What are the Challenges of CAR-T Cell Therapy?

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What are the Challenges of CAR-T Cell Therapy?

Chemotherapy, radiotherapy and surgery are the most traditional cancer treatments, but they are less effective and have serious side effects.

Therefore, over the past decade, researchers have developed new strategies to achieve complete remission of the disease.

Currently, immunotherapy has revolutionized cancer treatment, and there are several types of immunotherapy used to treat cancer, including adoptive cell therapy ( ACT ).

Chimeric Antigen Receptor ( CAR ) T cell therapy is an ACT in which autologous T cells are genetically engineered to express a CAR to specifically kill tumor cells.

CAR-T cell therapy is an opportunity to treat patients who do not respond to other first-line cancer treatments and has demonstrated superior antitumor efficacy in the treatment of hematological malignancies.

However, there are still many challenges to overcome for this type of therapy as first-line clinical treatment.

From a pharmaceutical point of view, this emerging technology is still classified as an advanced therapy, so certain requirements of pharmaceutical regulation must first be met before it can be applied.

Therefore, it is necessary to analyze the elements and challenges of CAR-T cell technology, and take into account basic, clinical, and practical factors, and adopt coping strategies, so that CAR-T technology can become an affordable treatment mode.

A brief history of CAR-T cell development

Reviewing the development history of CAR-T, we should first mention the first bone marrow transplantation of leukemia patients reported by Thomas and colleagues in 1957 and the subsequent discovery of the origin of T cells by Miller et al.

However, it wasn’t until Steven Rosenberg reported a study on tumor-infiltrating lymphocytes ( TILs ) in 1986 that people focused on the idea that “a patient’s own immune cells can fight their own cancer.”

In 1992, Sadelain, who successfully established a method of gene transfer to T lymphocytes retrovirus-mediated gene modification as a means to control the immune experimental or therapeutic setting.

Almost simultaneously, ZeligEshhar and colleagues used an antibody and T cell receptor binding domain of the immunoglobulin ζ or γ subunits, by fitting the single-chain design specifically activate cytotoxic lymphocytes, which developed the first Generation CAR-T cells.

Five years later, Dr. Sadelain’s group demonstrated that integrating costimulatory signals such as CD28 into CAR-T enhanced survival, proliferation, and maintenance of activity, leading to the development of second-generation CARs.

Subsequently, CAR-T cells carrying CD19 targeting were developed and phase I clinical trials for chronic lymphocytic leukemia ( CLL ) and acute lymphoblastic leukemia ( ALL ) were initiated.

Trial results demonstrate that CAR-T therapy induces effective remission in adults with chemotherapy-refractory ALL, followed by scale-up of bioprocessing manufacturing.

In 2017, the FDA approved a CD19 CAR-T cell therapy ( Tisagenlecleucel ) for ALL in children and young adults. So far, the FDA has approved five CAR-T cell therapies for cancer treatment.

What are the Challenges of CAR-T Cell Therapy?

Clinical Challenges of CAR-T Cell Therapy

The challenges facing CAR-T cell therapy are mainly related to side effects, toxicity, T cell depletion and the malignant tumor microenvironment ( TME ).

In addition, the manufacturing process in large-scale production is currently time-consuming and expensive, making it a greater challenge to get as many patients as possible to receive CAR-T cell immunotherapy.

What are the Challenges of CAR-T Cell Therapy?

Side Effects and Toxicity

Following CAR-T cell infusion, this immunotherapy could have potentially lethal toxicity. Some reported side effects include fever, inflammation, abnormally elevated liver enzymes, difficulty breathing, chills, confusion, dizziness, severe nausea, vomiting, and diarrhea.

All patients had long-term B-cell aplasia, which was relieved by administration of gamma globulin. There are two main categories of toxicity: cytokine release syndrome ( CRS ) and neurotoxicity ( NTX ) or CAR-T cell-associated encephalopathy syndrome ( CRES ).

CRS or “cytokine storm” is a systemic inflammatory response that is caused by extensive activation of lymphocytes ( B cells, T cells and natural killer cells ) and myeloid cells ( macrophages, dendritic cells and monocytes ) clinical symptoms, including fever, fatigue, headache, rash, arthralgia, and myalgia.

CRS was the most common adverse reaction that occurred within a few days after the first infusion of CAR-T cells ( 85% of patients experienced CRS of any grade ).

