June 25, 2022

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What are the challenges and solutions for CAR-T therapy?

What are the challenges and solutions for CAR-T therapy?



 

What are the challenges and solutions for CAR-T therapy?

Chimeric antigen receptor T cell (chimeric antigen receptor T cell, CAR-T) immunotherapy is to genetically engineer T cells isolated from patients or allogeneic donors to express chimeric antigen receptor (chimeric antigen receptor). receptor, CAR), an adoptive cell therapy that specifically recognizes and kills tumor cells.

CAR-T is one of the major breakthroughs in the field of cancer immunotherapy in recent years.

It has great advantages in the treatment of hematological malignancies and has broad development prospects.

This article reviews the basic structure and function of CAR-T cells, their applications in hematological malignancies, and the challenges they face.

 

 

What are the challenges and solutions for CAR-T therapy?

 

 

 


01 Structure and function of CAR-T cells

 

CAR-T uses gene modification technology to transduce genetic material with specific antigen recognition domains and T cell activation signals into T cells, so that T cells can target and recognize relevant antigens on the surface of tumor cells, thereby activating effector T cells. cells, thereby exerting an anti-tumor effect.

This target recognition process does not depend on the interaction between the traditional T cell receptor (TCR) and the major histocompatibility complex (MHC), and avoids tumor cells by downregulating MHC expression.

Immune escape. CAR-T can be divided into extracellular domain, transmembrane domain and intracellular domain according to its structure.

The extracellular region is usually a single-chain variable fragment (scFv) that targets and recognizes the antigen. The transmembrane region is usually derived from CD28 or CD8 and can affect the interaction between CARs.

The intracellular domain is the CD3ζ signaling domain, which contains three immunoreceptor tyrosine-based activation motifs (ITAMs), which can initiate intracellular signal transduction through phosphorylation activation, activate T cells, promote proliferation, Cytokine secretion and cytotoxicity.

 

The first generation of CARs, composed of scFv and CD3ζ signaling domains, possessed T cell killing toxicity in vitro, but showed no antitumor effect in clinical trials.

The second generation of CARs added a co-stimulatory domain derived from CD28 or 4-1BB on the basis of the first generation, which extended the survival time of CAR-T cells in vivo, and enhanced the proliferation ability and killing toxicity.

The third-generation CARs added two costimulatory molecules on the basis of the first-generation CARs, and their anti-tumor effects were further improved.

The fourth generation of CARs, also known as “armored” T cells (T cells redirected for universal cytokine killing, TRUCKs), enhances the efficacy of CAR-T by genetic modification to secrete specific cytokines or express additional costimulatory ligands, while introducing The suicide gene system controls CAR-T activity and is activated when necessary to reduce cytotoxicity.

 

At present, clinical trials carried out around the world mainly focus on the second-generation CAR-T composed of CD28 or 4-1BB co-stimulatory domains.

The CD28 or 4-1BB co-stimulatory domain is of great significance in promoting the proliferation and survival of CAR-T cells in vivo, and can significantly improve the complete remission rate of patients receiving CAR-T therapy.

Studies have shown that the CD28 co-stimulatory domain can induce a more rapid anti-tumor response, but less durable; while 4-1BB has a slower tumor clearance rate, but can induce a sustained high level of response, but clinical studies have shown that expression of CD28 co-stimulatory response.

The clinical efficacy of CAR-T cells in the stimulatory domain is similar to that of CAR-T cells expressing 4-1BB in the treatment of hematological malignancies.

In addition to CD28 and 4-1BB, research on CAR-T cells derived from other costimulatory domains is also being actively carried out, such as OX40 (CD137), ICOS, etc.

 

 


02 Clinical application of CAR-T Cells


CAR-T immunotherapy has made a breakthrough in the treatment of hematological malignancies.

At present, the U.S. Food and Drug Administration (FDA) has approved four CAR-T cell products targeting CD19, namely Tisagenlecleucel (tisa-cel, Kymriah), Axicabtagene Ciloleucel (axio-cel, Yescarta), Brexucabtagene Autoleucel (brexu-cel, Tecartus) and Lisocabtagene Maraleucel (liso-cel, Breyanzi).

