Chimeric antigen receptor T-Cell therapy: The way forward!

Sunil B Rajadhyaksha; Anisha A Navkudkar; Priti D Desai

doi:10.4103/gjtm.gjtm_88_21

REVIEW ARTICLE

Year : 2021 | Volume : 6 | Issue : 2 | Page : 118-126

Chimeric antigen receptor T-Cell therapy: The way forward!

Sunil B Rajadhyaksha, Anisha A Navkudkar, Priti D Desai
Department of Transfusion Medicine, Tata Memorial Centre, Homi Bhabha National Institute, Mumbai, Maharashtra, India

Date of Submission	25-Sep-2021
Date of Decision	15-Oct-2021
Date of Acceptance	19-Oct-2021
Date of Web Publication	30-Nov-2021

Correspondence Address:
Dr. Sunil B Rajadhyaksha
Department of Transfusion Medicine, Tata Memorial Centre, Homi Bhabha National Institute, Mumbai, Maharashtra
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/gjtm.gjtm_88_21

Abstract

Various cytotoxic approaches for cancer therapy have been developed over the years. However, due to their limited efficacy, there is a continual search for therapeutic approaches with better outcomes, such as immunotherapy that develops and augments the patient's immune system. Chimeric Antigen Receptor (CAR) T-cell immunotherapy involves genetic modification of patient's own T-cells to express CAR exclusive for a tumor antigen. It involves bioengineering, ex vivo cell expansion, and re-infusion back to the patient. The application of CAR-T therapy is seen as a potential mainstay treatment for hematologic cancers, while it is still being explored for solid-organ malignancies. In this review, the rationale for the development of genetically modified T-cells, its generations, the process of preparing CAR T cells, challenges and potential strategies, limitations, and various clinical applications are described. Information for review was obtained from available material in Google and PubMed.

Keywords: Chimeric antigen receptor-T, immunotherapy, bioengineering, cytokine release syndrome

How to cite this article:
Rajadhyaksha SB, Navkudkar AA, Desai PD. Chimeric antigen receptor T-Cell therapy: The way forward!. Glob J Transfus Med 2021;6:118-26

How to cite this URL:
Rajadhyaksha SB, Navkudkar AA, Desai PD. Chimeric antigen receptor T-Cell therapy: The way forward!. Glob J Transfus Med [serial online] 2021 [cited 2023 Mar 28];6:118-26. Available from: https://www.gjtmonline.com/text.asp?2021/6/2/118/331629

Introduction

The immune system which classically consists of the innate and adaptive parts has overlapping functions and is intimately related. The innate immune system act as the first line of defense, does not require prior stimulation by antigens, and includes dendritic cells, natural killer cells (NK), macrophages, neutrophils, eosinophils, basophils, and mast cells. The adaptive immune system requires the presentation by the antigen-presenting cells for its activation and includes B lymphocytes, helper T lymphocytes, and cytotoxic T lymphocytes.^[1] The adaptive immune system produces antigen-specific T- and B-cell lymphocytes. The immune system is highly variable between persons but relatively stable over time in an individual.^[2]

Methodology of review

The study methodology was a review of available information in Google and PubMed.

Cancer Immunotherapy

The idea to adopt the immune system to treat neoplastic diseases originated in the 19^th century which gave a basis for cancer immunotherapy.^[3] Cancer immunotherapy is rapidly evolving and can now be considered as one of the important pillars of cancer therapy, along with surgery, chemotherapy, and radiation therapy. Although the limited understanding of immune regulatory mechanisms hampers the implementation of immune-based protocols in cancer treatment, its promising effects bring us closer to a future where this disease can be successfully controlled. It uses the antitumor properties of the immune system to fight cancer. Different types of cancer immunotherapies include immune checkpoint inhibitors, monoclonal antibodies, oncolytic virus therapies, cancer vaccines, immune system modulators, and adoptive therapies which induce, augment, suppress, or release the suppression of the immune system response.^[4] Cancer immunotherapy focused on T-cells has surfaced as a powerful tool in the armory against cancer.

