WO2024145700A1

WO2024145700A1 - Method of expanding virus-specific lymphocytes, composition, and use of a composition

Info

Publication number: WO2024145700A1
Application number: PCT/BR2023/050384
Authority: WO
Inventors: Fernanda AGOSTINI ROCHE; Luciana CAVALHEIRO MARTI
Original assignee: Sociedade Beneficente Israelita Brasileira Hospital Albert Einstein
Priority date: 2023-01-02
Filing date: 2023-11-10
Publication date: 2024-07-11

Abstract

The present invention relates to a method for expanding virus-specific lymphocytes. The present invention also relates to a composition comprising virus-specific lymphocytes and the use of this composition to manufacture a cell therapy.

Description

METHOD OF EXPANSION OF VIRUS-SPECIFIC LYMPHOCYTES, COMPOSITION AND USE OF A COMPOSITION C^{AMPO DA} I^NVENTION The present invention relates to a method of expanding virus-specific lymphocytes. The present invention also relates to a composition comprising virus-specific lymphocytes and the use of this composition for the manufacture of a cell therapy. BACKGROUND OF THE INVENTION It is known that after bone marrow transplantation, transplant patients may be affected by various complications, including complications arising from the conditioning regimen prior to transplantation, complications related to graft-versus-host disease (GVHD/GvHD) acute and chronic, and conditions related to the patient's own health, such as recurrence of the underlying disease. 15 Among these complications, infections and reactivation of viral infections, for example such as human cytomegalovirus (hCMV), adenovirus, Epstein-Barr virus (EBV) and polyomavirus (BK virus) are frequent causes of serious complications in patients undergoing allogeneic liver transplants. bone marrow (BMT) (Düver et al., 2020). 20 hCMV reactivation/infection can lead to serious complications for patients, such as irreversible neurological changes, pulmonary, gastrointestinal and ophthalmological complications, among others, in addition to prolonged hospitalizations, leading to patient morbidity and mortality and the consequent increase in costs in health, both in the public and private sectors. 25 hCMV or human herpes virus 5 (HHV-5) is a virus belonging to the Herpesviridae family, Betaherpesviridae subfamily, of the Cytomegalovirus genus. hCMV infection typically follows an asymptomatic or oligosymptomatic course in immunocompetent individuals. However, in immunosuppressed patients, clinical manifestations related to viral proliferation in 30 different tissues (epithelial, endothelial, parenchymal and mononuclear cells) can compromise several systems (lung, gastrointestinal tract, central nervous system, reticuloendothelial system), causing deleterious damage due to sometimes irreversible to patients (Blümel et al., 2010). Specific considerations should be given to immunosuppressed patients infected with hCMV post-BMT, as this is an infectious agent whose prevalence rates are extremely high with seropositivity of almost 100% in African, Asian and Middle Eastern populations 5 and around 40 -70% on the American and European continents (Zuhair et al., 2019). Therefore, it is expected that post-transplant reactivation rates will also be the same. A reactivation rate of 70% was estimated in post-allogeneic BMT patients, which makes the control and treatment of this infection deserve special attention (Teria et al., 2015). 10 Pneumonia, gastrointestinal tract ulcers and retinitis are the main clinical manifestations in this subgroup of patients. Multiple factors favor hCMV reactivation in the post-transplant setting. In addition to the immunosuppressive agents used and the immunosuppression caused by the conditioning regimen, there are: (i) interaction with other viruses (for example: HHV-6); 15 (ii) increased production of cytokines during bacterial and fungal infections; (iii) the occurrence of graft-versus-host disease and its treatment, carried out mainly with corticosteroid therapy; and (iv) previous treatment with anti-lymphocytic immunoglobulin (ATG). Such clinical situations are very common in these patients and provide an immunological status that allows the spread of hCMV (Asano-20 Mori et al., 2005, Frere et al., 2006). To reduce the length of stay for bone marrow transplant patients suffering from hCMV infection, several strategies have been developed over the years. It is known that healthy individuals seropositive for hCMV develop a large number of cytotoxic T lymphocytes (CTL) 25 specific for the virus in peripheral blood. T lymphocytes are cells that differentiate in the human thymus and play a central role in the immune response. T lymphocytes are generally distinct from other cells because they have T cell receptors (TCRs) and the CD3 protein on their surface. Precursor lymphoid cells originate in the bone marrow, but only differentiate into the CD4 and CD8 subtypes of T lymphocytes after having migrated to the thymus. One of the functions of CD8 T lymphocytes, also known as CTL, is to cause immune-mediated death and the active component released by CD8 T lymphocytes are perforins, granzyme A and granzyme B. CD4 cells also Known as helper T cells, they use the secretion of cytokines to influence other cells of the immune system to fulfill their role, such as inducing B cells to differentiate into plasma cells and produce antibodies or attracting CD8 T lymphocytes to the site of infection. 5 Based on this principle, it was understood that lymphocytes could be used as an adoptive immunity reference for the treatment of hCMV infection and new techniques for the development and in vitro isolation of T lymphocytes specific against hCMV emerged. It has been demonstrated that protective immunity can be transferred to immunosuppressed patients through the infusion of clones of 10 virus-specific CD4 and CD8 T lymphocytes, leading to suppression of viremia (Einsele et al., 2002; Hanley et al., 2015). The efficacy of immunoadoptive transfer of specific CD8 hCMV T cell clones applied prophylactically to immunosuppressed patients was also demonstrated. This therapy protected patients against the onset of disease complications, with clonal cells being detected in patients up to 8 weeks after infusion (Blyth et al., 2013). Given the severity of the clinical infection, the lack of prophylactic strategies that definitively reduce the risk of infection and the