These products are mainly used for the treatment of children and young adults with relapsed/refractory acute B-cell lymphoblastic leukemia (B-cell acute lymphoblastic leukemia, B- ALL) and certain types of B cell non-Hodgkin lymphoma (B-NHL) in adults, including relapsed/refractory diffuse large B cell lymphoma (DLBCL) , relapsed/refractory mantle cell lymphoma (mantle cell lymphoma, MCL) and relapsed/refractory follicular lymphoma (follicular lymphoma, FL), and the curative effect is significant.

With the development of a large number of CAR-T clinical studies, CAR-T cells targeting different targets are also expected to be used in B-NHL, multiple myeloma (MM), chronic lymphocytic leukemia (chronic lymphocytic leukemia, CLL), acute myeloid leukemia (AML), T-cell lymphoma and solid tumors.

 

 

2.1 B-cell malignancies

CD19 is a B-cell-specific target that is widely expressed in almost all B-cell malignancies.

CD19-targeted CAR-T therapy demonstrated rapid and durable efficacy in clinical trials of B-cell malignancies such as children and adults with relapsed/refractory B-ALL, relapsed/refractory DLBCL, relapsed/refractory FL, and CLL Antitumor effect.

CD20 is expressed in B cells of all differentiation stages except plasma cells. CD20-targeted CAR-T cells are currently in the early clinical research stage and have a high response rate in patients with DLBCL and indolent non-Hodgkin lymphoma.

In addition, CD19, CD20 bispecific CAR-T cells have shown low toxicity and high efficiency in clinical trials, and may become an effective means to overcome the recurrence of drug resistance caused by antigen downregulation.

CD22, a member of the sialoadhesin family, is unique to B cells and is expressed in the vast majority of B cell malignancies.

Since about 50% of patients receiving CD19-targeted CAR-T therapy experience drug resistance relapse, and the main mechanism is immune escape caused by antigen loss, the application of CD22-targeted CAR-T cells may be beneficial to CD19-targeted CAR-T cells. Patients who relapsed after T therapy were effective.

In a clinical trial of B-ALL patients treated with CD22-specific CAR-T cells, the complete response rate was >70%, including a large number of patients who relapsed after CD19-targeted CAR-T therapy.

 

 

2.2MM

B cell maturation protein (BCMA) is a member of the TNF superfamily, expressed in some mature B cells, plasma cells and almost all MM cells.

BCMA-targeted CAR-T cells showed satisfactory complete response rates in MM treatment, but patients were prone to relapse within 12 months after treatment.

Ciltacabtagene Autoleucel (cilta-cel) is a CAR-T cell expressing dual BCMA epitopes, containing a co-stimulatory domain derived from 4-1BB and a CD3ζ intracellular signaling domain.

Studies have shown that a single infusion of cilta-cel can produce deep and durable responses in patients with relapsed/refractory MM, with a complete response rate of 67% within 1 month of infusion and 62% within 3 months.

In addition, other potential targets for the treatment of MM include SLAM7, CD38, CD138, immunoglobulin kappa light chain and GPRC5D, etc.

These targets have also shown initial efficacy in clinical trials.

 

 

2.3 Hodgkin lymphoma

CD30 is a member of the TNF receptor superfamily, ubiquitously expressed in Reed-Sternberg cells, and is a safe and effective target for the treatment of relapsed/refractory Hodgkin lymphoma.

In a phase I clinical trial, CD30-targeting CAR-T cells demonstrated significant antitumor activity and low toxicity in patients with relapsed/refractory Hodgkin lymphoma after high-intensity lymphodepletion preconditioning.

 

2.4 T/NK cell malignancies

The CAR-T research on T/NK cell lymphoma is still in the early stage, and the development is relatively slow.

The main limitation is that it is difficult to find suitable targets to avoid the “cannibalism” of CAR-T cells. The main targets currently in clinical research are CD5 and CD7.

Among them, CD5-targeted CAR-T cells avoid “cannibalism” by downregulating the expression of endogenous CD5.

In clinical trials, peripheral T cell lymphoma (PTCL) and acute T lymphocytic leukemia (T Cell acute lymphoblastic leukemia, T-ALL)) patients are effective.

CD7-targeted CAR-T cells cannot significantly downregulate their own CD7 expression, but CD7-targeted gene disruption can make CAR-T cells resistant to “cannibalism” without affecting antigen recognition, thus confirming CD7-targeted therapy feasibility.

