Prospects for implementation of CAR-T cell therapy for leukemia in Uzbekistan

Introduction

CAR-T cell therapy, or immunotherapy mediated by chimeric antigen receptors (CARs), is considered a promising method of leukemia treatment. By this approach the patient's own immune cells are genetically modified to enhance their anti-cancer activity, increase the effectiveness of treatment and improve the clinical outcomes of the disease. CAR-T cells are genetically engineered with modified artificial T-cell receptors which are able to recognize specific antigens on the surface of the target malignant cells and subsequently induce activation of T-cells in a manner independent of histocompatibility molecules [7, 11, 14]. They are able to guide T-cells and secure recognition of tumor antigens. The modified receptors can be directed at any antigen of choice, increasing its ability to kill target tumor cells. CAR-T cells therapy is considered a "living therapy" because once the cells are reinfused into the patient, they tend to persist in the body for a long time [2, 3, 5, 10, 17].

The first clinically available CAR-T cell-based drugs were produced back in the 1990’s. Several autologous CAR-T cell products targeting CD19 antigen were approved in Europe for the treatment of resistant/relapsing B-cell acute lymphoblastic leukemia (ALL), B-cell lymphoma, and mantle cell lymphoma [3, 9]. This method also received FDA approval for the treatment of these diseases, being commercially licensed. Recently FDA has approved CAR-T therapy for the treatment of multiple myeloma. Nowadays, CAR-T cells of different specificity are being intensively studied worldwide for the treatment of acute myeloid leukemia (AML) and solid tumors [1, 13, 16, 17].

CAR T-cell therapy is a complex and expensive treatment, e.g., $475,000 for one-time treatment with tizagenleucel (Kymriah, Novartis) and list price of over $373,000 for axicabtagen ciloleucel (Yescarta, Kite Pharma). This, however, does not include the pre-infusion treatment cost, drug administration and hospitalization costs, or costs associated with the treatment of complications and follow-up care. According to a survey from the Vizient's Inc. healthcare company (2019), the mean hospitalization time for such patients is about 15-19 days, which requires additional costs of $15,000 to $20,000 for the treatment of complications.

State of art

All new CAR-T cell products go through the established sequence of clinical trials to confirm their safety, improve cytotoxic profile, increase efficiency and durability of clinical effect. Based on the accumulated international evidence, the following promising areas of CAR-T cell usage may be outlined:

1. Bispecific CAR-T cells bear receptors which contain two distinct antigen recognition domains and two intracellular signaling domains expressed as two specific CARs on the cell surface. E.g., bi-specific CAR CD19/CD20 is a new synthetic molecule that can recognize and bind several targets of tumor antigens on malignant cells. Consequently, it can form a synergistic set of effector molecules when it encounters two antigens on malignant cells. Such an effector cell contains two CARs in a single T-cell. In other words, it seems to be "holding hands", consisting of two different recognition domains (scFvs) in the CAR structure [5, 11]. Such tandem CAR (Tuner) or Dual-signal CAR construct targets two tumor antigens [5].

2. Inhibitory CARs (iCARs). Another novel approach in cancer immunotherapy concerns activation of new immuno-inhibitory receptors expressed by T-cells, i.e., programmed death-1 (PD-1), programmed death ligand-1 (PD-L1) or cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). These molecules have been shown to modulate T-cell activity, such as attenuation or cessation of cellular immune response. E.g., some tumor cells contain high levels of PD-L1 to avoid T-cell attack. It has been found that PD-1 or CTLA-4 based iCARs can effectively control cytotoxicity, cytokine secretion and cell proliferation. CTLA-4 or PD-1 make up the intracellular domain of an i-CAR, triggering inhibitory signals on T-cells e.g., causing decreased lysis of target cells and reduced cytokine production. iCARs are designed to control the action of CAR-T cells through inhibitory receptors. These effects combine the action of two chimeric receptors; one of them produces a dominant-negative signal limiting the response of CA-T cells activated by the appropriate receptor [3, 14, 15, 16].

iCARs can limit the response of CAR activator to the antigens expressed by normal cells only, having the ability to distinguish between cancer and normal cells. Therefore, inhibition of T-cells and usage of genetic engineering to regulate the response of T-cells may be used in antigen-selective manner [15].

3. Physiological CAR (CAR structure combines ligand-receptor molecule). This CAR construct contains a ligand and receptor molecule rather than highly immunogenic murine scFv, in order to recognize paired target molecules on tumor cells. This CAR-T version can persist in recipient much longer. In addition to conventional physiological CARs, a modified version has been developed, also known as a receptor-ligand CAR – it can recognize tumor antigens such as HER3 and HER4 and bind to them. The CARs of this type contain an antigenic receptor and an intracellular CD3 signaling domain with or without the transmembrane domain. This construct may also be engineered in immune cells to target ligands expressed on tumor cells. This novel approach increases the ability of T-lymphocytes to distinguish between tumor-related targets and destroy cancer cells [9, 13].

