Latest research progress of allogeneic CAR-T cell therapy

Latest research progress of allogeneic CAR-T cell therapy.  In recent decades, great progress has been made in the field of tumor treatment. In particular, the cell therapy technology with tumor-associated antigen (TAA) as the target has been developed by leaps and bounds.

By generating CAR constructs consisting of genes encoding single-chain antibodies (scFv), costimulatory domains (CD28 or TNFRSF9), and CD247 signaling domains for T cell proliferation and activation, T cells have the ability to attack tumor cells .

Most importantly, CAR-T cells are activated by recognizing TAA by scFv on the surface of T cells, and then activate the intracellular signal domains connected by scFv to induce downstream signaling pathways involving T cell proliferation, activation and cytokine production. Scientists have made a lot of efforts to improve efficacy and durability and reduce T cell failure.

In general, the allogeneic and general CAR-T generation has attracted much attention due to its wide and rapid use of patients. Dong Wook Kim and Je-Yoel Cho of Seoul National University in South Korea published an article on Biomolecules, reviewing the current generation technology of allogeneic and universal CAR-T cells and their shortcomings and limitations to be overcome.

01Introduction

Initially, CAR-T therapy was tested on patients with blood system cancers, especially for B-cell acute lymphoblastic leukemia (B-ALL) patients who had not improved with known therapies such as hematopoietic stem cell transplantation (HSCT) and chemotherapy. -T therapy is the last treatment option.

Currently, two CAR-T cell drugs YESCARTA (axicabatagene ciloleucel) and KYMRIAH (tisagenlecleucel) have been approved by the FDA for use in certain adult patients with large B-cell lymphoma that do not respond or relapse after at least two other treatments, and Treat patients with B-cell precursors under the age of 25, all of whom are refractory or relapse for the second time or later.

Three other CAR-Ts with international non-proprietary names (INN) are also cited in the International Immunogenetics Information System®IMGT/mAb DB, namely vadacabtagene-Leraluecel, idecabtagene-Vicluecel and Liscabatagene-Maraluecel.

Although there are still side effects such as cytokine release syndrome (CRS) and neurotoxicity during CAR-T treatment, recent studies using modified CAR-T cells through various advanced technologies have shown that CAR-T cells can be used more effectively and safely. The prospect of T cells. The effective and safe use of CAR-T cells may include the following concepts:

(1) CAR-T cells should be produced quickly before injecting CAR-T cells back into the patient to avoid further development of the disease.

(2) CAR-T cells can be used for allogeneic and widely used. Gene editing is the most widely used technology for the manufacture of universal CAR-T cells. One of the main targets of this system is the T cell receptor (TR) to minimize the occurrence of graft-versus-host disease (GvHD) during allotransplantation.

In order to minimize GvHD, chemotherapy can be performed before allogeneic treatment, including immunosuppressive combination therapy of Fludarabine and Cyclophosphamide, and serum therapy with anti-CD52 monoclonal antibody. When using CAR-T cells as drugs, another point that needs to be carefully considered is the careful handling of side effects.

Since the curative effect of CAR-T cells mainly comes from various released cytokines, common side effects are often related to the uncontrollable release of cytokines, and may be very harmful; for example, some cytokines can penetrate the blood-brain barrier ( BBB) and cause neurotoxicity.

In order to prevent this problem, various safety switches have been developed, such as the incorporation of suicide genes, the expression of known target genes of therapeutic antibodies, and the addition of molecular switch proteins between CAR and tumor cells.

02General CAR structure

In the early experiments to create CAR-T cells, VHsp6 was fused with Cα or Cβ, and it was found that the VHCα or VHCβ chimeric chain could form a heterodimer with the β or α chain of the recipient T cell.

This is the first system to get rid of MHC-restricted T cell activation. Then by adding the signal domain of CD247 (first generation CAR), adding a costimulatory domain of TNFRSF9 or CD28 plus CD247 domain (second generation CAR), adding two costimulatory domains of CD28 and TNFRSF9 plus CD247 domain (first generation CAR) Third-generation CAR) to design the signal domain of CAR, add a co-stimulator plus CD247 domain and IL12 expression system (fourth-generation CAR), and add a co-stimulator, cells that bind cytokine receptors with STAT3 and CD247 domains Inner domain (eg, IL2RB chain fragment) (fifth generation CAR).