In a clinical trial of donor-derived CD7-targeting CAR-T cells, CAR-T cells had higher remission rates and less toxicity in patients with relapsed/refractory T-ALL, although CD7-positive T-cell Cells and NK cells were rapidly cleared, but CD7-negative T cells and NK cells expanded dramatically and were effector to fungal and viral stimuli, suggesting that these cells were immunoprotective.

 

2.5 AML

AML cell surface antigens are shared with normal myeloid cell surface antigens, so direct targeting of AML antigens would have severe toxic side effects on normal myeloid cells, thereby limiting the therapeutic potential of CAR-T cells.

A preclinical trial used gene editing technology to transfer autologous CD33-knockout hematopoietic stem and progenitor cells into animal models, established a hematopoietic system resistant to CD33-targeted therapy, and enabled CD33-targeted CAR-T cells Locate to AML.

Although it is currently difficult to advance research into the clinic, the results of this trial provide a new way to solve the problem.

Relevant research to find AML targets that are tolerated by normal tissues is also actively carried out.

 

 

 

 


03 Challenges of CAR-T Cell Therapy

 

Understanding the challenges faced by CAR-T cell therapy, identifying the mechanisms that lead to limitations and overcoming these obstacles can enable CAR-T cells to better realize their potential, optimize treatment strategies, and improve patient outcomes.

Several key factors that have been found to affect the efficacy of CAR-T cell therapy include the manufacture of CAR-T cells, the management of toxic side effects, and the recurrence of drug resistance.

 

 

3.1 Problems in CAR-T Cell Manufacturing

The challenges faced by CAR-T cell manufacturing involve multiple links such as T cell acquisition, isolation and screening, transduction, culture expansion, and initial T cell phenotype selection.

Through the optimization of methods in each link, CAR-T cell products can be realized Higher clinical efficacy and less toxic side effects.

At present, the CAR-T cells approved by the FDA are all autologous, and there is no risk of allogeneic rejection and graft-versus-host disease (GVHD), but it is difficult to obtain, and the cell quality is often not available. 

Using cells from healthy donors to produce CAR-T products is one solution to the problem of low-quality CAR-T cell sources.

Early clinical studies have demonstrated the feasibility of using donor-derived CAR-T cells in patients with disease relapse after allogeneic transplantation with a lower risk of GVHD.

In addition, donor-derived T cells facilitate the development of universal CAR-T products, which is of great significance to overcome the existing problems of insufficient CAR-T cell sources, poor quality, and long production cycles, but additional genetic modifications are required to reduce Risk of immune rejection and GVHD.

In addition, CAR structures often contain exogenous sequences.

Due to the difficulty of preparation, most scFvs of CAR-T cells are of murine origin and are immunogenic.

Human anti-mouse antibodies against scFv have been detected in treated patients .

 

Studies have shown that the initial T cell phenotype of CAR-T cell products plays an important role in subsequent clinical responses.

Specific T cell phenotypes, such as central memory T cells, stem-like memory T cells, and precursor T cells, may improve the expansion capacity and persistence of CAR-T cells.

A study of CD19-targeted CAR-T cell therapy in CLL patients found that the CAR-T cell population of responders had abundant memory T cell-related gene expression compared with non-responders.

Another research team induced the CAR protein to enter a quiescent state by forcing down-regulation of the CAR protein through a drug regulatory system or dasatinib, thereby obtaining a memory-like phenotype, successfully reversing the phenotype and transcriptional characteristics of exhausted CAR-T cells, and then restoring Antitumor function of CAR-T cells.

 

In addition, the timing of infusion of CAR-T cells also has an important impact on treatment response.

By shortening the production cycle of CAR-T cells through technological optimization, it is expected to reduce the delay of patients’ illness and benefit more patients.

In addition, the genes encoding CAR structures are usually transduced into T cells by retrovirus or lentivirus, but with the development of transposon systems, it is more economical to use transposons instead of viral vectors for CAR-T cell production.

At present, the Sleeping Beauty transposon system has been applied to the manufacture of CD19-targeted CAR-T cells.

 

 

3.2 Toxic and side effects of CAR-T cell therapy

Almost all patients treated with CD19-targeted CAR-T cells developed toxic side effects of varying degrees, including cytokine release syndrome (CRS) and immune effector associated neurotoxicity syndrome , ICANS), etc., the latter also known as neurotoxic side effects.

