method significantly increases cost and labor. In addition, in preclinical models, this approach is not as effective as engineering multispecific recognition into a single cell, and in the only clinical trial reported so far, the response rate is similar to that of patients treated with a single CAR T cell product. Several methods are being developed to design a single cell that can target two antigens, where the combination of either antigen triggers CAR T cell activation (the “OR” gate in Boolean logic). One approach is to use vectors encoding two CARs to co-transduce a single T cell population, while related methods combine bicistronic vectors to express two separate chimeric receptors on each cell.

Another approach is to create a bivalent or “tandem” construct in which the recognition of the antigen by either of the two binding domains on the extracellular part of the CAR can trigger effector functions. In 2016, a HER2/IL13Rα2 tandem CAR for the treatment of glioblastoma was reported, and protection of antigen escape and a synergistic effect on CAR T cell activation when both antigens were present were observed. A number of tandem CAR CD19/20 and CD19/22 CAR treatments for lymphoma and CD19/22 CAR treatment for leukemia trials have been developed and studied in preclinical and clinical models. Interestingly, all tandem CAR designs require systematic testing of various configurations to determine the best design for each antigen. For example, CD19/20CAR needs to test linkers of different lengths between specificities, of which only short linkers retain function, while the best CD19/22 CAR requires a loop structure, in which two variable regions CD19 are dispersed in the CD22 binding component. In the variable region.

Dual-target or multi-target OR CARs can also be generated using so-called “adapter” CARs, which have extracellular domains that can bind to multiple different antigen-specific binders (Figure 3). A soluble adaptor must be used to activate CAR T cells. When multiple binding agents are used at the same time, OR gating can be achieved. This platform theoretically provides a safety switch because CAR T cell function is ablated by the removal of the soluble adaptor. The ability to regulate CAR activity depends not only on the kinetics of T cell expansion and persistence, but also on the half-life and stability of the adaptor protein that confers antigen specificity. Another approach involves engineering CAR T cells to secrete a bispecific antibody-like molecule that triggers T cell activation at one end and binds to a second antigen on the tumor at the other end. This method was recently pioneered in the context of glioblastoma, in which CAR T cells targeting the oncogenic tumor antigen EGFR variant III also secrete bispecific molecules that target EGFR.


Enhanced security “AND” and “NOT” gating strategies

CAR T cells can also be designed to be activated only in response to target cells expressing two antigens at the same time, thereby being able to distinguish tumor cells expressing an antigen pair from healthy tissues expressing only one target (Figure 3). In one strategy, one receptor binds to the CD3 zeta inner domain, while the other binds to the costimulatory domain. In order to prevent OR gates, CARs containing CD3 zeta must be designed to have very low affinity so that only substandard activation is induced when the antigen binds. A different approach involves the use of synthetic Notch (synNotch) receptors, where the induction of antigen 1 by the synNotch receptor induces CAR transcription specific to antigen 2. This strategy is effective in the context of preclinical models of anatomically isolated solid tumors, in which one tumor expressing two antigens is transplanted to one side, and the second (control) tumor expressing only one antigen It is transplanted to the second side. However, in the liquid tumor model, normal matrix expressing antigen 1 (ROR1) and tumor cells expressing antigen 1 (ROR1) and antigen 2 (EpCAM or B7-H3) are anatomically mixed together, and the synNotch logic gate fails Survived by healthy cells that only express antigen 1. Some researchers have also tried to develop a NOT gate by binding CTLA-4 or the more effective intracellular domain of PD1 on CAR targeting antigen 2, in which antigen 1 is only targeted without antigen 2. This has been shown to be effective in a preclinical model of fibroblast allogeneic rejection, but it has not yet entered clinical trials.


Targets T cell exhaustion and tumor microenvironment to enhance effectiveness

Chronic antigen stimulation leads to a state of T cell exhaustion, which is characterized by dysfunction; surface expression of multiple inhibitory receptors, including PD-1, TIM-3 and LAG-3, etc.; and unique transcription and epigenetic profiles are sufficient The preclinical and clinical evidence indicates that CAR T cells are easily depleted, which limits the efficacy. Compared with patients who showed complete response (CR), the typical depletion markers of tumor-infiltrating CD19 CAR T cells were higher in non-responders, and the expression of high depletion markers on CAR T manufactured products was found to predict non-response. The tonic signal phenomenon caused by antigen-independent accumulation of CAR receptors in the cell membrane or exposure to high tumor burden may also lead to exhaustion. If there are depleted T cells before engineering, selecting a T cell subset with greater proliferation capacity before genetic manipulation can improve results. Interestingly, preclinical and clinical studies have shown that small molecule drugs such as dasatinib or ibrutinib can prevent or reverse T cell depletion. Finally, by regulating the transient destruction of the tonic CAR signal by CAR protein, the depleted CAR T cells can be epigenetically reprogrammed and the efficacy of preclinical models can be enhanced.