4. Universal CAR-T cells (Universal CAR, uCAR). Universal CARs (uCARs) refer to the mAbs labeled with avidin-biotin, and not to scFvs, and which can recognize almost any antigen targeted by the given mAb in the extracellular space. While scFv specifically targets tumor-associated antigens, CAR-T cells may have insufficient specificity potential for their recognition. Universal CARs (uCARs) were developed to overcome this limitation. To create a uCAR, biotin or antifluorescent isothiocyanate (FITC) scFv is used as a targeting region, which is fused to a transmembrane domain with one or two endo-domains. T-cells expressing uCAR may effectively recognize cancer cells by binding FITC-labeled or biotinylated antigen-specific monoclonal antibodies. This activates T-cells and stimulates their proliferation and cytokine production. Human clinical trials involving uCAR T-cells are ongoing [3, 8, 12, 14].

5. NK CAR-T cells. Natural killer cell (NK) is a type of cytotoxic T-cells necessary for innate immunity and plays an important role in host immunity against cancer. NK cells are the only immune cells that trigger a rapid, MHC-independent immune response once they detect infected/malignant cells. They are known as "natural killers" because they do not require activation to destroy cells lacking MHC Class I molecules. NK cells produce various cytokines that act as immunosuppressants, including tumor necrosis factor α, interferon γ and IL-10 [4, 7, 9]. The activation of NK cells leads to the gradual formation of cytolytic effector cells such as dendritic cells, macrophages and neutrophils, and promotes antigen-specific responses of T- and B-cells. NK-mediated tumor cell lysis involves a variety of receptors, including NKp44, NKp46, NKG2D, NKp30 and DNAM. Tumor cells usually express NKG2D in addition to ULBP and MICA. NK CAR cells target NKGD2 and CD16 antigens. Upon detection of tumor antigens on cancer cells, they secrete perforin and granzyme, which directly cause tumor destruction [10, 14, 16, 17].

Clinical efficacy of CAR-T cells has also been demonstrated in acute lymphoblastic leukemia. This, however, was not the case for acute myeloid leukemia, where new solutions are needed [6]. Hypothetically, NK-CAR cells are less toxic than CART cells, especially when it comes to side effects such as cytokine release syndrome. Moreover, in contrast to T-cells, donor NK cells do not target non-hematopoietic cells, which suggests that NK-cell-mediated antitumor activity may be activated in the absence of GvHD [11, 14].

According to the EMA Guideline on the follow-up on patients administered with gene therapy products, post-registration pharmacological surveillance must include a 15-years follow-up after CAR-T cell infusion to ensure continuous evaluation of the effectiveness and safety of the licensed CAR-T therapy through the EBMT registration bureau. A similar procedure is followed by CIBMTR (USA) [1, 4, 9]. Another initiative – the PASS (Post-Registration Study) – provides an assessment of the CAR-T cell with respect to the standards of care [3].

Pre-requisites for CAR-T cell therapy in Uzbekistan

Since 2008-2010 sufficient experience of high-dose chemotherapy for leukemias has been accumulated at the Research Institute of Hematology and Blood Transfusion (RIHBT, Tashkent, Republic of Uzbekistan), resulting in increased frequency of clinical remissions and number of patients with 5-year relapse-free survival (from 32% to 64% in AML; and from 45% to 78% in ALL). Once high-dose chemotherapy was introduced in routine clinical practice, rapid technology development has been seen in other related areas at the national level, i.e., blood transfusion support, DNA-based diagnostics, proteomic studies, conventional cytogenetics, immunophenotyping. The latter are required mainly for successful performance and follow-up in hematopoietic stem cell transplantation. In 2014, for the first time in Uzbekistan, a Bone Marrow Transplantation Department was opened at the RIHBT, offering autologous bone marrow transplantation (BMT) for patients with myeloma and lymphomas. After conversion of RIHBT to the Republican Specialized Research and Practical Medical Center of Hematology in early 2023, allo-HSCT was launched together with the National Medical Research Center of Hematology (Moscow). Implementation of allo-HSCTT into our clinical practice has expanded the list of locally available laboratory tests for hematological patients, which now includes FISH-based cytogenetic analysis, HLA typing, MRD molecular monitoring. Moreover, the Cell Technology Laboratory is planned for opening. Appropriate documentation has already been submitted for international accreditation under the Joint Commission International Accreditation Standards for Clinical Laboratories. That makes the Centre fully equipped to perform diagnostics, treatment and monitoring of all types of hematological diseases. Doctors and nurses have completed internship and training in the Russian Federation, Turkey and Germany and have full support by the Ministry of Health of the Republic of Uzbekistan.