Severe cases of CRS are characterized by tachycardia, hypotension, pulmonary edema, cardiac insufficiency, hyperthermia, hypoxia, renal impairment, liver failure, coagulation disorders, and irreversible organ damage.

Fortunately, the effects of CRS can be alleviated by reducing the number of infused T cells and/or by taking anti-IL-6 receptor monoclonal antibodies and steroids.

NTX is another common complication of CAR-T cell immunotherapy, occurring in more than 40% of patients. It usually occurs within 1 to 3 weeks after CAR-T cell infusion and is usually associated with CRS.

Patients present with various symptoms such as confusion, dullness, tremor, delirium, difficulty finding words, and headache; other symptoms such as aphasia, cranial nerve abnormalities, and epilepsy have also been reported.

Timely management of toxicity is critical to reduce immunotherapy-related mortality, therefore, researchers have developed different safety strategies to overcome and prevent CAR-T cytotoxicity, such as designing a new generation of CARs.

Toxicity management has become a critical step in the success of CAR-T cell immunotherapy.

CAR-T cell depletion

Despite the high rate of complete remission with CAR-T cell therapy, most patients who achieve remission show disease relapse within a few years, with B-ALL relapse rates ranging from 21% to 45% and increasing with longer follow-up .

Part of the reason for treatment failure is the depletion of CAR-T cells by TME produced by solid tumors.

CAR-T cell depletion refers to a dysfunctional state characterized by the loss of antigen-specific T cells due to persistent antigenic stimulation and increased expression of the costimulatory domains and inhibitory receptors of the CAR construct.

In vitro CAR-T cell studies have shown that the upregulation of inhibitory receptors ( such as PD-1, Lag3, Tim3, and TIGIT ) and the inhibition of the PI3K/AKT pathway by CTLA-4 during CAR-T cell depletion are important The main reason for the loss of anti-tumor function. Cytokines also play an important role, as depleted CAR-T cells reduce the ability to express and secrete IL-2, TNF-α, and IFN-γ.

Other factors, such as transcription factors, metabolism, and epigenetic modifications, also play important roles in the development of CAR-T cell exhaustion.

A possible way to delay exhaustion is to construct exhaustion-resistant CAR-T cells. Recent reports suggest that the discovery of certain transcription factors such as TOX and NR4A, and the loss or overexpression of the AP-1 family transcription factor c-Jun increase the resistance of CAR-T cells to exhaustion.

Recently, PD-1 knockout via CAR-T cell engineering ( CRISPR/Cas9 ), or the use of PD-1-blocking antibodies have been used to enhance CAR-T therapy and avoid exhaustion.

Tumor microenvironment

CAR T-cell immunotherapy has not been successful in solid tumors. One possible reason is that the immunosuppressive properties of TME affect the efficacy of adoptive immunotherapy.

Solid tumors present highly infiltrating stromal cells such as cancer-associated fibroblasts ( CAFs ) and suppressive immune cells, including myeloid-derived suppressor cells ( MDSCs ), tumor-associated macrophages ( TAMs ), tumor-associated neutrophils ( TAN ), mast cells, and regulatory T cells ( Treg ), which help establish an immunosuppressive TME that can interfere with the efficacy of CAR-T cell therapy.

Strategies to overcome the effects of the TME include enabling T cells to resist tumor suppression in the TME, such as transgenic expression of dominant-negative receptors or signal converters, which can convert inhibitory signals into stimulatory signals.

Another opportunity to overcome CAR-T cell persistence and depletion is to improve drug delivery to the tumor site. For CAR-T cells, local injections are an ongoing endeavor.

Genetic alteration

Some reports show lack of efficacy and relapse in patients treated with CAR-T cells with genetic alterations.

Orlando et al. integrated exome-wide DNA-seq and RNA-seq to investigate the extent to which CD19 mutations contribute to relapse.

They found de novo genetic alterations in exons 2–5 of the CD19 gene in all 12 patient samples, and loss of heterozygosity in 8 of 9 patients, concluding that CD19 homozygous mutations are acquired anti-CAR- The main reason for T cell therapy.

Similar findings were reported by Asnani et al., who described skipping of exons 2 and exons 5–6 in patients with relapsed leukemia after CAR-T cell therapy.

Exon 2 is critical for the integrity of the CAR-T CD19 epitope, while exons 5-6 are responsible for the CD19 transmembrane domain. However, further studies are needed to explore the impact of genomic analysis.