side effects of antivirals, from the 90s onwards the development of alternative methods for the expansion of lymphocytes for the treatment of viral infections, mainly hCMV. Some of these methods are described below. Riddell and colleagues (Riddell et al., 1992) generated cytotoxic T lymphocytes from peripheral blood cells from CMV-positive bone marrow donors by culturing these cells for 12 weeks with fibroblasts infected with 25 hCMV (5 MOIs) from the virus. live whole, IL-2 and anti-CD3 and anti-CD28 in RPMI culture medium supplemented with human AB serum. The cells were infused intravenously in 3 patients. Cells were infused 4 times in staggered doses for 4 weeks (3.3x10⁴, 1x10⁵, 3.3x10⁵ and 1x10⁶). The work of Walters et al., 1995, generated cytotoxic T lymphocytes from peripheral blood cells from CMV-positive bone marrow donors by culturing these cells with hCMV-infected fibroblasts (5 MOIs) from whole virus, IL- 2 and anti-CD3 in RPMI culture medium supplemented with human AB serum. The cells were infused into 14 patients via central catheter in 4 doses staggered weekly 3.3x10⁷, 1x10⁸, 3.3x10⁸ and 10⁹ cell./m^two 4.1x10⁵, 3.3x10⁵ and 1x10⁶/kg) in 100% compatible allogeneic patients and patients maintained CMV-specific responses for up to 8 weeks (60 days) after cell infusion. No serious adverse effects were noted in patients. Vital signs of all patients remained unchanged after all infusions and 3/14 patients developed grade I and II GvHD/GvHD after cell infusion. For GVHD/GvHD, patients were treated with prednisone 1.0 to 1.5/kg per day for 3 weeks, patients had no other reactions and additional doses of cells were well tolerated and there was no new recurrence of GVHD /GvHD. In the work of Einsele et al., 2002, CMV-specific lymphocytes were isolated from 200 mL of peripheral blood from stem cell donors. Cells were cultured and stimulated with viral peptides for 21 days of culture using RPMI culture medium supplemented with human AB serum, IL-2 and viral lysate/viral peptide. The work mentions that after 4 stimuli with specific peptides in vitro, cultured cells lose their initial alloreactivity against the receptor, even for partially compatible receptors. Cells were obtained from donors and 8 patients whose HLA compatibilities between donor and recipient were variable, 5 had complete compatibility, while 3 had partial compatibility. Of the 8 patients, 7 received a single dose of 1x10⁷ cell./m^two (equivalent to 1x10⁴/kg) intravenously and 5 patients out of 8 resolved CMV infection after an infusion of cells. One of the patients needed an extra infusion of cells and after the second infusion the infection was controlled. Two patients did not respond to cell treatment. No serious adverse effects or GvHD occurred in patients who received these cells. Peggs and colleagues (Peggs et al., 2003) isolated hCMV-specific lymphocytes from CMV-positive donors from peripheral blood and cultured them with autologous dendritic cells pulsed with CMV antigen for 14 to 21 days using X-Vivo 20 medium. with 10% human serum AB and IL-2. Cultures were restimulated on day 7 of cultivation. In this protocol, 16 patients were included who received intravenously around 1x 10⁵ cells/kg after the first episode of CMV viremia. Of the 16 treated patients, 3 developed grade I GVHD/GvHD and 14/16 treated patients did not reactivate viremia. In the work of Feuchtinger and his collaborators (Feuchtinger et al., 2010), hCMV-specific lymphocytes were isolated from leukapheresis or from 500mL of whole blood. Cells were cultured in RPMI medium with 10% human AB serum and 10 μL/mL pp65 protein (Miltenyi Biotec) for 16 hours at 37 °C. Cell enrichment occurred using the capture of IFN-γ-producing cells in the CliniMACS System (Miltenyi Biotec). The purity of the selections performed averaged around 65% of IFN-γ cells. In this study, 18 patients were treated post-haploidentical transplants or transplants with unrelated donors. The cells were infused intravenously and the average dose of cells infused was ¹⁰ 2.1x10⁴ CD3/kg (ranging between 1x10³ and 1.3x10⁵ cell./kg) of specific T lymphocytes against CMV capsid protein (pp65). The results observed were that 83% of cases of infections had a complete response with a greatly reduced viral load, including 2 cases of hCMV encephalitis. In this study, no cases of GVHD/GvHD or acute side effects were observed. 15 In the 2011 work by Peggs and colleagues (Peggs et al., 2011), cells were obtained by leukapheresis and cultured in RPMI medium with 10% human AB serum and 600 ng of pp65 peptides (CMV Peptivator; Miltenyi-Biotec ) for 6 hours at 37 °C. Cell enrichment occurred using the CliniMACS IFN-γ-producing cell capture system (Miltenyi Biotec). The cells obtained from ²⁰ selection by IFN-γ were cryopreserved in two doses (1.3x10⁴ and 3.3x10⁴ CD3 lymphocytes/kg of the recipient). The cells were infused after the negative result of the microbiological cultures and the average purity of the cells obtained in the capture system was 41.7%. In total, 18 patients received the selected cells and all received a single dose of cells, the second dose was not necessary. The average number of cells infused was about 2840 CD4/kg (280-6880 CD4/kg) and 630 CD8/kg (60-3990 CD8/kg). No infusion toxicity was detected, five patients developed grade I GVHD/GvHD and responded to topical corticosteroids. Another 2 patients developed grade II and III GVHD/GvHD and received oral and intravenous treatment. 30 Meij and colleagues (Meij et al., 2012) cultured peripheral blood cells in IMDM culture medium, supplemented with L-glutamine, antibiotics and 10% human AB serum. These cells were cultivated at a concentration of 1x10⁷ cell./mL in the presence of pp65 peptides from the hCMV capsid (1 mg/mL). After incubation for 12 h, cells were captured using the CliniMACS Cytokine Capture System (IFN-γ) (Miltenyi Biotec, Bergisch Gladbach, Germany). After capture, the cells were further cultured with culture medium and IL-2 for 14 days. Six cells generated in this system were infused into 5 transplant patients with hCMV reactivation. Patients received a dose variation of 0.9x10⁴ – 3.1x10⁵ T cell./kg administered intravenously and these 6 patients responded to treatment. Patients did not experience any serious adverse effects or GVHD/GvHD. Leen and colleagues (Leen et al., 2013) used an adenoviral vector carrying the hCMV pp65 protein. Dendritic cells/monocytes were transfected with this vector, placed in culture with lymphocytes from CMV, EBV and Adenovirus positive donors and then these cells were frozen. The intensity of the CMV response of the expanded cells was measured by ELISPOT assay for all specific viruses. In this protocol, 50 patients were treated for CMV, EBV and adenovirus, 23 for CMV. Of the 23 patients, 17 were male and HLA compatibility was partial. 