Adoptive immunotherapy

Adoptive immunotherapy also called adoptive cellular therapy (ACT) typically refers to a cellular infusion product.^[4] It implies the infusion of immunocompetent cells and is a robust form of immunotherapy for the treatment of established tumors.^[5] It encompasses the isolation and in vitro expansion of autologous or allogeneic tumor-specific T-cells, followed by infusion back into the patient.^[3] ACT bestows the T-cells with the ability to recognize and kill cancer cells through gene engineering. To a certain extent, the manipulation improves or alters the intrinsic immune capacity and utilizes its efficiency in the treatment of cancer diseases.^[6] The various kinds of immune cells which have been used in adoptive cell therapy include tumor-infiltrating lymphocytes, NK cells, cytokine-induced killer cells, T-cell receptor (TCR) T-cells, and chimeric antigen receptor (CAR) T-cells.^[7] One of the most important and promising examples of ACT is chimeric antigen receptor T-cell immunotherapy for the treatment of B-cell hematologic malignancies.^[8]

Chimeric antigen receptor T-cell therapy

CAR T-cell therapy is novel immunotherapy for cancer treatment involving the adoptive transfer of autologous T-cells bioengineered by gene transfer to express receptors that target molecules expressed on malignant cells.^[5],[9] The efficacy of CAR T-cells for the treatment of acute B lymphocytic leukemia (B-ALL) has been revolutionary and numerous clinical trials using CAR-T-cell therapy in the treatment of various types of tumors have been stated.^{[10],[11],[12]} CARs are engineered receptors that redirect most commonly the T lymphocytes to recognize and eliminate cells expressing a specific target antigen. The adaptability of CARs stems from the fact that, unlike innate TCRs, they are independent of the major histocompatibility complex (MHC) receptor resulting in T-cell activation and potent antitumor responses.^[13],[14]

The first CARs were made around 30 years ago and have subsequently undergone a gradual development.^[14],[15]

Structure of Chimeric Antigen Receptor

CARs are integrated synthetic receptors that consist of the following main components [Figure 1]: ^{[16],[17],[18]}

Figure 1: Structure of chimeric antigen receptor

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target antigen binding domain (extracellular)
a hinge region
a transmembrane domain, and
one or more signaling domain (intracellular).

Extracellular target antigen-binding domain

It is the extracellular portion of the CAR that identifies the target antigen and redirects the specificity of CAR-expressing lymphocytes accordingly. CARs typically contain a single-chain variable fragment (scFv) targeting extracellular antigens of proteins expressed by cancer cells on their cell surface, thus MHC independent T-cell activation. Prominently, scFv affinity for the target antigen is an important determinant of CAR function and should effectively recognize tumor cells and induce CAR signaling and T-cell activation. However, extremely high affinity can lead to activation-induced cell death of the CAR cells and thus potential toxicities.^{[19],[20],[21],[22],} Molecules other than scFv which have been used as alternative antigen-binding domains for CARs are ligands, nanobodies, cytokines, peptides, etc.

Hinge region

The hinge region connects the extracellular antigen-binding domain to the intracellular signaling domains through transmembrane domains of CARs. It provides acceptable flexibility to overcome steric hindrance and enough distance to enable access to the target antigen. The differences in the length and composition of the hinge can affect antigen binding and signaling.^[23] The characteristics of the hinge and transmembrane domain also influence CAR T-cell cytokine production and activation-induced cell death (ACID).^[24]

Transmembrane domain

The transmembrane domain anchors the CAR in the T-cell and is a derivative of various proteins such as CD3 ζ, CD28, CD4, or CD8α.^[18] It influences the stability and functions of the CAR.^[25],[26] CAR containing the CD3 ζ transmembrane domain mediates CAR dimerization and incorporation in endogenous TCRs, which might assist CAR-mediated T-cell activation.^[25] CAR T-cells with CD8α hinge and transmembrane domains have been shown to release less IFN γ and TNF and are less susceptible to AICD than those in which these domains are derived from CD28.^[24]

Intracellular signaling domain

The intracellular signaling domain comprises an activation domain and one or more co-stimulatory domains. The majority of CAR activates CAR T-cells via CD3 ζ derived immunoreceptor tyrosine-based activation motifs which alone is insufficient to induce productive T-cell responses. This results in in vivo T-cell persistence and activity. Therefore, a co-stimulatory signal is necessary for optimal T-cell function, metabolism, and persistence. T-cells with CARs containing co-stimulatory domains in adjunct to activation domains produce interleukin (IL-2) and may proliferate upon repeated antigen exposure.^[27] CD28 or 4-1BB (CD137) is the most extensively studied co-stimulatory domain.