All CAR structure modifications are to enhance the activity of CAR-T cells, increase cell proliferation and cytokine release, and reduce T cell exhaustion.

In theory, various modifications to the CAR structure are possible, but the basic structure used so far may not change significantly. The general functions and descriptions of the various signal domains used in the CAR structure are as follows, and are summarized in Figure 1.

figure 1. The structure of CAR-T cells. CAR-T cells are composed of single-chain variable fragments (scFv), transmembrane domains and signal transduction domains. The first CAR-T cell has only the cd247itam (immunoreceptor tyrosine-based activation motif) domain, which activates the ZAP70/Syk tyrosine kinase and activates the downstream signal cascade.

The second CAR-T cell has an additional costimulatory domain derived from CD28 or TNFRSF9. Various proteins containing SH2 domains such as PI3K, GRB2 and GADs are recruited to the costimulatory domain and induce IL2 production.

In the third CAR-T cell, it contains two costimulatory domains, which produces more cytotoxic activity on tumor cells. In the fourth CAR-T cell, an extra gene that produces IL12 is introduced into the CAR-T cell. The expression of IL12 is controlled by the NFAT transcription factor, which is activated by binding to the CD247 domain.

This system was developed to activate the natural immune system when CAR-T cells cannot recognize tumor cells due to low expression of surface antigens. In the fifth generation CAR-T cells, the JAK-STAT activation domain derived from IL2RB is integrated between CD28 and CD247. This domain stimulates cell proliferation, prevents terminal differentiation, and shows better persistence.

2.1 CD247 domain

CD247 (called CD3ζ) is a transmembrane adaptor in the CD3 complex of the TR-co receptor. CD247 has three tyrosine-based immune receptor activation motifs (ITAMs) to achieve T cell activation. In addition to T cell activation, CD247 also participates in the signal transduction of Nkp46/Nkp30, and is a low-affinity Fc receptor for IgG CD16 in NK cells. In the construction of CAR-T, ITAMs are usually integrated into the CAR structure to induce T cell proliferation and activation. The role of ITAM in CAR-T cell activation was identified as mediated by heterodimerization with the natural CD247 partner in the cell. In addition, among the three ITAMs in CD247, the third ITAM has been determined not to affect T cell activation, but the second ITAM has been determined to be a key factor in T cell activation.

2.2 TNFRSF9 domain

Although the successful trials of the first generation of CAR-T cells showed anti-tumor activity, the efficacy of T cells was not sufficient due to the low persistence of T cells. To overcome this problem, a costimulatory domain was added. TNFRSF9 (called 4-1BB and CD137) is a member of the tumor necrosis factor (TNF) receptor family and is known to be expressed in activated T cells. By comparing CAR-T cells with and without the TNFRSF9 signal domain that targets CD19, the importance of the costimulatory domain for anti-tumor activity is demonstrated. Compared with CAR-T cells without TNFRSF9 domain, CAR-T cells with TNFRSF9 domain show strong and specific cytotoxicity to B-ALL and produce more IL12, which indicates that the additional signal domain is important for B-ALL. It is necessary to enhance the activity of CAR-T cells.

2.3 CD28 domain

Another costimulatory domain of CAR-T activity comes from CD28, a receptor called CD80, which stimulates the production of various interleukins during T cell stimulation [19]. The phosphorylation of tyrosine 170 in the intracellular domain of CD28 is the reason for its binding to SH2 domain-containing proteins (such as PI3K, Grb2, and Gads), leading to increased IL2 production and T cell activation [20-22]. Comparative analysis of different CAR structures to observe the effects of TNFRSF9 or CD28 costimulatory domains, and found that when co-cultured with cancer cells expressing target antigens, the expansion rate of CAR-T cells with either costimulatory domain is higher than that of those with either costimulatory domain alone. CAR-T cells with CD247 domain. In addition, there is no significant difference in tumor lysis activity between CAR-T cells containing both CD28 and TNFRSF9 domains and cells containing either domain alone [23], resulting in 4th generation cells using CD28 or TNFRSF9 alone.