6 alleles were also analyzed and the number of cells infused was approximately 1x10⁴ cell/kg). Of the 23 patients included, 5 died from causes unrelated to cell infusion and 18 patients who remained alive responded to treatment. The response against CMV was evaluated in vitro by the response of the patients' peripheral blood cells. The infusion was well tolerated, with no reactions at the time of infusion and grade I GvHD occurred in 2/50 partially compatible and 3rd party patients (neither the patient nor the marrow donor). Pei and colleagues (Pei et al., 2017) used cells from 25 CMV-positive blood donors. Cells were cultured with 1,000 IU/mL IFN-γ, followed by the addition of 500 IU/mL IL-2 and anti-CD3 (100 ng/mL). After 4 days of culture, the pool of 138 synthetic peptides from the hCMV viral capsid (pp65) was added to the culture. Two days later, a second stimulus with peptides was performed. Cells were expanded in vitro for another 6-10 days. The expanded cells were selected by the automatic cell capture system (CSS) (Miltenyi Biotec). The dose of cells infused intravenously was on average 4x10⁵/kg for CD8 and 3x10³/kg for CD4. These cells were infused into 32 patients refractory to antiviral treatment and with hCMV reactivation after Bone marrow transplant. The cells were mostly from transplant cell donors, so compatibility was total or partial. Of the patients who received the cells, only 1 patient developed grade II GVHD/GvHD 2 weeks after cell infusion, however this patient already had grade 5 I GVHD/GvHD before infusion. Of the 32 patients treated, 3 patients died after infusion of hCMV-specific cells from causes unrelated to the infused cells, such as disease relapse, graft failure, and bacterial infection. Of the 32 patients treated, 27 had a complete response. In Whiters et al., 2017, cells from CMV, adenovirus (ADV) and Epstein-Barr (EBV) positive blood donors were used. Cells from these blood donors were used to create a bank of 46 monovalent cells, 17 hCMV, 14 EBV and 15 ADV. Dendritic cells derived from donor monocytes were generated and pulsed with MACS GMP PepTivators (Miltenyi Biotec) pool of hCMV pp65, AdV5 Hexon, or EBV BZLF1/LMP2A/EBNA-1. The cells were cultured for up to 21 days and restimulated with the specific peptide on day 7 of culture and the cultures were also supplemented with IL-2. After cultivation, the cells were frozen. Participants were treated with partially compatible cells at a dose of approximately 1x10⁴/Kg intravenously and compatibility was partial up to 1 in 6 alleles for HLA A, B and C. Of the 30 patients 20 included in the study, the vast majority (28) were treated for hCMV reactivation and only 1 for ADV and another for EBV. Patients were infused with different doses of the cells, with 17 patients receiving a single dose, 9 patients receiving 2 doses, 2 patients receiving 3 doses and 2 patients receiving 4 doses. No patient presented immediate toxicity to cell infusion. One patient 25 was admitted 24 h after the infusion with a fever, but it was determined that this was due to bacterial contamination of the central catheter. Patients were followed for 12 months and no adverse effects were attributed to the lymphocyte infusion. The biggest cause of problems unrelated to cell infusion were related to infection or disease relapse. Acute GvHD occurred in 2 patients, 1 patient developed grade II GvHD but was treated and responded to treatment, and the other patient who received the ADV-specific cells developed GvHD and died from the adenovirus infection. Of the 27 patients treated, 26 responded to treatment with a total or partial response. Kallay and colleagues (Kallay et al., 2018) collected donor lymphocytes by leukapheresis and stimulated them. The cells were subjected to separation using the CliniMACS PRODIGY Cytokine Capture System (IFN-γ; CCS) equipment. Lymphocytes were obtained by capturing cells secreting IFN-γ upon specific stimulation with pp65 for hCMV. In this protocol, 9 pediatric patients were treated, 4 females and 5 males. However, only 3 patients were treated for CMV, the other patients were treated for EBV and adenovirus. The number of cells infused was 9.9x10³/kg (ranging between 6.7 and 25) of CD4 and 32.6x10³/kg (ranging from 16-125) of CD8. In this protocol, no cases of rejection, GVHD/GvHD or relapse of infection were observed. Document US 2005/0221481 relates to a method of ex vivo expansion of neonatal T cells from umbilical cord blood which comprises obtaining mononuclear cells from a sample of umbilical cord blood and then culturing said cells mononuclear cells in a serum-deprived culture medium supplemented with various combinations of cytokines including stem cell factor (SCF), interleukin-7, interleukin-4, and interleukin-2. These results indicated that it is possible, through modulation of cytokines added to cultures, to expand specific subtypes of T lymphocytes for adoptive immunotherapy without losing the myeloid progenitor cells necessary for the recovery of 20 neutrophils after umbilical cord blood transplantation. Document EP 2718426 relates to a method for generating T lymphocyte preparations that are specific for at least one target antigen, comprising the steps of expanding lymphoid cells in vitro in the presence of a target antigen or peptide fragments thereof in a expansion step, 25 isolating cells that secrete IFN-γ, IL-2 or TNF-α or express the activation markers CD137 or CD40L. After selection by cultivating these cells in the presence of IL-2 or IL-7, other examples being IL-15 or IL-21, with preference for the combination of IL-2 and IL-7 and an mTOR complex 1 inhibitor ( Rapamycin). Document WO 2009/053109 deals with a method for generating 30 preparations of antigen-specific T lymphocytes for adoptive therapy, comprising the steps of obtaining lymphoid cells from a patient's bone marrow in a first stage of stimulation, where the cells are incubated with the antigens of interest and followed by the selection stage of stimulated cells that secrete IFN-γ, IL-2 or TNF-α or that are positive for a tetramer related to the target antigen. The selected cells are subjected to a second stage of lymphoid cell expansion in ex vivo cell culture medium in the presence of IL-2 and/or IL-7 and one or more antigens. 