Generations of chimeric antigen receptor T-cells

With progressively more effort put into cancer adoptive cell research, CAR-T therapy has gone through generations as mentioned in [Figure 2].^[8] First-generation CARs contain only a CD3 ζ signaling domain but are insufficient to initiate CAR T-cell expansion and generate continuous antitumor activity in vivo. Second- and third-generation CARs hold one or more costimulatory domains, respectively.,^[28] An additional co-stimulatory signal amplifies the original signal and increases T-cell proliferation and cytokine secretion, promoting the secretion of anti-apoptotic proteins thus facilitating the antitumor activity.^[29] After the CAR recognizes the target antigens, fourth generation can activate the downstream transcription factor to induce cytokine production and enhance T-cell activity. The fifth- or next-generation CARs use gene editing to inactivate the T-cell receptor alpha constant (TRAC) gene, leading to the removal of the TCR alpha and beta chains. This has been intended to knock out the human leukocyte antigen (HLA) and TCR genes of T-cells obtained from healthy donors to avoid graft-vs-host disease against transplanted CAR T-cells.^{[30],[31],[32]} Since it does not need to be modified according to the patient, this strategy can be used for the treatment of multiple patients.^[31] The synthetic nature of CARs permits targeting a variety of cancers.

Figure 2: Generations of chimeric antigen receptor T-cells

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Steps in Chimeric Antigen Receptor T-Cell Therapy

The steps involved can be divided into roles by different departments as described in [Figure 3]:

Figure 3: Steps in chimeric antigen receptor T-cell therapy

Click here to view

Department of transfusion medicine
Laboratory
Department of oncology.

Department of transfusion medicine

Pre-apheresis checkup

Clinical checkup: Medical history, current medications, physical examination, and evaluation of comorbidities
Venous access assessment including skin status
Complete blood count
Screening for transfusion transmissible infections markers
Informed consent for apheresis.

Leukapheresis/collection of lymphocytes

Performed on apheresis equipment to collect lymphocytes and remaining blood components, i.e. red cells, plasma, and platelets, are returned back
Approximately 2–4 times' total blood volume may be processed^[33]
Anticoagulant (acid citrate dextrose) is used
Monitoring of ionized calcium levels pre and during procedure must be done and if required calcium infusion to be given to prevent hypocalcemic symptoms
Procedure duration may be around 2–5 h.^[33]

Laboratory

Bioengineering

The T-cells collected by leukapheresis are sent to a laboratory where they are activated by positive selection with a CD3 antibody ± a CD28 antibody.^[34] T-cells are then genetically engineered to express CARs on their surface.^[35] CARs are proteins that permit the T-cells to recognize an antigen on targeted tumor cells. Some of the viruses commonly used for the development of modified T-cells include a γ-retrovirus or lentiviral vector constructs. The genetically modified T-cells are cultured (expanded/multiplied) in vitro in the laboratory and stimulated by cytokines to produce numerous CAR T-cells.^[35] The expansion is done using complex and expensive cell processing methodologies in highly specialized good manufacturing practice facilities.^[6] It is ideal that protocols utilize closed processing to curtail the risk of contamination.^[36] This process takes a few weeks and when there are enough of them, the CAR T-cells are cryopreserved.

Cryopreservation

The processed cells are cryopreserved using a controlled rate deep freezer (minus 80°C) using a cryoprotectant such as dimethyl sulfoxide.

Department of oncology

Conditioning therapy

Before infusion of the CAR T-cells, patients may receive lymphodepleting chemotherapy (low dose chemotherapy with cyclophosphamide or fludarabine) to create space in their immune system for the infused CAR T-cells to proliferate. Effects of conditioning therapy include eradication of immunosuppressive cells, modulation of the tumor, elimination of homeostatic cytokine sinks (IL-2, IL-7, IL-15), increased expansion, function, and persistence of CAR T-cells.^[37],[38]

Infusion of chimeric antigen receptor T-cells

Infusion is done after thawing the cryopreserved product. It takes about 30–60 min for the infusion to be completed.

Recovery of patient

The recovery time for CAR T-cell therapy is shorter and less intense as compared to stem cell transplant. Most patients have complete recovery (complete remission) or partial recovery by 2 to 3 months.

Quality Control Parameters for the Use of Chimeric Antigen Receptor T-Cells

The most relevant parameters for CAR T-cell batch release are provided in [Table 1].^[39]

Table 1: Quality control parameters for the release of chimeric antigen receptor T-cells

Click here to view

Side Effects/Clinical Challenges with Chimeric Antigen Receptor T-Cell Therapy

Following side effects can be associated with CAR-T cell therapy.^{[10],[11],[40],[41],[42]}

Cytokine release syndrome

The most common adverse effect of CAR T-cell therapy is cytokine release syndrome. Signs and symptoms associated with side effects of CAR T cell therapy can be fever, low blood pressure, hypoxia, with or without multi-organ toxicities. The trigger for this condition is the activation of T-cells on the engagement of their T-cell CARs with surrogate antigens expressed by the tumor cell. It characteristically occurs within the 1^st week of CAR T-cell therapy and typically peaks within 1 to 2 weeks of CAR T-cell infusion. The management of CRS includes supportive care (i.e., paracetamol for fever, intravenous fluid for dehydration or hypotension, supplemental oxygen for hypoxia, etc.), corticosteroids, and interleukin 6 antagonists such as tocilizumab and siltuximab.