2.4 IL12 expression system

Adding a costimulatory domain seems to be sufficient to make CAR-T fully active, but some studies have shown that another factor may be needed to enhance the tumor-killing effect of CAR-T cells. A key problem in using CAR-T cells is that they can only be activated when the antigens in target tumor cells are exposed and captured, which means that some tumor cells without antigen may escape from CAR-T cells. To overcome this problem, we developed CAR-T cells expressing IL12 and named them T cells for cytokine-mediated universal killing (TRUCK). Its main functions include differentiation of primitive T cells into Th1 cells, activation of natural killer cells and T lymphocytes, and anti-angiogenic activity. The fourth-generation CAR-T system that uses NFAT transcription factors to stimulate IL12 production has been tested on a variety of tumors, including solid tumors, and has shown promising results.

2.5 IL2RB chain fragment

Recently, newly developed fifth-generation CAR-T cells are used to induce cytokine signaling, in which IL2RB chain fragments are inserted between TR signaling (CD247) and costimulatory domains (CD28). Since the IL2RB fragment has a STAT3 binding YXXQ motif, these CAR-T cells induce antigen-dependent activation of the JAK-STAT pathway, thereby promoting cell proliferation and preventing terminal differentiation. In addition, this CAR-T cell shows better persistence and better effect in treating leukemia than CAR-T cells containing the costimulatory domain of TNFRSF9 alone.

03 Effectiveness of allogeneic CAR-T cell therapy

Although autologous CAR-T cells are safe and effective to treat hematopoietic tumors, there are some limitations, such as the time required to prepare sufficient CAR-T cells. Therefore, allogeneic therapy has been strongly tested and has shown promise in some studies. Since autologous CAR-T cell therapy has shown significant toxicity problems in clinical trials, the toxicity of allogeneic CAR-T cells is also considered to be significant.

Nevertheless, allogeneic CAR-T cell therapy has been applied to many patients due to its great advantages. The source of CAR-T cells can be autologous or allogeneic. CAR-T cells of allogeneic origin are found in patients undergoing allogeneic hematopoietic stem cell transplantation and can be derived from donors or recipients. Studies have shown that compared with autologous CAR-T cell therapy, receptor-derived CAR-T cell therapy has an increased complete remission rate (CR) in CRS patients.

Although the recipient-derived CAR-T cells are not truly allogeneic, this study reveals the potential efficacy of allogeneic CAR-T cells in the treatment of patients with relapsed/refractory ALL. At the same time, patients with ALL, NHL (B-cell non-Hodgkin’s lymphoma) and CLL (chronic lymphocytic leukemia) were treated with CAR-T cells and allogeneic hematopoietic stem cell transplantation was performed to assess the toxicity after transplantation.

In this study, all patients received allogeneic hematopoietic stem cell transplantation, and NHL/CLL patients received myeloablative pretreatment, but the mortality rate was higher.

04 GvHD related to allogeneic CAR-T cell therapy

When allogeneic CAR-T cell therapy is applied, GvHD is always related to a problem that needs to be overcome or minimized. Early studies have shown that donor-derived CAR-T therapy for patients with B-cell-related diseases shows very low GvHD levels, despite the toxicity problems such as hypotension and fever.

In addition, a mouse study investigating the role of donor-derived CD19 CAR-T cells in allogeneic hematopoietic stem cell transplantation showed that allogeneic CAR-T cells have strong graft anti-lymphoma activity, reduce GvHD, and confirm CAR-T cells co-stimulated with TNFRSF9 increased the occurrence of GvHD, although TNFRSF9 co-stimulation has been shown to be effective in reducing T cell failure through repeated CAR signals.

In addition, in a study of patients with ALL, NHL, and CLL, a 25% incidence of acute or 10% of chronic GvHD was reported. Therefore, allogeneic CAR-T cell therapy requires further technological advances to overcome GvHD.

05 Engineered allogeneic CAR-T cells

Avoiding GvHD is a major obstacle that needs to be overcome in allogeneic CAR-T cell therapy, especially when CAR-T cells used in patients are produced by healthy donors with mismatched HLAs.

5.1 TR delete

Most graft-versus-host disease occurs due to the diversified recurrence of allogeneic T cell receptors (TR). In order to overcome this problem, various technologies have been used to develop new engineered CAR-T cells on TR.

More effective CAR-T cells can also be produced by destroying TR and β-2 microglobulin, which are responsible for GvHD and host versus graft effect (HvGE) respectively, as well as destroying PDCD1 (PD1) and CD274 (PDL). -1) to improve the effectiveness of CAR-T cell therapy.

Another way to close the signal pathway triggered by TR is to use the endoplasmic reticulum retention signal to transduce the single-chain variable fragment transmembrane domain bound by CD247.

