CREATION OF A TECHNOLOGY FOR OBTAINING AUTOLOGICAL GENETICALLY MODIFIED T-LYMPHOCYTES EXPRESSING CHIMERIC ANTIGEN RECEPTORS (CAR-T) AND THE APPLICATION OF CAR-T-LYMPHOCYTES FOR ADAPTIVE IMMUNOTHERAPY OF PATIENTS WITH LEUKEMIA

within the framework of the implementation of the scientific and technical program “National program for the introduction of personalized and preventive medicine in the Republic of Kazakhstan”

Relevance

The frequency of new cases of leukemia in the Republic of Kazakhstan is 4.4 cases per 100 thousand people per year. In the Republic of Kazakhstan, the treatment of patients with hematological tumors relies on chemotherapy. In some cases, myeloablative or lymphoablative therapy followed by bone marrow transplantation is used as the last frontier in saving the patient’s life, but the number of such patients is small. None of the listed therapeutic approaches is a way to completely cure patients with acute leukemia, but leads to a temporary remission.

The ongoing worldwide search for new cancer treatments has led to the creation of innovative therapies using targeted tumor delivery or immune-mediated responses (BNCT, CAR-T). Experimental therapeutic approaches include the use of control pathway inhibitors, cancer vaccines, antibodies conjugated to effector molecules, and adaptive cell therapy. Immunotherapy is one of the methods that are now increasingly used in cancer therapy. Modern cancer immunotherapy uses the possibilities of cell engineering to direct the patient’s own cytotoxic (CD8+) T-lymphocytes (CTL) to tumor cells, as well as to significantly increase the number of tumor-specific CTL in the patient’s body. An important advance has been the ability to alter the immunological specificity of CTLs in a heterogeneous population of donor lymphocytes such that the altered CTLs are able to specifically recognize a selected known marker antigen on the surface of tumor cells and kill cells expressing the marker antigen. The latest achievement was made possible by the advances in genetic engineering. The synthesis of two technologies – cell engineering and genetic engineering – has led to the emergence of an innovative method, called “chimeric T-lymphocyte antigen receptor technology” or CAR-T, which shows high therapeutic efficacy, measured by hematological parameters and by increasing life expectancy. In a number of cases, CAR-T therapy has led to many years of remission in patients with B-cell neoplasia, especially acute lymphoblastic leukemia. Marked improvement is observed in 70-90% of patients after CAR-T therapy. The US Federal Drug Administration (FDA) has approved the widespread clinical use of CAR-T for the treatment of acute lymphoblastic leukemia.

Adaptive cellular therapy for CAR-T is now perceived as a revolutionary treatment because some patients with refractory (not responding to conventional treatments) cancer experience persistent remissions after CAR-T. CAR-T therapy is an example of an increasingly popular area of ​​personalized medicine.

An important problem with CAR-T therapy is the huge price that this technology currently has on the market. In the US, CAR-T is currently marketed by two providers: Gilead (the therapy is sold under the Yescarta brand), and Novartis (the therapy is called Kymriah). Both therapeutic options are the most expensive known cancer treatments, respectively: $373,000 per course and $475,000 per patient. At such prices, the vast majority of patients with leukemia in the Republic of Kazakhstan will not be able to receive CAR-T therapy unless their own technological base for CAR-T technology is developed. So far, no work is underway in Kazakhstan to develop or implement foreign experience in CAR-T therapy and no alternative methods of adaptive cell therapy are used. Taking into account the fact that living (cellular) targeting systems in the world are among the priorities for the development of oncohematology of the future, it is necessary to develop adaptive cell therapy in the Republic of Kazakhstan. A particularly significant goal should be to reduce the price of CAR-T therapy, with the maximum localization of key stages, so that CAR-T therapy becomes financially affordable for patients in the Republic of Kazakhstan.

As a result of the third task, the country’s first protocol for CAR-T therapy at the stage of phase I clinical trials will be created. The first planned result of the project is the technology for the production of cytotoxic T-lymphocytes directed against tumor cells in acute lymphoblastic leukemia, which is a necessary step in the development of a clinical protocol for the introduction of adaptive cell therapy in domestic oncohematology.

The purpose of this study

By creating a technology for obtaining autologous genetically modified T-lymphocytes expressing chimeric antigenic rec

eptors (CAR-T), application of CAR-T-lymphocytes for adaptive immunotherapy of patients with leukemia.

Research objectives

Isolation of CD8+ lymphocytes by immunomagnetic separation, activation of T-lymphocytes.
Carrying out the transduction of T-lymphocytes with a CAR-T vector.
Measurement of cytotoxic activity of CAR-T-lymphocytes in vitro. Cultivation of lymphocytes in the station CliniMACS Prodigy (Miltenyi Biotec).
Evaluation of the antitumor activity of CAR-T-lymphocytes in animal experiments.