A recent case study reported the enrichment of a single T cell clone in which the CAR transgene was integrated into the TET2 locus, resulting in a loss-of-function mutation. This mutation gives CAR T cells higher potency, expansion, persistence, and memory-like phenotypes, and ultimately leads to complete remission in 5 years at the time of reporting. It improves the prospects of CAR T cells. Cells can be designed to avoid or resist exhaustion ( image 3). This has become an active and promising research field in this field. The insertion of the CAR transgene into the TRAC locus and the endogenous control of CAR expression prevented the failure of preclinical leukemia models. Overexpression of the transcription factor c-Jun has been shown to protect T cells from the most tiring CAR design. In murine models, the expression of nuclear receptor transcription factors NR4A1, NR4A2, and NR4A3 are also associated with CAR T cell exhaustion, but there are no available inhibitors of these drugs, and it is unclear how this strategy can be applied to human T cells. Three separate genes need to be knocked out.

Many methods to alter the tumor microenvironment or confer resistance to CART cells are also under investigation. Some teams have focused on secreting anti-PD1 nanobodies, gene editing to completely delete PD-1 protein, or designing a “switch” receptor, which consists of extracellular PD-fused with the intracellular domain of costimulatory molecules such as CD28. 1 composition, which can transform the inhibitory signal induced by tumor PD-L1 into an activation signal. Similarly, in order to overcome the death signal imposed by the tumor overexpressing Fas ligand, the researchers expressed a dominant negative Fas receptor, which increases the expansion and persistence of CAR T cells that target CD19. Brentjens has published a series of publications on “armored” CAR T cells, in which the second transgene is designed to change the tumor environment; this includes the secretion of the inflammatory cytokines IL-12 or IL-18 or CD40L to enhance antigen cross-presentation And promote the spread of epitopes. Other groups have also used a non-signaling form of the transforming growth factor beta (TGF-beta) receptor, which outperforms the endogenous receptor due to its constitutive and high expression. The transgene was first applied to human EBV-specific T cells, and then included as a second transgene in human CAR T cells targeting prostate cancer in preclinical models and clinical trials (NCT03089203). Finally, preclinical models indicate that targeting tumor stroma and/or vasculature can enhance the efficacy of CAR-T and may limit the escape of antigen-negative variants.


Alternative immune cells (natural killer [NK] cells, γ-δ [γδ] cells, NK T [NKT] cells and induced pluripotent stem cell [iPSC]-derived immune effector cells) and allogeneic immune cell therapy

Although this review focuses on the management and modification of αβ T cell therapies, there is an emerging work that shows progress has been made in using similar techniques to modify other immune effector cells, which can confer certain advantages. The main advantage of NK, γδ T cells and NKT cells is that they all have cytotoxicity, but none of them express endogenous TCR; therefore, they will not mediate GVHD when given to a host that does not match the MHC. However, adult peripheral blood NK cells are relatively resistant to retroviral and lentiviral transduction, and show poor persistence in the absence of high levels of IL-2 or IL-15. To avoid this, a method was created by which NK cells contained in unrelated umbilical cord blood were transduced to express CD19 targeting CAR and transgenes encoding IL-15. A recent report showed , 7 of 11 patients who received this therapy mediated CR. This method can provide ready-made CAR-NK cell products, thereby achieving an unprecedented scale of CAR-NK therapy. γδ T cells are a rare population in peripheral blood and require a large amount of enrichment and bisphosphonates during in vitro culture (Xiao et al., 2018). Nevertheless, researchers have successfully used CARs to transduce γδ T cells, which have shown activity in preclinical models. Similarly, unchanged NKT cells are a very rare population, but show preclinical efficacy in solid tumors and show enhanced persistence when they are designed to secrete IL-15.

Improved methods for generating immune effector cells from iPSCs are emerging, further opening up the scalability potential of engineered immune cell populations. It is not clear how iPSC-derived cells exhibit cytotoxicity and persistence compared to mature αβ T cell engineered cell products, and whether artificial cell culture systems can be used to generate clinically relevant cell numbers; however, effective The successful method of engineered immune effector cells derived from iPSC provides attractive possibilities for the production of hundreds of therapeutic cells from inexhaustible sources.

The main obstacle to the success of this method is the rejection of allogeneic products. However, progress is being made in this area. Recent preliminary reports have demonstrated the feasibility and some clinical activities of allogeneic CAR T cells designed to delete TCR and CD52, which can selectively remove lymphocytes from the host to use CD52-directed mAbs to prevent rejection. It has also recently been reported to reduce rejection by deleting HLA class I and class II combined with CD47 overexpression. If the dual challenges of GVHD and rejection can be overcome, the availability of immune effector cell banks produced by healthy donors can change the field of immune cell therapy by achieving more cost-effective therapies, reducing the need to provide such therapies to patients time. For patients, it provides a platform for more complex multi-projects and achieves cross-product quality standardization, which is beyond what can be accomplished using an own platform.

Immune cell therapy is a rapidly emerging therapeutic agent. The FDA has approved CAR T cell therapy for the treatment of B-cell malignancies and has made significant progress. However, it has been proved that it is extremely challenging to transform it into solid tumors. The effects of therapeutic drugs on other diseases are limited. Complex bioengineering methods using gene deletion, ectopic overexpression of transcription factors, multispecific binding agents, Boolean gating, and other synthetic systems will ultimately determine the extent to which next-generation immune cell therapy becomes an effective alternative to traditional drugs.



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