This CD247-specific single-chain antibody overexpressed in the endoplasmic reticulum can capture CD247, thereby inhibiting the interaction between CD247 and TR, thereby inhibiting TR signal transduction. More advanced allogeneic CAR-T cells are produced by adding safety options.

In this system, over-activated CAR-T cells can be targeted by rituximab, which recognizes the CD20 mimotope and blocks the activation signal, thereby eliminating these cells.

5.2 Potential inhibitors to prevent GvHD

Another possible way to inhibit the allogeneic GvHD effect of CAR-T therapy is to treat with drugs that inhibit the TR signaling pathway, as shown in Figure 2. Recently, intravenous immunoglobulin (I5. VIg) has been proven to inhibit TR-mediated signaling pathways. IVIg is usually used to treat autoimmune and infectious diseases. Lysophosphatidic acid (LPA5) G protein-coupled receptor (GPCR) is identified as expressed in CD8 T cells and binds to the biologically active serum lipids of lysophosphatidic acid that is usually increased in chronic inflammatory diseases, thereby inhibiting early TR signaling. Including calcium mobilization and ERK activation.

figure 2. Potential inhibitor of T cell activation. To reduce GvHD, inhibitors of T cell activation may be useful. Intravenous immunoglobulin (IVIg), ceramide synthase 6 (CERS6) inhibitors, and fucosylation inhibitors can inhibit T cell activation and can be used to improve GvHD in CAR-T cell therapy. Another potential inhibitor may be N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienyl)-benzamide hydrochloride Salt (AMTB) and lysophosphatidic acid G protein-coupled receptor (LPA5-GPCR), which are known to inhibit calcium-mediated T cell activation.

Compound 211 is an allosteric non-competitive selective CD45 tyrosine phosphatase inhibitor, which can inhibit TR-mediated expression and activation of LCK, ZAP70, ERK and IL2. Notch signaling inhibitors can reduce the activation of T cells by reducing the activity of RAS (H-, K-, N-), ERK and NF-κb.

In an experiment to study the relationship between GvHD development and CERS6 activation, specific inhibitors of CERS6 were applied to mouse and human T cells, and it was shown that T cell activation was significantly reduced, indicating that CERS6 inhibitors may be one of the controls for GvHD. A promising reagent. In addition, the small molecule c-Rel inhibitor weakens the negative feedback produced by IL2, showing that the allogeneic activation of T cells is reduced without affecting the anti-tumor activity. Notch inhibition also showed an anti-GvHD effect induced in CD4+ or CD8+ T cells, and showed a decrease in RAS/ERK and NF-kappaB activity when re-stimulated by TR.

In general, through genetic engineering, various experiments to reduce GvHD mainly through TR inactivation and TR signal-mediated specific inhibitors have been carried out. It is important to reduce the allogeneic GvHD effect of CAR-T cells, but before applying it to patients, it is necessary to carefully verify whether CAR-T cells can mediate toxicity through genetic engineering or inhibitors.

06 Realize the heterogeneous application of universal CAR-T by switching molecules

By adding switch molecules between CAR-T cells and tumor cells, more powerful and wider applications of allogeneic CAR-T cells can be realized, which are called UniCAR-T cells (UniCAR-T). The specific switch molecule is responsible for connecting the CAR structure of CAR-T cells with the TAAs-recognizing antibodies in tumor cells, so that CAR-T cells can target various tumor cells. Switch molecules can be produced through protein-protein interactions or protein-chemical interactions, where each part should be connected to an antibody or CAR. Therefore, the UniCAR-T system has the huge advantage of controlling the activity of CAR-T cells by changing the number of switch molecule-antibody complexes to be injected.

Various antibodies targeting TAAs can be easily connected to molecular adaptors through chemical or genetic means. For example, an antibody-based bifunctional switch has been developed. The switch is composed of a peptide neo epitope (PNE) linked to a tumor antigen-specific Fab region of TAA, and can be combined with an anti-PNE single-chain antibody linked to a CAR. Combine. This switch model allows the control of CAR-T cell activity, tissue homing, cytokine release and phenotype in a dose-dependent manner. Another type of switch molecule is the FITC-folate system.