Immune effector cell-associated neurotoxicity syndrome

The causal pathophysiology and mechanisms in ICANS are incompletely understood. Clinical manifestations range from delirium, seizures, aphasia, cerebral edema, headache, intracranial hemorrhage, or transient coma. In general, at the first sign of neurological symptoms, the patient's bed (head) should be elevated by >30° to minimize the risk of aspiration and to improve cerebral blood flow. Steroids are typically used as first-line therapy and tapered over 2 to 3 weeks.

Other side effects reported are cytopenia, allergy/anaphylaxis, hypogammaglobulinemia, infections, aplasia, etc.^[41]

Advantages and Disadvantages of CAR T-Cell Therapy

One of the major advantages of CAR T-cell therapy is the short treatment time, mostly a single infusion and few weeks of inpatient care, while disadvantages may include production time, cost, manufacturing delay, and dependence on functionality of patient T-cells, which is often reduced by the disease or previous therapies.

Detailed advantages and disadvantages are enumerated as below:^{[6],[16],[43],[44]}

Advantages

High antigen affinity, specificity
HLA independent antigenic recognition
Recognizes proteins, carbohydrates, and glycolipids
Repetitive, serial killing of tumor cells
Living drug and the benefits may last for years.

Disadvantages

Off-target CAR T-cell activation in presence of antigen cross-reactivity
On target CAR T-cell activation in presence of soluble antigens (it may block the ability to recognize cell surface antigen)
Poor trafficking and tumor infiltration
Immunosuppressive microenvironment
Limited efficacy against solid tumors
Antigen escape
Inhibition and resistance in B-cell malignancies
Limited persistence.

Approved Chimeric Antigen Receptor T-Cell Therapies

Currently following CAR T-cell therapies have been approved by the FDA:

Tisagenlecleucel (Kymriah): Approved for the treatment of adults with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) and young adult patients with relapsed or refractory ALL.
Axicabtageneciloleucel (Yescarta): Approved for the treatment of adults with certain types of B-cell lymphoma
(Brexucabtagene autoleucel (Tecartus): Approved to treat patients with mantle cell lymphoma
Lisocabtagene maraleucel (Breyanzi): Approved to treat adult patients with relapsed or refractory large B-cell lymphoma
Idecabtagene vicleucel (Abecma): Approved to treat patients with Multiple myeloma.

While this technology is not yet routinely available in India, several groups are working to establish the technology and make it locally available.^[45] CAR T-cells have to follow all the necessary steps and regulatory requirements of HSCT along with additional standards for the manufacturing and release of CAR T-cell products for clinical use.

Applications of Chimeric Antigen Receptor T-Cell Therapy

In various hematological malignancies

Chimeric antigen receptorT-cell therapy in acute lymphoblastic leukemia

CAR T-cell therapy has demonstrated efficacy in treating acute lymphoblastic leukemia (ALL), especially for relapsed or refractory B-ALL. CD19, an important molecular marker of B-cells, is almost an ideal target in treating B-ALL for its higher expression on the surface of tumor cells. Some patients gradually became insensitive to CD19-specific CAR T-cells because of “antigen escape” of cancer cells, for which additional molecule targets on tumor cells such as CD 20 and CD 22 can be used.^[46] Kymriah (Novartis) is a CD19-specific CAR T-cells FDA-approved drug for the treatment of ALL.

Chimeric antigen receptor T-cell therapy in chronic lymphoblastic leukemia

Chronic lymphoblastic leukemia (CLL) can generate developing immune deficiency and lead to more complex clinical symptoms than ALL. CAR T-cells were explored for therapy of relapsed CLL in which CD19 CAR T-cells were mostly used.^[47] Some clinic trials have shown decent curative effect but with limited ability of cell expansion and proliferative response for the immune deficiency of CLL patients.^[48]

Chimeric antigen receptor T-cell therapy in lymphoma

Although the most traditional therapy for lymphoma including non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) was chemotherapy regimens and monoclonal antibodies, still, some patients experienced disease deterioration after these therapies.^[49] CAR T-cell therapy is the immunotherapy that is used for intractable B-cell lymphoma or patients with poor prognosis after primary therapies.^[50] Besides CD19, CD20 or CD30 is also critical in treating lymphoma by using CAR T-cells. CD20 CAR T-cells have produced remarkable clinical benefits in innumerable NHL treatments.^[51] FDA-approved anti-CD19 CAR T-cell products for the treatment of DLBCL are axicabtagene ciloleucel/Yescarta.