5 Despite the growing interest in the development of methodologies for the expansion of cytotoxic T lymphocytes that can be used as an adoptive immunity reference for the treatment of viral infections in immunosuppressed patients, the methods available to date present undesirable characteristics such as the way of obtaining , cost and low 10 availability of starting materials; the use of whole live viruses or viral vectors, compromising the safety of the method; the need to co-culture lymphocytes with other cells, such as dendritic cells and antigen-presenting cells (APCs); the non-use of IL-7 and/or the absence of restimulation in the expansion stage or even the absence of the expansion stage, which reduces the efficiency of the method in relation to the number of cells produced and in relation to the cell purity obtained ; the lack of selection that compromises the safety and specificity of the lymphocytes obtained; the use of low-resolution HLA-1 typing which can compromise the efficiency of the treatment given the rejection of the lymphocyte by the patient. In this scenario, there remains a need to develop an efficient, virus-specific and safe lymphocyte expansion methodology, as well as an efficient and safe product to treat bone marrow transplant patients affected by hCMV infection, aiming to reduce the length of hospital stay. of patients, reducing their morbidity and mortality, in addition to reducing healthcare costs. 25 Based on the above, the objective of the present invention is to provide a more efficient, virus-specific and safe hCMV-specific T lymphocyte expansion methodology to treat bone marrow transplant patients who do not respond to conventional therapies such as antivirals, using cells that are capable of specifically responding to the patient's hCMV infection that is not capable of combating the infection and reducing the patient's hospitalization time. Thus, the inventors surprisingly managed to overcome the problem of the state of the art and developed a methodology for expanding hCMV-specific T lymphocytes from lymphomononuclear cells obtained by apheresis of healthy platelet donors who are immune to hCMV and who were isolated from the buffy-coat (cell concentrate) of the collection kit, through successive stimulation with a pool of peptides derived from the phosphoprotein 65 (pp65) of the hCMV capsid in the presence of the cytokines IL-2 and IL-7, and selected according to their ability to secrete interferon-γ (IFN-γ) in response to the stimulation of the pp65 peptides for which they were primed, that is, exposed in culture. The lymphocytes obtained by the method of the present invention can be expanded for a prolonged period of time and cryopreserved, and can be used in the treatment of different bone marrow transplant patients who do not respond to conventional therapies, as long as they are HLA class I related. The inventors further believe that the present invention will be the basis for the development of therapies against other viruses and, in the future, will be the basis for alternative therapies for the treatment of viral reactivations in the context of bone marrow transplantation and immunosuppression. 15 BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a method of expanding virus-specific lymphocytes. The present invention also relates to a composition comprising virus-specific lymphocytes and the use of this composition for the manufacture of a cell therapy. BRIEF DESCRIPTION OF THE FIGURES Figure 1 identifies the leukocyte subpopulations (%) present in the starting material after processing (n=5). Figure 2 represents the flow cytometric analysis of the leukocyte populations present among the cells isolated from the spinning top: (A) shows the selection of CD45 cells (leukocytes) versus granularity; (B) shows the selection of T lymphocytes (CD3+), B lymphocytes and Natural Killer (NK) cells (CD3-); (C) shows the separation of T lymphocytes into CD8 and CD4 subtypes (T lymphocyte subpopulations); (D) shows the selection of B lymphocytes (CD19+) within CD3- cells; and (E) shows the selection of 30 NK cells (CD16+CD56+) within the CD3- cells. Figure 3 demonstrates the viability of isolated cells after separation by Ficoll-Paque^TM (n=5). Figure 4A-C shows the validation of the initial cell number and dose of cytokines for cell expansion. (A) CD4 T lymphocyte proliferation assay with different concentrations of cytokines. (B) CD8 T lymphocyte proliferation assay with different concentrations of cytokines. (C) Lymphocyte proliferation assay with different cell concentrations (IL-2500 UL/mL + IL-710 ng/mL). 5 Figure 5A-D graphically shows the pattern of leukocyte expansion and metabolite detection in the Xuri and G-REX bioreactors over 15 days. (A) Expansion of leukocytes. (B) pH assessment. (C) Glucose assessment. (D) lactate assessment. Figure 6A-D graphically shows the pattern of 10 lymphocyte subpopulations in the Xuri and G-REX bioreactors over 15 days. (A) CD3+ lymphocytes. (B) CD3/CD8+ lymphocytes (CTLs). (C) CD3+CD45RO+CD45RA- Memory Lymphocytes. (D) CD3+CD45RA+CD45RO- naïve lymphocytes. Figure 7A-B shows the comparison between the selected cells (TCB, target cell bag) in the column systems and PRODIGY system. (A) Total number of cells obtained in both systems. (B) Percentage of CD3 T lymphocytes obtained in both systems. Figure 8 shows analyzed purity data of TCBs from manual column and CliniMACS PRODIGY system. Figure 9A-B shows the average number of T lymphocytes (CD3) and CD3 20 T lymphocytes positive for INF-γ in the depleted (NTCB - non target cell bag) and selected (TCB) fractions captured by the CliniMACS PRODIGY system. Figure 10A-D demonstrates the analysis of cells after the IFN-γ capture assay. NTCB - Analysis of the cellular fraction discarded in the capture assay. (A) Selection of cells by SSC vs. FSC. (B) Selection of other cells due to the absence of CD3 labeling (21%), selection of T lymphocytes by the expression of CD3 (78.4%) and then of CD4 (48.6%) and CD8 (50) T lymphocytes ,two%). (C) Selection of