In addition, FITC-HM-3 bifunctional molecule (FHBM) was also created as a switch molecule for the new switchable dual receptor CAR engineering T cell. Two CARs were created, and each CAR has the ability to bind to the TAA or FITC of the FHBM respectively. Because every CAR has a CD247 and TNFRSF9 domain as signal inducer in the cell, both CARs should be activated. Therefore, one CAR should be combined with TAA, and the other CAR should be combined with FITC of FHBM to activate CAR-T cells. This dual activation system with switch molecules can effectively control the activity of CAR-T cells.

Another switch molecule is the leucine zipper domain, which is an endogenous nuclear-molecular interaction domain widely involved in protein interactions. The SUPRA (split, universal, and programmable) CAR system uses the leucine zipper domain as a switch molecule.

The monomeric streptavidin 2 (mSA2) biotin binding domain was also introduced as a switch molecule. Biotinylated antibodies that recognize TAA can be captured by CAR-T cells in the mSA2 domain linked to the CAR structure. The switch molecule described in this article is shown in Figure 3.

image 3. Various switch molecules. In order to universally use CAR-T cells, various switch molecules have been developed. (A) The general CAR structure is composed of intracellular signal domain, transmembrane domain and extracellular single-chain antibody.

The single-chain antibody portion can be further modified by adding switch molecules. (B) Peptide neo-epitope (PNE), (C) fluorescein (FITC), (D) 10 amino acids (5B9 tag), (E) FITC-HM-3 bifunctional molecule (FHBM) and single chain antibody, (F) Leucine ZipFv is linked to the antibody, and (G) streptavidin 2 (mSA2) biotin binding domain was developed as a switch molecule.

These molecules can be recognized by antibodies or ZipCAR linked to CAR, thereby inducing and activating the signaling pathway of CAR-T cells. This system controls the activity of CAR-T cells by changing the number of switch molecules connected to the antibody, which has great advantages.

During development, the switch molecule should be selected so that it does not induce additional immunogenic reactions, such as graft-versus-host disease, and its specificity should ensure minimal reactivity.

07 CAR-T cell safety system

Although the effectiveness of CAR-T therapy has improved, side effects such as neurotoxicity and CRS can still be observed when applied to patients. One of the safety switches is the use of a kinase inhibitor to block the TR-mediated signaling pathway. Dasatinib is a well-known lymphocyte-specific protein tyrosine kinase (LCK) inhibitor, which can block LCK-mediated phosphorylation of CD247, thereby blocking the signal mediated by CARs containing CD28+CD247 or CD247 Pathway TNFRSF9 plus CD247 activation domain. Another way to safely activate CAR-T cells is to use a suicide gene induction system. The activation of MyD88 and CD40 induced by rimiducid can eliminate over-activated CAR-T cells.

Rituximab has been used to treat many cancers, such as non-Hodgkin’s lymphoma, rheumatoid arthritis, and chronic lymphocytic leukemia. By generating CAR-T cells that overexpress CD20, rituximab treatment can be As a safe reagent when CAR-T cells are over-activated. This is a very fast control method that can reduce the release of cytokines and even cause CAR-T cell death. The truncated form of epidermal growth factor receptor (EGFRt) is also expressed as a safe suicide gene in cd19car-T cells, which can be targeted by cetuximab. Cetuximab treatment showed that CD19 CAR-T cells were effectively eliminated in the early and late stages after adoptive transfer in mice. Various safety systems that regulate CAR-T cell activity are still under development, but it is important to predict potential side effects and should be tested before being fully applied to patients.

08 Conclusion

CAR-T cell therapy is called a living drug, used to treat B-ALL patients with no improvement in other therapies (such as HSCT and chemotherapy). It has been widely used in the past ten years and has shown good results. However, YESCARTA and KYMRIAH are the only CAR-T cells approved by the FDA for the treatment of adult patients, which are the last resort after all other treatments.

Autologous CAR-T cell therapy still has many obstacles to overcome in shortening the patient’s reinjection time. For those patients who are not easy to obtain autologous T cells and low-cost universal CAR-T cells, the development of allogeneic universal CAR-T therapy is an urgent need. In CAR-T treatment programs, it is necessary to reduce GvHD by adding safety systems. In order to solve these problems, people have made many technological advances, such as gene editing.

In addition, recent research has focused on the application of CAR-T cells in solid tumors, which proves the wide application of CAR-T cell therapy, and will open up its application in other diseases such as metabolism, heart and rare diseases in the near future. The possibility.

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