The American Society for Transplantation and Cellular Therapy (ASTCT) has developed and published standardized grading recommendations for CRS and ICANS, which have guiding significance for the management and treatment of CAR-T toxicity.

 

The clinical symptoms of CRS often start with fever, and severe cases can lead to systemic inflammatory response, hypotension, hypoxia, and organ failure.

ICANS is mainly manifested as toxic encephalopathy, severe cases can lead to seizures, cerebral edema and coma. Most of the patients with ICANS had a history of CRS, suggesting that CRS may act as an initiating factor or promoting factor for ICANS.

If the early symptoms of CRS and ICANS can be detected and effectively intervened, the clinical course of both is reversible, but severe CRS and ICANS can be fatal.

Understanding the pathophysiological mechanisms of CRS and ICANS is helpful for the development of targeted drugs to suppress the toxicity of CAR-T cells on the basis of retaining the anti-tumor activity of CAR-T cells as much as possible.

CRS is associated with elevated levels of various cytokines, among which IL-6 is an important immune molecule mediating CRS.

Tocilizumab, which blocks the IL-6 receptor, is currently the main treatment for CRS. Preclinical trials have shown that CRS is triggered by a multicellular network of CAR-T cells and host cells, with the monocyte-macrophage system playing a central role in the activation process.

IL-1 is one of the main cytokine products of the monocyte-macrophage system, and may be involved in the driving link of CRS, and blocking this target is effective in relieving CRS.

TNF, interferon-γ (IFN-γ), granulocyte/macrophage colony stimulating factor (GM-CSF) and other pro-inflammatory cytokines are also involved in the process of CRS, which may be potential ‘s target.

Currently, low-grade CRS is mainly treated with antipyretic and supportive treatment, and other complications that may lead to fever, such as infection, are actively prevented.

For moderate to severe CRS, tocilizumab is generally used, and steroids are selectively used as adjuvant therapy according to the patient’s condition, and the effect is more significant.

In patients with severe CRS, steroids are generally used to inhibit the proliferation and cytokine secretion of CAR-T cells and other “bystander” cells.

It should be noted that steroids cannot be used in large doses, and their inhibitory effect on the immune system will lead to a decrease in the efficacy of CAR-T.

Some small molecule inhibitors such as ruxolitinib and ibrutinib can extensively inhibit the production and signal transduction of a variety of cytokines, and can bind to multiple targets, thereby regulating the immune function of CAR-T cells and reducing side effects.

 

The mechanism of ICANS may be related to the accumulation of CAR-T cells and pro-inflammatory cytokines in the central nervous system.

Preclinical trials have observed a correlation between the number of CAR-T cells in cerebrospinal fluid and cytokine levels and the severity of ICANS.

The incidence of ICANS in CD19-targeted CAR-T therapy is higher than that in CD22-targeted CAR-T cell therapy, which may be due to the expression of CD19 in human brain parietal cells.

The clinical treatment of ICANS patients is to give steroids, and the dosage should be the lowest to avoid the impact on the efficacy of CAR-T and serious immunosuppression.

Tocilizumab has achieved good results in the treatment of CRS, but its effect on ICANS is very limited, which may be related to its difficulty in passing through the blood-brain barrier.

 

Toxic side effects are currently an important factor limiting the efficacy of CAR-T, hindering the enhancement of the anti-tumor effect of CAR-T cells by increasing the dose of CAR-T cells or enhancing effector activity.

High tumor burden, advanced age, and high-intensity lymphodepleting preconditioning are thought to be associated with the occurrence of immunotoxic side effects.

With the increase of treatment cases and the extension of follow-up time, more toxic and side effects appeared, such as hemophagocytic lymphohistiocytosis/macrophage activation syndrome-like toxicity, B cell aplastic anemia-related immune dysfunction Damaged state complicated by fatal infection, fatal cerebral edema, etc.

Existing studies have found that adding suicide genes, such as inducible caspase-9 or herpes simplex virus thymidine kinase, to CAR is a possible way to reduce the cytotoxic side effects of CAR-T, but it will cause irreversible clearance of CAR-T cells and reduce resistance. Tumor efficacy.

 

 

3.3 Relapse of drug resistance after CAR-T cell therapy

Although CAR-T cells have made great breakthroughs in the treatment of hematological malignancies, among patients receiving CD19-targeted CAR-T cell therapy, the rate of drug resistance recurrence is as high as 30% to 50%, and most recurrences occur 12 years after treatment. within a month.