T lymphocytes secreting INF-γ CD4 (0.44%) and CD8 (0.20%). (D) Selection within other cells: monocytes (30.4%), NK (36.4%) and B lymphocytes (21.8%). TCB - Analysis of the cellular fraction enriched in the capture assay. (A) Selection of cells by SSC vs. FSC. (B) 30 Selection of other cells due to the absence of CD3 marking (6.52%), selection of T lymphocytes by expression of CD3 (92.7%) and then CD4 (33.7%) and CD8 T lymphocytes (59.7%). (C) Selection of T lymphocytes secreting INF-γ CD4 (99.2%) and CD8 (33.7%). (D) Selection within other cells: monocytes (32.6%), NK (13.5%) and B lymphocyte (31.5%). Figure 11A-F demonstrates the analysis performed by the Clinical Pathology Laboratory of cells after the INF-γ capture assay. TCB - Analysis of the cellular fraction enriched in the capture assay. (A) Cell selection by FSC-H 5 vs. FSC-A. (B) Selection of other cells labeled for CD45 - Leukocytes (80.1%). (C) Selection of viable cells by exclusion of cells labeled with 7-AAD (74.4%) and then (D) selection of lymphocytes by expression of CD3 (99.5%), followed by (E) T lymphocytes CD4 (4.7%) and CD8 (92.2) and (F) Selection of T lymphocytes (CD3) secreting INF-γ (100%). 10 Figure 12 demonstrates the analysis of cell death with Propidium Iodide by product flow cytometry. (A) Distribution of cells by size (FSC) vs. granularity (SSC). (B) Variation in the percentage of viable cells (propidium iodide negative) according to the thawing time of cells from Donor 15IVN9083 that had been frozen for around 7 days. (C) 15 Variation in the percentage of viable cells (propidium iodide negative) according to the thawing time of cells from Donor 18FS4945 that had been frozen for approximately 6 months. (D) Variation in the percentage of viable cells (propidium iodide negative) according to the thawing time of cells from Donor 24MAPM6923 that had been frozen for around 11 months.20 Figure 13 shows the ELISpot result of the product: B300121394945- donor : 18FSH4945. Figure 14 shows the ELISpot result of the product: B300121391223- donor: 59TPAA1223. Figure 15 shows the ELISpot result of the product: B300121327279- 25 donor: 60GDD2727. Figure 16 shows the flow cytometry result regarding the percentage of MCR-5 fibroblasts positive for CMV infection. Figure 17 depicts the specific production of IFN-γ by T lymphocytes in co-culture for 24 and 48 h with fibroblasts infected with the AD169 strain of cytomegalovirus. Product: B300121394945/Donor: 18FSH4945. Figure 18 details the percentage of MCR-5 killing by T lymphocytes of 3 products after 48 h in culture. D^WRITING D^{DETAILED OF} I^NVENTION The present invention relates to a method of expanding virus-specific lymphocytes, which comprises the steps of: (I) pre-expansion from a sample of lymphomononuclear cells, which comprises the steps of: 5 (a) separating the cells peripheral blood mononuclear cells (PBMCs); (b) centrifugation and discarding the supernatant; and (c) resuspension of PBMCs; (II) expansion of cells in vitro, 10 (III) selection of lymphocytes positive for INF-γ. In a preferred embodiment, the method of expanding virus-specific lymphocytes additionally comprises the step of (IV) freezing at -80 ° C. In another preferred embodiment, the sample(s) of lymphomononuclear cells is(are) obtained from the buffy-coat (cell concentrate) generated in the donation of platelets by apheresis, which are retained in the center of the kit. collect. Preferably, the lymphomononuclear cells are obtained from a whole blood sample. More preferably, the platelet donor suitable for the present invention is immune to hCMV (IgM negative, IgG positive). Particularly, said donor is a regular platelet donor. 20 To carry out the separation step (a), common separation techniques can be used. In a preferred embodiment, step (a) of separating peripheral blood mononuclear cells (PBMCs) from the sample is carried out by gradient separation. Preferably, step (a) is followed by a step of counting 25 cells and checking cell viability, wherein the sample acceptance criteria are a number of at least 6x10⁸ total cells and at least 90% viability, such as 90% to 100%. Cell counting and cell viability analysis can be carried out according to various protocols and equipment available in the art, such as by means of microscopy, serial dilution, automatic counting in a blood count reader, staining and flow cytometry. From the sample obtained in (a), step (b) of centrifugation and discarding of the supernatant is carried out. In step (c), part of the sample obtained in step (b) is resuspended in culture medium and/or buffer, such as, but not limited to, phosphate-buffered saline (PBS), and the remainder of the sample is resuspended in cryoprotectant solution for freezing at -75 °C to -95 °C. In a preferred embodiment, the cryoprotectant solution comprises Voluven® 6% or a mixture of hydroxyethyl starch and 6% sodium chloride, 20% human albumin and dimethyl sulfoxide (DMSO). In an even more preferred embodiment, the cryoprotectant solution comprises 50% Voluven® 6%, or a mixture of hydroxyethyl starch and 6% sodium chloride, 40% 20% human albumin and 10% dimethyl sulfoxide (DMSO), relative to the volume of the cryoprotectant solution. 10 In a preferred embodiment, step (II) of expanding cells in vitro comprises the steps of: (d) inoculating the cells obtained in step (I) in a bioreactor comprising culture medium comprising IL-2 and IL-7 at a temperature of 37ºC+1^OC (may have greater variations when collecting samples 15 for the analyzes described here), pH between 7.0 and 7.5 and 5% CO2; (e) stimulation of cells with the hCMV virus capsid peptide; and (f) restimulating the cells with the hCMV virus capsid peptide and PBMC cells from the same sample previously frozen in step (c). 