Manufacturing process challenges of CAR-T cells

Traditional techniques for making CAR-T cells include:

1) Isolation of patient T cells by apheresis ( autologous );

2) Shipping the recovered cells to a central production site;

3) Genetically modifying them to express CAR;

4) Expansion in the laboratory;

5) CAR-T cells are sent back to the hospital and injected into the patient.

What are the Challenges of CAR-T Cell Therapy?

The logistics involved in traditional manufacturing and treatment with autologous CAR-T cells have added complexity to clinicians and patients, and today, this therapy presents some major manufacturing challenges, including:

Packaging, Shipping and Storage of CAR-T Cells

The clinical manufacturing of CAR-T cells is currently a complex process involving multiple steps, spanning different geographic locations, employing multiple technologies and logistics.

Any errors in timing, transportation method, cold chain, or storage can lead to cell damage that directly affects treatment efficiency, so every step requires careful management, accurate sample tracking, and adequate preservation techniques to freeze patient samples.

During the entire CAR-T cell manufacturing process, different transports at different temperatures are required, therefore, the cryopreservation during the production process must ensure quality control.

Good Manufacturing Practice (GMP)

CAR-T cells are a complex preparation process, and cGMP is the key and bottleneck of CAR-T cell production.

The purpose of cGMP is to provide a framework to ensure high-quality production in well-controlled facilities and equipment by well-trained and regularly trained employees.

Likewise, it provides rigorous documentation processes covering all operational aspects to demonstrate ongoing and adequate compliance.

According to the International Organization for Standardization ( ISO ), CAR-T cell manufacturing requires a GMP facility as a cell processing clean room, which must be equipped with

1) facility systems ( such as air handlers, 24/7 alarm monitoring systems );

2) environmental monitoring equipment ( such as particle counters );

3) manufacturing process equipment ( such as cell washers, bioreactors );

4) analytical equipment ( such as automated cell counters, flow cytometers ).

Another key factor in maintaining a GMP compliant production environment is highly skilled staff with extensive knowledge of GMP production, quality control and quality assurance.

Preparation of lentiviral vector (LV)

The production of LVs faces many challenges, such as their inherent cytotoxicity, low stability, and dependence on transient transfection effects, in addition, the upstream and downstream processes are low-yield and cost-effective.

Part of this successfully commercialized product is the establishment of standardized and stable cell lines to generate LVs that facilitate a GMP-compliant process that can provide easier scale-up, reproducibility, biosafety, and cost-effectiveness.

Staff and training

Given the complexity of the treatment and its associated high risk of side effects, the use of CAR-T cells is highly regulated and can only be used in accredited centers and administered by trained staff.

All employees involved in CAR-T cell manufacturing ( from T cell collection to manufacturer to clinical unit ) require extensive training with a satisfactory level of competence.

This capability manages the complexities that may arise in the process so that the product can be delivered.

Today, there are only a few qualified professionals in this field, multidisciplinary collaboration and exchange are required to create more knowledge in this field, and academic participation is also an important aspect.

Quality Control

As a living “drug”, CAR-T cells have a complex preparation process that requires “whole process quality control”.

During production, well-controlled cold chain transportation and storage play an important role in ensuring the quality of cell products and preventing bacterial and mycoplasma contamination.

Requirements for quality control of CAR-T cells include checking T cells transduced in vitro for viral replication and residual production materials.

In addition, considering the characteristics of CAR-T cells as biological products, cell products, and gene therapy products, release testing of finished products should also be included to confirm their identity, purity, safety, and potency.

What are the Challenges of CAR-T Cell Therapy?

In addition, stability studies are required to verify storage conditions and their shelf life.

The generation of CAR-T cells requires more in-depth studies to assess T cell quality in relapsed and reinfused patients.

These studies should provide data on the distribution of lymphocyte populations. In conclusion, quality control is critical to the success of CAR-T therapy.

Production scale up

CAR-T cell manufacturing should be scalable ( i.e., equip each patient with multiple single bioreactors ) to expand the reach of patients without sacrificing product quality and reproducibility.

Personalized therapy ( such as autologous cell therapy ) does not just increase the volume like ordinary biopharmaceuticals, but requires more delicate scale-up, that is, having multiple bioreactors to amplify each patient’s CAR-T cells. Furthermore, it depends on the ability to implement multiple independent products in parallel.

Manufacturing time and repeat dosing

The manufacture of CAR-T cells can take up to 4 weeks, during which time patients are extremely vulnerable to disease progression and death.

In addition, CAR-T cell manufacturing does not allow for volume scaling, therefore, cells must be prepared as a single batch, limiting the number of products available.