However, this kind of relapse is not just for the CD19 target, and related studies on other targets such as CD22 and BCMA have also proved that drug resistance relapse is a major challenge commonly faced by CAR-T cell therapy.

At present, relapse events are usually divided into antigen-negative relapse and antigen-positive relapse.

 

The primary mechanism of antigen-negative relapse is antigen loss.

Currently recognized mechanisms of antigen loss include splicing mutations, epitope cryptic, and cell lineage changes due to loss of target epitopes.

However, even if the antigen is not completely lost, reduced antigen expression or density through immunomodulation is sufficient to allow tumor cells to escape.

In a clinical trial using CD22-targeted CAR-T cells to treat leukemia patients, it was found that leukemia patients with positive antigens relapsed, suggesting that the maintenance of CAR-T cell activity requires a minimum antigen expression threshold.

Combinatorial multi-molecular-targeted CAR-T cells are expected to overcome tumor cell escape from antigen loss or downregulation mechanisms.

For patients who relapse after CD19-targeted CRA-T cell therapy, CD22 is an ideal target because most CD19-negative patients remain positive for CD22 expression.

In clinical trials, CAR-T cells targeting CD22 were effective in the treatment of patients with CD19-negative B-cell lymphoma and leukemia recurrence, but in the process of sequential immunotherapy, drug resistance recurrence caused by down-regulation of CD22 expression by tumor cells was also found.

Therefore, the development of CAR-T cells that simultaneously target CD19 and CD22 may have greater potential in overcoming drug resistance. In addition to the selection of target antigens, it is also necessary to pay attention to the precise mechanism of CAR-T in the formation of immune synapses and the killing of target cells.

Natural TCRs can recognize antigens at low density levels, and it is speculated that structural differences between CARs and natural TCRs may lead to differences in the requirements for antigen recognition density.

 

It is worth noting that not all relapsed patients are CD19 negative, which also shows that in addition to antigen loss and tumor cell escape, there are other factors that lead to CAR-T resistance.

The main cause of antigen-positive relapse is CAR-T cell exhaustion, which leads to self-function decline due to long-term exposure to high levels of antigen.

It is generally believed that the antigen-independent signal transduction of CAR-T cells is closely related to cell exhaustion, and high tumor burden is also an important factor leading to exhaustion.

Immune checkpoint blockade technology combined with CAR-T cells holds promise in overcoming exhaustion and enhancing the effector and persistence of CAR-T cells.

Co-expression of IL-7 receptor with CAR can avoid stimulation of “bystander” cells and improve the proliferative capacity, antitumor activity and persistence of CAR-T cells.

The CAR structure of CAR-T cells contains non-self components, which are immunogenic and may induce humoral and cellular anti-CAR immunity, thereby limiting efficacy and affecting the proliferative capacity and persistence of CAR-T cells.

Studies have shown that 5% of DLBCL patients and 36.7% of B-ALL patients have increased levels of anti-CAR antibodies after infusion of CAR-T cells.

Cyclophosphamide or fludarabine pretreatment is considered to be an important factor in reducing the degree of anti-CAR cellular immunity.

The development of humanized CAR-T products is an effective means to solve this problem, which has shown durable efficacy in relapsed/refractory B-ALL in clinical trials.

The co-stimulatory domain in the CAR structure also affects the persistence of CAR-T cells. It is generally believed that compared with the 4-1BB costimulatory domain, CD28-derived CARs are less durable and more prone to exhaustion; whereas CAR-T cells containing the 4-1BB costimulatory domain have higher levels of anti-apoptotic proteins BCL-2 and BCL-XL, and there may be a mechanism to promote the formation of memory phenotype T cells.

Improvements currently in clinical trials include the use of artificial antigen-presenting cells to activate CAR-T cells, modulate CAR-T phenotypes, and jointly inhibit immune checkpoint molecules.

The results are worth looking forward to.

 

 

 

 


04  Methods to improve the properties of CAR-T cells

 

4.1 Improvements in CAR-T cell design

4.1.1 Improvement of CAR structure

Small changes in any part of the CAR structure can have an impact on the antitumor activity of CAR-T cells.