20 Preferably, step (II) of the method further comprises the step of: (g) adding culture medium in a volume suitable for maintaining cell concentration. More preferably, step (II) of the method further comprises the step(s) of: 25 (h) evaluating the number of T lymphocytes and the viability of the cells, in which expansion is considered adequate when the number of lymphocytes final is at least double the initial number of lymphocytes and viability is at least 80%; and/or (i) reducing the culture volume, where the reduction is preferably carried out by filtration, centrifugation and/or plasma extractor. Other techniques for reducing the culture volume are also known in the art and can be used alternatively and/or in conjunction with filtration, centrifugation and/or plasma extractor techniques. The bioreactor can be an open or closed system. Preferably, the bioreactor is a closed system. In a preferred embodiment, cell cultivation is carried out at a preferred temperature of 37°C+1^OC (may have greater variations when collecting samples for the analyzes described here), 5 more preferably at a temperature of 37°C. Cell culture can occur at neutral pH. In a preferred embodiment, the pH is between 7.0 and 7.5. In a preferred embodiment, the CO2 concentration of the cell culture in the bioreactor is kept stable. Preferably, the CO2 concentration is 5% (v/v). In a preferred embodiment, step (e) of stimulating cells 10 with the hCMV virus capsid peptide is carried out with the hCMV virus pp65 peptide (UniProt ID: P06725). Even more preferably, stimulation of cells with the hCMV virus capsid peptide is performed with the PepTivator CMV pp65 peptide obtained commercially from Miltenyi Biotec®. PepTivator CMV pp65 is a peptide pool consisting mainly of 15-mers with overlapping 11 amino acids (aa), covering the complete sequence of the cytomegalovirus pp65 protein (UniProt ID: P06725). Preferably, step (f) of restimulating the cells is carried out through successive stimulation with the hCMV virus capsid peptide and with PBMC cells from the same sample previously frozen in step (c), followed by the addition of culture medium in adequate volume to maintain cell concentration and (h) assessment of the number of T lymphocytes and cell viability, in which expansion is considered adequate when the final number of lymphocytes is at least twice the initial number of lymphocytes and viability is at least 80%. In a preferred embodiment, step (i) of reducing the culture volume is followed by an additional sampling step for cell counting, viability analysis and karyotype analysis; and/or carrying out a sterility test. Sterility tests can evaluate the presence of aerobic and anaerobic microorganisms, such as Mycoplasma pneumoniae. Furthermore, endotoxin tests can be performed. Several methodologies 30 are available in the art for karyotype analysis and carrying out sterility and endotoxin testing that are known to professionals in the field. Preferably, the determination of viability and cell counting uses the Countess II Automatic Cell Counter equipment (Thermo Fisher), in which the cells are stained with 0.4% trypan blue, transferred to a disposable slide and automatically counted by the equipment. Counting can also be done using the ABX Micros ES60 equipment (Horiba) or by microscopy. In another preferred embodiment, cell viability is determined by flow cytometry. In a preferred embodiment, the karyotype analysis is carried out by karyotyping 10 metaphases and analyzing the others, with or without fluorescence in situ hybridization (FISH). Preferably, if abnormalities are found, 10 more metaphases are analyzed. In a preferred embodiment, the sterility test is carried out by submitting cellular samples to microbiological tests, which can be complemented by a pyrogenicity test through the evaluation of endotoxins. Several sterility tests are known in the art and can be used in an alternative or additive way to prove the safety of the methodology, composition and product obtained by the method of the present invention. Preferably, microbiological tests are carried out in Bactec flasks (BD), which are added with media for the growth of specific microorganisms: BD Bactec plus aerobic medium (for aerobic microorganisms), BD Bactec plus 20 anaerobic medium (for anaerobic microorganisms) and BD Bactec Myco/F Lytic (for special microorganisms). Particularly, the tests are carried out on the BD Bactec FX equipment. Preferably, for Myco-F vials, the incubation time is 42 days. Alternatively or jointly, samples can also be analyzed using the compendial sterility test in fluid thioglycolate media (THIO) and casein-soy broth (TSB) cultured for 14 days. The specific conditions for each medium are known to a person skilled in the art and are part of routine laboratory activities. Preferably, samples incubated in THIO medium are cultured at 32.5±2.5°C for 14 days and samples incubated in 30 TSB medium are cultured at 22.5±2.5°C for 14 days. In a preferred embodiment, the endotoxin test is performed using the Endosafe nexgen-PTS (Charles River) equipment and the results are analyzed in the LIMS or Charles River Cortex software. The acceptance criteria in international units is < 5 IU/mL. In a preferred embodiment, testing for Mycoplasma pneumoniae contamination is performed by molecular biology. Preferably, DNA from the initial and final culture medium samples is extracted using Qiagen's DSP 5 Virus/Pathogen mini kit (catalog number 937036) and the nucleic acids can be used directly for PCR analysis. The RT-PCR process can be carried out automatically using the Universal Disc (Focus) and the samples can be processed on the 3M Integrated Cycler equipment (Focus/3M). In a preferred embodiment, step (III) comprises the steps of (j) capturing INF-γ-producing cells in a capture system, (k) optionally, enriching INF-γ-positive cells; and (l) cellular immunophenotyping by flow cytometry. The capture of INF-γ-producing cells can be carried out in an open and/or closed system. The capture can also be carried out manually or automatically. In a preferred embodiment, step (j) of capturing INF-γ-producing cells in a capture system is carried out in an open and manual system. In another preferred embodiment, step (j) of capturing INF-γ-producing cells in a capture system is carried out in a closed and automatic system. A further 20 cell capture techniques are known in the art and can be used as an alternative to the technique described here. Preferably, capture in an open and manual system is carried out after expansion of CMV-specific cells in a bioreactor, through the isolation of IFN-γ-producing cells using the detection and enrichment kit for 25 IFN-γ-secreting cells (MACS Miltenyi Biotec ) using a disposable LS column (Miltenyi Biotec). Preferably, new separation is performed on another LS column to ensure purity and enrichment of IFN-γ positive cells (step (k)). The result of positive selection is the TCB fraction, and the unselected cells are in the NTCB fraction. 