Chimeric antigen receptor T-cell therapy in multiple myeloma

Clinical trials of CAR T-cell therapy for multiple myeloma have demonstrated promising clinical activity, providing exceptional response rates. The B-cell maturation antigen is an alluring target for CAR T-cell therapy as it is only expressed by normal and malignant plasma cells and by a subset of mature B-cells.^[52],[53] Patients with multiple myeloma who have been treated with CAR T-cells generally report improvements in quality of life during the treatment-free interval. Thus, it focuses on the potential benefit of CAR T-cell therapies for patients, especially if the endurance of responses to CAR T-cell therapy can be eventually improved.

In solid tumors

Although CAR T-cells are transforming the management of hematologic malignancies, there are still numerous hurdles to efficaciously apply these therapeutic approaches more broadly to solid tumors. As compared to hematological tumors, CAR T-cell therapy is limited in solid tumors as CAR T-cells may be unable to enter the tumor tissue through the vascular endothelium. The major difference between solid tumors and hematological tumors is that it is more difficult to find an ideal target antigen.^[54]

Other nononcologic conditions where chimeric antigen receptor T-cell therapy is being explored

The application of CAR T-cell technology is eagerly anticipated in different fields like for the treatment of viral infections in patients with primary immune deficiency.^[55] This includes selection and expansion of antigen-specific T-cells for the treatment of human cytomegalovirus, infectious mononucleosis (Epstein–Barr virus disease), and adenoviral infections of respiratory and intestinal tracts.

Universal Chimeric Antigen Receptor T-Cells

Universal CAR T-cells are “off-the-shelf” allogeneic products, whose production can be industrialized, evolved, and thereby standardized with consistent pharmaceutical release criteria, over time and from batch to batch. Universal immune receptors offer the ability to improve upon many of the drawbacks that may be associated with CAR T-cell therapy. It creates the opportunity to significantly reduce the cost burden associated with the personalized medicine manufacturing approach of current CAR T-cells. These cells could then be engineered with further enhancing elements to provide an optimized universal T-cell therapy.

Conclusion

CARs are modular synthetic receptors and CAR T-cells have transformed the treatment of certain hematological malignancies however, as outlined obstacles persist. To meet the demands of this complex and progressing field, innovative curriculum development and trained workforce is required. In the future, CAR T-cell therapy may even replace most types of transplants but at present, CAR T-cell therapy is approved for the treatment of patients in whom transplant is not likely to be curative or in patients who relapse after transplant.

Current Limitations and Potential Strategies of Chimeric Antigen Receptor T-Cell Therapy

Antigen escape

It can occur when development of tumor resistance is to single antigen targeting CAR structures. Over the time, malignant cells of a significant fraction of patients treated with CAR T-cells display either partial or complete loss of target antigen expression. Targeting multiple antigens and enhancing the selection of target antigens will improve the antitumor response and also reduce antigen escape mechanisms to avert relapse.

On-target, off tumor effects

In solid tumor, the antigens are often also expressed on normal tissues at differing levels. Therefore, selection of antigen is crucial in design of CAR to ensure therapeutic effectiveness and to reduce “on-target off-tumor” toxicity. A potential chance to overcome this is the targeting of tumor-restricted post-translational modifications.

Chimeric antigen receptor T-cell trafficking and tumor infiltration

Solid tumor CAR T-cell therapy is limited by CAR T-cells' ability to travel to and infiltrate solid tumors as compared to hematological malignancies. The immunosuppressive tumor microenvironment and the tumor stroma may limit the penetration and mobility of CAR T-cells. Local administration as compared to systemic delivery may help overcome this limitation.

Immunosuppressive microenvironment

In the tumor microenvironment, several cell types that lead to immunosuppression can infiltrate solid tumors and may cause the production of tumor facilitating cytokines, chemokines, and growth factors. In addition, immune checkpoint pathways can serve to decrease antitumor immunity.

One of the main causes of no or a weak response to CAR T-cell therapy is short-term T-cell persistence and poor T-cell expansion. Combination of immunotherapy with CAR T-cells and checkpoint blockade can help with this situation.

Chimeric antigen receptor T-cell-associated toxicities

Cytokine release syndrome and neurotoxicity are the common CAR T cell-associated toxicities which can be overcome by altering or modifying the CAR structure.^[16]

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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