Studies have found that different scFvs, hinge regions, transmembrane regions, costimulatory domains, and ITAMs can all affect the effectority of CAR-T cells.

 

The most direct way to overcome antigen loss is to develop multi-targeted antigen CARs.

Currently, CAR-T cells using CD20 and CD22 targets in tandem or in combination with CD19 have shown certain safety and feasibility in clinical trials. Another promising multi-target development direction is the modification of CAR-T cells with BiTEs.

BiTEs are usually composed of a CD3-targeting scFv and a tumor-associated antigen (TAA)-targeting scFv connected by a linker.

The design of replacing the scFv with a tumor-specific domain is also in the preclinical and early clinical evaluation stage.

The change in the length of the scFv linker can affect the in vivo activity of CAR-T cells.

Studies have found that short-chain scFv linkers can increase antigen-independent receptor signal transduction and downstream T cell activation by promoting autonomous co-stimulatory signals, promote immune synapse formation, and enhance anti-tumor effects.

Bactrian camel heavy chain CAR replaces traditional scFv, shows strong targeting affinity and efficacy in preclinical models, and has certain advantages due to the elimination of the linker part and the reduction of one immunogenic target.

Replacing murine structures with humanized or fully human scFvs can also reduce the immunogenicity of CARs, avoid host immune rejection, and improve CAR-T cell persistence.

 

The costimulatory domain is an important part of the CAR-T cell effect, but excessive costimulatory signals may promote exhaustion and reduce CAR-T cell persistence.

By mutating specific sites in the CD28 domain, it is beneficial to control costimulation intensity, reduce CAR-T toxicity and improve persistence.

Modulating the strength of intracellular signaling can also affect CAR-T effect, a CD3ζ truncated signaling domain CAR-T cell containing only one ITAM structure showed better responsiveness and persistence in animal models.

Differences in hinge length and composition can also affect the binding and signaling of CAR-T cells to tumor cells.

Modifications to the hinge and transmembrane regions can also reduce immunogenicity and regulate cytokine secretion.

 

 

4.1.2 Gene editing

The application of gene editing technology to optimize CAR-T cells has great potential for development. CRISPR/Cas9-guided genome editing can integrate the CAR gene into the T-cell receptor alpha constant domain (TRAC), which reduces antigen-independent signal transduction by destroying endogenous TCR. Enhance the antitumor activity of CAR-T cells.

Another benefit of disrupting endogenous TCR is GVHD inhibition, this approach not only enhances the persistence of CAR-T cells in vivo, but also facilitates the construction of general-purpose CAR-T cells, overcoming the long production cycle of autologous CAR-T cells The problem.

Programmed cell death 1 (PD-1) is an immune checkpoint molecule expressed on a variety of immune cells and plays an important role in the process of immunosuppression.

PD-1 induces T cell dysfunction and exhaustion by binding to programmed cell death-ligand 1 (PD-L1) on tumor cells. Combining CAR-T cells with PD-1/PD-L1 antibodies is a feasible way to improve the efficacy of CAR-T.

The application of scFv fragments that secrete PD-1 antibody and the use of CRISPR/Cas9 to ablate PD-1 gene CAR-T cells also showed the feasibility of enhancing the efficacy of CAR-T by inhibiting PD-1.

Overexpression of basic leucine zipper ATF-like transcription factor (BATF) and interferon regulatory factor 4 (IRF4) in CD8 + CAR-T cells promotes CAR -T cells survive and expand, and generate long-lived memory T cells that suppress tumor recurrence, and this response is dependent on BATF-IRF interaction, possibly by altering the phenotype and transcriptional pattern of CAR-T cells away from exhaustion , enhance the anti-tumor effect.

 

 

4.1.3 Allogeneic CAR-T cells

The use of donor-derived allogeneic CAR-T cells has many potential advantages, such as facilitating product standardization, improving production efficiency, reducing manufacturing costs, and ensuring product quality.

At present, allogeneic CAR-T cells are mainly derived from peripheral blood mononuclear cells, and a few are derived from umbilical cord blood.

Umbilical cord blood-derived T cells have a unique antigen-naive state, and the use of cord blood-derived CAR-T cells can reduce the risk of GVHD.

Inserting CAR into TRAC through CRISPR/Cas9 gene editing technology can inhibit GVHD by disrupting TCR signal transduction, providing the possibility for the development of allogeneic CAR-T cells and universal CAR-T cells.