In this case, patients may not have the opportunity to receive new CAR-T cell infusions quickly and easily.

Pricing and Availability

Pricing and patient accessibility are the most important constraints to the widespread use of CAR-T cells worldwide.

The current CAR-T cell manufacturing model is highly centralized and the process at each step is complex, resulting in a cost of up to $373,000 to $475,000 per treatment ( treatment-related hospital costs are not included in these average costs ) , neither patients nor the healthcare system can afford it.

This prohibitive cost limits patient access to treatment, especially in socioeconomically underdeveloped countries, which further limits the widespread use of CAR-T cell therapy.

Until CAR-T cell therapy becomes affordable, its therapeutic potential will not truly be realized.

Regulatory requirements

Another important bottleneck for cellular products is regulation. CAR-T cells are globally considered Advanced Therapeutic Drug Products ( ATMPs ), which require a license.

Regulatory agencies are highly relevant to standard therapies, but cell products have special requirements. U.S. or EU regulators are working to define the best guidelines to harmonize requirements for the clinical manufacture of ATMPs globally.

At the same time, less developed countries face greater challenges, as the use of CAR-T therapies in the clinic is greatly restricted, resulting in a lack of understanding of regulatory requirements by the authorities.

Strategies to enhance the use of CAR-T cell technology

Discovery of new biomarkers

Biomarkers are of great significance for the clinical treatment of cancer, and they can be used to identify patients suitable for CAR-T therapy, prognosis, prediction of treatment response, and monitoring of disease progression.

The first biomarker for CAR-T therapy is CD19, a B-cell surface protein expressed primarily on malignant B cells.

Currently, different biomarkers are being sought depending on the stage of immunotherapy, including biomarkers to determine the baseline status of patients, CAR-T cell function, CAR-T cell depletion, biomarkers of CAR-T cytotoxicity and cancer prognosis, Biomarkers of Response and Relapse.

Baseline biomarkers include cytokines such as IL-2, IL-5, IL-7, TNF-a, etc.; lactate dehydrogenase ( LDH ) and CD9 cells have been widely used.

For CAR-T cell function, the following biomarkers were proposed: CD45RA, CD45RO, CD62L, CCR7, CD27, CD28, CD25, CD127, CD57, and CD137.

Currently, there are no well-established biomarkers available to assess CAR-T cell depletion after infusion in patients. Some indirect parameters may contribute to this goal, such as high-level expression of inhibitory receptors such as PD-1, LAG-3, TIM-3, etc.

Despite significant progress in CAR-T therapy, there is still a need to continue exploring different cancer cell type-specific biomarkers to develop more specific treatments.

Allogeneic CAR-T cells

Currently, most CAR-T cell immunotherapies are generated using autologous T cells.

This presents several disadvantages at different levels, such as the potentially time-consuming and complex production process leading to increased costs, and furthermore, due to the use of patient-derived T cells for CAR therapy, challenges include weaker proliferation of CAR-T cells, expansion of Limited growth and poor sustainability.

One opportunity to ameliorate these problems is the use of allogeneic CAR-T cells, thereby reducing the time delay in autologous cell production. In addition, generating generic CAR-T cells from allogeneic healthy donors is easier to obtain and of higher quality.

This is important for patients with aggressive cancer who need urgent treatment.

This strategy will expand the number of patients who can receive this immunotherapy, making CAR-T cell therapy an off-the-shelf treatment that is inexpensive, readily available, and will improve the quality properties of T cells.

Generic CAR-T cells also face some challenges and problems. For example, an immune mismatch between donor and recipient could lead to life-threatening graft-versus-host disease ( GVHD ) if the subject’s allogeneic T cells attack healthy recipient tissue if the subject’s immune system Identify and respond to allogeneic T cells that may be rapidly eliminated by the host immune system. A possible solution is to eliminate GVHD by knocking out or disrupting the TCR gene and/or HLA class I locus on the donor.

Compared to CAR-T cells, CAR-NK cells offer additional advantages, such as reduced cytokine release syndrome and neurotoxicity in an autologous environment; unlimited “off-the-shelf” NK cells can be provided through the use of IPSCs, Rapid response to malignant cells without causing GVHDs; another advantage is the activation of multiple mechanisms of cytotoxic activity ( NKG2D, KIR, CD16, NKp30, NKp44, NKp46 ), still maintaining its target for solid tumors and drug resistance Permeability of the tumor microenvironment.