Future perspectives

The implementation of CAR therapy for leukemias in Uzbekistan will open up great horizons in the treatment of distinct lymphoproliferative disorders for which this technology has proven to be effective. The country has secured sufficient capacities for this: we have highly qualified specialists in the field of hematology and oncology, capable of carrying out this therapy; there are also opportunities to conduct the necessary genetic research and counselling. Our resident doctors and researchers closely follow the emerging CAR-T cell applications in order to be ready to implement the appropriate techniques at the earliest convenience, since CAR therapy today is a perspective approach to the treatment of oncological diseases.

In Uzbekistan there are plans to introduce CAR-T for multiple myeloma – this disease has already been successfully treated with autologous hematopoietic stem cell transplantation (HSCT) (with more than 100 transplants performed overall since 2014 and more than 10 HSCTs in lymphoma patients). Given that CD19-targeted CAR-T cells are widely used in the treatment of recurrent/resistant (R/R) B-cell malignancies, including B-cell acute lymphoblastic leukemia (B-ALL), diffuse large-cell B-cell lymphoma (DVCCL) and mantle cell lymphoma, these pathologies have been chosen for introduction in the program scheduled at the Republican Specialized Research and Practical Medical Center of Hematology. Since in CAR-T cell therapy, autologous T cells are usually isolated from the patient's own peripheral blood, this technology is being developed to create "ready-made" allogeneic CAR-T cells [6, 12].

For patients with myeloma anti-BCMA/CD19 CAR-T cells have shown positive clinical results. The B-cell maturation antigen (BCMA) is detected in 80-100% of MM patients, whereas being rarely detected in healthy patients. Considering that it plays an important role in the "immortality" of pathological myeloma cells, BCMA targeted therapy in our vision may be the most effective.

CAR-T therapy aimed at BCMA receptor is based on the combination of mAb therapy and cytotoxicity of T-cells. More than 10 drugs aimed at BCMA have passed CT phase I. Most of them have shown good efficacy even in the high-risk subset of patients.

The Hematology Center of Uzbekistan is considering utilizing BCMA directed CAR-T cells of both "industrial" and "academic" production. At the same time, the utilization of "academic" CAR-T cells in clinical trials is far more acceptable. There are several reasons why. Firstly, in Uzbekistan, as well as in the neighboring countries [18], there is no regulatory basis for the utilization of "industrial" biotechnology products. In-country production of CAR-T cells requires setting up the legal and production base from scratch. Secondly, to set up the production of CAR-T cells trained biotechnologists and genetic engineers are needed, which is time and labor intensive.

From this point of view, the utilization of "academic" products seems to be less complicated. Here Uzbekistan is planning to cooperate with one of the "academic" manufacturers of CAR-T cells and, after adopting the necessary legal framework, to set up the logistics for the production of cell products from the patient’s own cells. This could be done at the Republican Center of Hematology, which has a transplantation department with the necessary conditions to perform the isolation of patient cells and then utilize the cell product for the treatment of MM.

It should be kept in mind that CAR-T cell therapy is associated with the risk of life-threatening immunological toxicity and alarmingly high frequency of neurotoxic complications, which requires careful training of personnel involved in this procedure, including ITU specialists and neurologists [2, 7].

The Hematology Center is committed to prepare and redesign the transplantation service to create all the necessary conditions to implement in Uzbekistan such breakthrough therapy as CAR-T.

Conclusions

The onset of chimeric antigen receptor T-cell therapy has revolutionized cancer treatment and has promising perspectives in the view of reducing the toxic burden of chemo- and radiotherapy for malignant disorders. However, the high cost and overall complexity of this procedure imposes certain limitations on its development and routine implementation. Successful implementation of CAR therapy in Uzbekistan requires comprehensively addressing a range of problems. First and foremost, there are strong financial implications – CAR-T therapy is expensive. The introduction of this technology will require making big investments in medical infrastructure and procuring the necessary equipment. Second, regulatory and quality control algorithms for all stages of the procedure must be developed. Next is the development and implementation of the appropriate clinical practice protocols and standards, as well as regular professional training for medical staff and setting up a system for routine monitoring of treatment outcomes.

But in spite of all these problems, CAR-T cell therapy has great potential and Uzbekistan stands committed to implement it, at least in the form of clinical research protocols, to improve the outcomes of leukemia treatment and patients’ quality of life. That said, further research and improvement in this area will be continued, as well building the investment case and obtaining technical support from international organizations and foundations.

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