Research methods:

Isolation of T-lymphocytes from whole blood. Whole blood will be collected in heparin solution tubes. The erythrocytes will be destroyed by lysis in hypotonic buffer. The leukocytes will be pelleted by centrifugation and fractionated by centrifugation in a ficoll gradient, the monocyte fraction is taken. Isolation of CD8+ lymphocytes by immunomagnetic separation: cells will be mixed with biotinylated anti-CD8 antibodies, excess antibodies will be removed and the cells will be adsorbed onto magnetic microparticles coated with streptavidin. Further isolation will be carried out on a MACS LS column (Miltenyi Biotec). The viability of the primary culture of CD8+ lymphocytes will be assessed using the MTT test.
Activation of T-lymphocytes. Microparticles coated with antibodies against CD3 and CD will be added to the primary culture of T-lymphocytes. IL2 (40 U/ml) will be added to the incubation medium. Cells will be incubated for 72 h to activate proliferation.
Lentiviral transduction of T-lymphocytes. To package the transducing vector, a mixture of plasmids: a vector with CD19 CAR, an apoptosis induction gene, and other desired genes will be co-transfected in a mixture with packaging plasmids pVSV-G and pGAG-POL into a HEK293T culture. Transfection in preliminary experiments will be performed using cationic liposomes. Transfection to obtain preparative amounts of packaged vector (>5×10^7 FFU) will be performed in T150 culture flasks using polyethyleneimine. The collection of the virus will be carried out 1-3 days after transfection. Titration will be performed by quantitative PCR as well as packaging of a transducing vector expressing fluorescent protein (GFP), followed by infection of a naive culture with the packaged vector and counting of GFP-positive cells.
Preparation of culture of CAR-T-lymphocytes. T-lymphocytes activated for proliferation will be transduced with a vector packaged in lentivirus virions pseudotyped with the VSV-G protein (the latter provides a wide tropism and the ability to infect lymphocytes). A multiplicity of infection (MOI) of 5 will be used. Production of preparative quantities of CAR-T lymphocytes (>10^7 cells) will be achieved by infecting the primary lymphocyte culture in T flasks. Polybrene will be added to the incubation medium to increase transduction efficiency. Cells will be incubated in the presence of cytokines (IL2, IL6) to maintain proliferative activity. 24 h after transduction, puromycin (1–10 μg/mL) will be added to the culture [18]. The culture will be grown in puromycin media to eliminate non-transduced (non-CAR-T) lymphocytes prior to in vitro and in vivo experiments.
Measurement of cytotoxic activity of CD19 CAR-T-lymphocytes in vitro. In experiments with mouse CAR-T lymphocytes, mouse B-lymphoma A20 will be used as a test culture. In experiments with donor human CAR-T-lymphocytes, B-lymphoblasts of human origin: K562 and Raji will be used as a test culture. Test cultures of B-lymphoblasts and CAR-T-lymphocytes will be co-plated in culture plates in a ratio of (CAR-T/B) 1:1. As a control for non-specific reactions, unmodified T cells will be used instead of CAR-T lymphocytes. The co-cultures will be incubated for 24 hours, after which the incubation medium will be collected for the measurement of IFNγ and IL12 using commercial ELISA kits. Culture density (a direct indicator of cytotoxic activity) will be measured in the MTT assay.
Cultivation and propagation of the resulting therapeutic cells for use in clinical trials. The resulting cell populations will be cultured in the CliniMACS prodigy automated station (Miltenyi biotec). Consumable reagents and components necessary for growing cells will comply with GMP requirements. Sterility will be observed and production standards will be developed for the obtained CD19 CAR-T lymphocytes in the laboratory.
Evaluation of the antitumor activity of CD19 CAR-T lymphocytes in animal experiments. BALB/c mice will be inoculated with B-lymphoma. Mice (6-8 weeks) will be administered cyclophosphamide (100 mg/kg). The next day, mice will be injected with A20 cells (5 x 105 cells/mouse). Lymphoma develops within ~17 days. Hematological parameters will be monitored by counting peripheral blood cells. Mice with confirmed lymphoma will be immediately used for CAR-T therapy: CAR-T lymphocytes (10^7 cells/kg) will be injected into the tail vein. Mouse Survival Monitoring

it will be held within 60 days. Survival is recorded compared to the untreated control group. Animals will be withdrawn from the study in accordance with a protocol approved by the local ethics committee.
Evaluation of the antitumor activity of CD19 CAR-T-lymphocytes in experiments on patients will be carried out according to the developed protocol and standard operating procedures approved by the local ethical committee. It is planned to train a separate team of doctors of the Department of Oncohematology of the NREC (Nur-Sultan) to apply and develop the tactics of treating patients with acute lymphoblastic leukemia. It is planned to perform therapy using CAR-T technology for six patients of the NCEC who meet the developed inclusion criteria.

Research executor: RSE on REM “National Center for Biotechnology” of the Ministry of Education and Science of the Republic of Kazakhstan