Preclinical studies of CAR-NK have shown that it is effective against hematological malignancy targets ( CD19 and CD20 ) as well as solid tumor targets, proving its potential for allogeneic therapy.

Additional strategies to improve CAR-T cell therapy

Dual-targeting, or tandem CARs, consist of the co-expression of two separate CARs in each T cell that recognize two different antigens.

Some dual CARs have entered clinical trials targeting CD19/CD20 hematologic malignancies and solid tumors.

HER2/MUC1 bispecific CAR showed promising results in in vitro tests in breast cancer models. Dual CAR is a very promising approach to address antigenic heterogeneity and prevent relapse.

In addition, synthetic Notch ( synNotch ) receptors have been applied to CAR-T cells to improve safety.

SynNotch receptors recognize a specific tumor antigen and then release the transcriptional activation domain to promote local expression of the CAR.

Furthermore, synNotch-regulated CAR expression prevents constitutive signaling and depletion, leaving a higher proportion of T cells in a naive/stem cell memory state.

Inhibitory chimeric antigen receptors ( iCARs ) contain inhibitory receptors, such as PD-1 and CTLA-4, that play a key role in attenuating or terminating T cell responses, therefore, they are considered a safe strategy that enables T cells are able to differentiate between target and non-target cells.

Furthermore, for the effective treatment of solid tumors, innovative combinatorial strategies such as vaccines, biomaterials, and oncolytic viruses are promising as they can directly enhance T cell function or recruit endogenous immune cells and remodel the TME.

Fully automated manufacturing process

Worldwide, the number of patients requiring CAR-T cell immunotherapy is rapidly increasing, and automated and closed manufacturing platforms have been developed in the industry to accommodate this situation.

Examples of this work include automated platforms from Cocoon® ( Lonza ) and CliniMACS Prodigy ( Miltenyi Biotec ), both of which allow replication and rapid production of cells with rigorous documentation of each step.

In 2020, Lonza and Sheba Medical Center announced that the first patient received CD19 CAR-T cell immunotherapy at Sheba Medical Center using the Lonza Cocoon platform.

Collaborative network

Due to high cost and high technical requirements, CAR-T cell therapy is still unaffordable for most patients at present.

In order to achieve universalization of CAR-T cells, it is necessary to establish a collaborative network between different stakeholders such as academia, industry and hospitals, in order to develop adequate and robust protocols for such products in each country where this technology is applied. legislation.

Therefore, universities should provide future professionals with the knowledge and innovation to study new biomarker discovery, develop stable cell lines, develop new assays to validate product quality, and understand different production systems.

Industries that apply GMP, quality control, and automated processes can support the creation of more standardized, reliable, and higher-quality products, as well as develop on-site production units that reduce transportation and storage costs.

Hospitals must have adequate facilities to manage this technology, staffed with personnel trained in the management of on-site production units, as well as medical staff and health professionals who can effectively treat patients.

Including all these activities, strong and specific legislation in this area must be enacted to guarantee the quality of this type of advanced treatment.

Furthermore, regulation is an important part of this process. Today, there is little information on regulatory guidelines in different countries, and EMA and FDA guidelines have established a baseline of such requirements for these technologies, but the conduct and outcomes of clinical trials at different stages will involve new considerations.

Summary

From a pharmaceutical point of view, CAR-T cells are considered as advanced therapeutic products, the quality of which must be demonstrated by their properties, safety and efficacy. Each of these three areas presents significant challenges.

The scientific community as well as the industry need to continue their efforts, in the clinical aspect, to continue to find new biomarkers, improve the development of CARs, reduce the adverse reactions related to CRS and NTX, and the application of CAR-T cells in solid tumors to improve the Safety and efficacy of this therapy.

In terms of manufacturing and processes, the need for lower cost production systems, the stability of production, and the necessary quality requirements are ongoing challenges.

In addition, these processes must fully comply with the requirements set by international and domestic regulatory authorities.

Finally, multidisciplinary involvement of professionals from all fields at all stages is required.

Through close collaboration between academia, industry, hospitals and governments at the international and regional levels, it will be possible to make this novel technology more widely available to more patients.

references:

1. Chimeric Antigen Receptor-T Cells: A Pharmaceutical Scope. Front Pharmacol. 2021; 12: 720692.

What are the Challenges of CAR-T Cell Therapy?

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What are the Challenges of CAR-T Cell Therapy?

What are the Challenges of CAR-T Cell Therapy?

A brief history of CAR-T cell development