30 Preferably, capture in a closed and automatic system is carried out after expansion of CMV-specific cells in a bioreactor, using the CliniMACS PRODIGY equipment (Miltenyi Biotec). The equipment sterilely and automatically performs the selection (enrichment) of cells positive for IFN-γ and the separation of fractions with discarded cells and cells enriched in quality control bags. In a preferred embodiment, step (l) of cellular immunophenotyping is done by flow cytometry for the expression of IFN-γ to verify the 5 isolated and depleted cell populations. In a preferred embodiment, cell markers are used in the panel: 7-Actinomycin D (7-AAD for cell viability), and for cell populations the antibodies: CD45, CD3, CD4, CD8, CD11b and INF-γ, in which the Antibodies are qualified through titration to verify their function and specificity. Other cellular immunophenotyping techniques are known in the art and can be used as an alternative to the technique described here. Preferably, at the end of the selection, the TCB presents a value above 80% of IFN-γ positive cells and above 80% of T lymphocytes with viability also above 80%. In a preferred embodiment, the supernatant of the NTCB fraction is sent for microbiological tests of aerobic, anaerobic, fungal and special microorganism cultures, as described above. Additionally, the supernatant can be sent for endotoxin testing and sterility testing. The supernatant of the TCB fraction can be sent for serological tests, including, but not limited to, serology, NAT, PCR and in situ hybridization, 20 and may also include testing for impurities through the quantification of IL-2 and IL-7 in the supernatant of the cells and potency testing, for example, through quantification of IFN-γ in the co-culture supernatant of hCMV-specific lymphocytes and hCMV-infected fibroblasts. Several serological tests are available in the technique and are part of the routine activities of a professional in the field, such as the NAT test, from the English “Nucleic Acid Test”. In particular, the amplification and detection of nucleic acids can be carried out by polymerase chain reaction (PCR). The technique can be used for any pathogen with a known genome, including, but not limited to: human immunodeficiency virus (HIV), human T-cell lymphotropic virus (HTLV-I), hepatitis C virus (HCV), hepatitis C virus (HCV), hepatitis B (HBV), cytomegalovirus (CMV), Epstein–Barr virus (EBV), parvovirus B19, Mycoplasma pneumoniae etc. NAT can be performed manually or automatically. Kits for performing NAT are also commercially available. for one or more pathogens of interest. Alternatively or in a complementary way, the safety of lymphocytes at the end of each step of the method of the present invention can be assessed by traditional serology that investigates the presence or absence of antibodies in the samples. In a preferred embodiment, TCB supernatant samples are analyzed by NAT serology for HIV, HTLV-I, HCV, HBV and by PCR for CMV, EBV and Parvovirus B19 and Mycoplasma pneumoniae. In a preferred embodiment, step (IV) of freezing the TCB and NTCB fractions is carried out in a cryoprotectant solution and the cells are kept in a freezer at -80 ºC. In a preferred embodiment, the method further comprises a thawing step (V). Even more preferably, the defrosting step takes place at a temperature of 20 to 40 °C, preferably 37 °C+1^OC (may have greater variations when collecting samples for the analyzes described here). In a preferred embodiment, thawed lymphocytes are selected by HLA compatibility and infused into patient 15 intravenously. The present invention also relates to a composition comprising virus-specific T lymphocytes and one or more additional components. Particularly, the composition is a pharmaceutical composition. The expression “pharmaceutical composition” designates a preparation that is in a form that allows the biological activity of an active ingredient contained therein to be effective. Preferably, the virus-specific T lymphocytes contained in the composition are obtained by the method of the present invention. Even more preferably, the additional components comprise, but are not limited to a pharmaceutically acceptable carrier, a mixture of hydroxyethyl starch and sodium chloride 6%, Voluven 6%, human albumin 20%, dimethyl sulfoxide (DSMO) and mixtures thereof. The term “pharmaceutically acceptable carrier” means an ingredient in a pharmaceutical composition, other than the active ingredient, that is non-toxic to the patient. The definition of a pharmaceutically acceptable carrier includes, but is not limited to, buffer, excipient, stabilizer or preservative and are known in the art. Professionals in the field are highly capable of determining the pharmaceutically acceptable vehicle for each composition and purpose. Additionally, the present invention relates to the use of a composition comprising virus-specific T lymphocytes and one or more additional components to manufacture a cell therapy to treat viral infections. Preferably, viral infections are selected from human cytomegalovirus (hCMV), adenovirus, Epstein-barr virus (EBV), polyomavirus (BK virus) and human herpesvirus 6 (HHV-6). 5 Preferably, the patients to be treated are bone marrow transplant patients who do not respond to conventional therapies such as antivirals. Preferably, the quality grade of the reagents is clinical grade or GMP (Good Manufacturing Practice), complying with Good Production Practices and can be produced in accordance with the ISO 13485 quality system, with the standards determined by the National Agency of Health Surveillance (ANVISA) and/or with the standards determined by the Food and Drug Administration (FDA) for medical devices. DEFINITIONS 15 Unless otherwise indicated, terms used in the present application should be understood in accordance with conventional usage by those skilled in the art. The term “cell therapy”, as used herein, is interchangeable with the term “medicine”, and can be understood as a product used to treat diseases through the alteration or restoration of cells that may or may not belong to the patient being treated. By “medicine” or “cell therapy”, the subject matter of the present invention must be understood in any pharmaceutically acceptable form, which may be a solid or liquid form; Non-limiting examples are tablets, capsules, solution, suspension, among others. The