UCART19 constructed by gene editing also demonstrated safety and efficacy in a multicenter clinical trial against highly pretreated relapsed/refractory B-ALL, and consolidation therapy with allogeneic stem cell transplantation after UCART19 infusion can produce Long lasting effect.

 

 

4.1.4 Other effector cell types

Due to the limitations of autologous CAR-T cells, the development of CAR constructs for other effector cells has been in preclinical and clinical stages, including NK cells, iNKT cells, γδT cells, macrophages, and dendritic cells, among others.

Compared with CAR-T cells, CAR-NK cells are safer, less likely to produce serious toxic side effects such as CRS, ICANS, etc., and will not induce GVHD.

These characteristics make CAR-NK cells suitable for AML and relapsed/refractory treatment. B-cell malignancies become possible.

The addition of transgenic IL-15 to CAR constructs helps to improve the persistence and antitumor activity of CAR-NK cells.

Another option is cytokine-induced memory-like (CIML) NK cells, which can increase persistence through preactivation of IL-12, IL-15, and IL-18.

The CIML-NK cells exhibited potent antitumor effects in AML xenografted mice in preclinical trials and were also found to induce remission in 44% of AML patients in clinical trials.

Although NK cells and T cells can share some signaling and co-stimulatory domains, such as CD3ζ, 4-1BB, more specific co-stimulatory domains of NK cells, such as DAP10, DAP12, and 2B4, help to improve the CAR-NK cells Antitumor effect.

Previous studies have found that CAR-NK cells using the 2B4 intracellular domain to replace 4-1BB enhanced cytotoxicity, which provides the possibility for the treatment of T-cell malignancies.

 

 

 

4.2 Improving the CAR-T cell microenvironment

Before reinfusion of CAR-T cells, pretreatment with cyclophosphamide and fludarabine to promote lymphocyte exhaustion in vivo is beneficial to increase the effector and persistence of CAR-T cells.

Lenalidomide, ibrutinib, and immune checkpoint inhibitors in combination with CAR-T cell therapy can also enhance their antitumor effects.

However, it should be noted that although increasing the activity of CAR-T cells is beneficial to tumor clearance, the enhancement of CAR-T cell expansion and effector also often leads to enhanced antigen-independent signal transduction, thereby promoting CAR-T cells. exhaustion.

In preclinical trials, lenalidomide in combination with CAR-T cells can enhance the antitumor activity of CAR-T cells by enhancing cytotoxicity, promoting Th1 cytokine production and immune synapse formation.

The combination of ibrutinib and liso-cel can promote the transition of CAR-T cells to a memory-like cell phenotype and improve the effector and persistence of CAR-T cells.

It is generally believed that CAR-T cell exhaustion is an irreversible state of T cell dysfunction. However, recent studies have found that intermittent suspending of CAR-T cell signaling early in treatment can keep cells in a less differentiated stage, reverse the progression of exhausted CAR-T cells, and induce epigenetic reprogramming.

Blockade of proximal TCR signaling with tyrosine kinase inhibitor dasatinib to inhibit CAR-T cell activation can reverse antigen-independent signaling and preserve the antitumor function of CAR-T cells.

Based on this finding, regulating the “rest” of CAR-T cells by intermittent treatment with dasatinib may effectively alleviate the exhaustion of CAR-T cells and improve the anti-tumor ability of CAR-T cells.

Immune memory is critical for the persistence of antitumor effects. Preclinical studies have found that cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) can enhance the effector and persistence of CAR-T cells, promote the differentiation of memory phenotype, and induce anti-blockade Immune checkpoint favorable T cell phenotype.

 

 

 

 


05 Summary

 

CAR-T cell immunotherapy has brought new hope to patients with hematological malignancies, making it possible to cure refractory and recurrent hematological malignancies.

Although there are still many challenges in CAR-T cell therapy, such as immunogenicity, drug resistance, toxicity, etc.

With the development of gene editing technology and the development of other immune target drugs, CAR-T cells are believed to be one of the most promising cancer immunotherapy methods through continuous optimization of design and combination with other immunotherapies.

There is hope in the future to break through the limitations of solid tumors, expand indications, and cure more diseases

 

 

 

 

What are the challenges and solutions for CAR-T therapy?

(source:internet, reference only)


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