term “apheresis” is related to the separation of blood components by centrifugation, using automated equipment. The donor's blood is collected and the machine separates the components of interest. The blood returns to the donor's body through the same venous access and the collected material undergoes serological tests. During platelet donation by apheresis, there is an accumulation of lymphomononuclear cells in a compartment located in the central region of the collection kit 30, which we call the “spinning top”. In this compartment, the volume of accumulated blood varies between 8 and 10 mL, which after dilution, can result in an average volume of 30 mL and the number of cells present in the compartment is on average 6.6x10⁷ /mL, with the total number of leukocytes averaging 2x10⁹ with about 79% lymphocytes, while the rate of lymphocytes in a whole blood sample is about 30%. In addition to the high rate of lymphocytes in the sample, the starting material collected in the spinning top does not involve the cost of donation, as it is waste material from the platelet donation procedure. Furthermore, the material can be collected in a safe and sterile way. Preferably, donors are repeat donors who have a history of sequential serology. Furthermore, during the platelet donation procedure, a clinical screening questionnaire, screening assessment and serological tests on donors are carried out to ensure the health and safety of both the donor and the recipient of the collected material. 10 The term “serological test”, as now used, refers to any and all tests based on serology, that is, on the presence or absence of antibodies specific to pathogens, as well as biological materials and the pathogens themselves, in samples biological samples, particularly blood, to concretely determine their presence in the samples. The biological materials of pathogens including, but not limited to, antibodies, peptides, proteins, nucleic acids and antigens can be identified by techniques well known in the art such as immunochromatography, immunohistochemistry, polymerase chain reaction (PCR), hybridization of labeled probes, immunoenzymatic tests such as enzyme-linked immunosorbent assay (ELISA), nucleic acid test (NAT), chemiluminescence, among others. The term “whole blood” refers to a blood sample in which all components are present: plasma, red blood cells, white blood cells and platelets. White blood cells, also known as leukocytes, are the defense cells that the body produces so that the body can defend itself from foreign elements that may pose a threat and include, particularly, peripheral blood mononuclear cells. The term “peripheral blood mononuclear cell” (PBMC) as well as the term “lymphomononuclear cell” refer to any peripheral blood cell with a round nucleus, consisting of lymphocytes (T cells, B, Natural Killer (NK) cells) and monocytes, while erythrocytes and platelets lack a nucleus, and granulocytes (neutrophils, basophils, and eosinophils) have multilobed nuclei. The term “allogeneic transplant,” as used here, refers to the transplant in which the cells or organs come from another individual (donor), according to the level of compatibility of the blood material. The term “sterile” and its variants as used herein refer to environments, objects or surfaces in which microorganisms are absent or are incapable of reproducing. There are several techniques for sterilizing materials, such as autoclave, flame, oven, bactericidal or bacteriostatic chemicals, UV radiation, ultrafiltration, etc., as well as several techniques for checking suitable sterility are also known in the art. The term “about” used herein refers to a value of 5 to 10% to 10 plus or minus the values to which it refers. The term “cell viability” as used herein refers to the analysis of metabolically active cells in a cell culture and/or sample in order to evaluate their activity qualitatively and/or quantitatively. Several techniques for determining cell viability are known in the art and can be used alternatively or concomitantly. “Cytokines” are proteins that regulate the immune response, including but not limited to interleukins and interferon-γ (IFN-γ). Interleukins (IL) are produced by leukocytes in response to microorganisms and other antigens. IL-2 is the main T cell stimulating factor as a growth and activation factor for all subpopulations of T lymphocytes. IL-7 is a cytokine that stimulates the growth and maturation of B lymphocytes and activation of lymphocytes T, being secreted by stromal cells of the bone marrow and thymus. INF-γ is responsible for attracting macrophages, which help remove cell debris and promote healing and reorganization of areas with inflammation. 25 INF-γ is the main cytokine released after the induction of the adaptive immune response, being produced by effector T lymphocytes. Other lymphocyte stimulators are known in the art and can be used alternatively or concomitantly. Among them are anti-CD3 and anti-CD28 antibodies, which induce the activation of T lymphocytes because the T lymphocyte receptor is associated with complexes of CD3 and CD28 molecules. The term “naïve lymphocyte” as used here corresponds to mature T cells originating from lymphoid organs that have never recognized the antigen to which they are specific, that is, they have never been activated by cells presenting antigens (APCs). As used herein, the term “lymphocyte expansion” corresponds to the clonal expansion of lymphocytes. Clonal expansion begins with the activation of lymphocytes through the presentation of the antigen of interest through histocompatibility complex class II (MHC II) molecules from APCs to T lymphocyte receptors (TCRs). This presentation promoted by the APC is called “stimulus”. Antigen recognition allows the interaction of co-stimulatory molecule complexes and the secretion of cytokines, which in turn ensure the survival of T cells specific to the antigen and activate factors that promote the exposure of specific gene segments leading to cellular differentiation of the antigen. lymphocytes subdivided into subtypes (Th1, Th2 and Th17), which can be identified according to the cytokine they produce most (INF-γ, IL-4 and IL-17, respectively). In the present invention, the “stimuli” are preferably carried out by direct exposure of the lymphocyte to peptides from the human cytomegalovirus viral capsid that can be presented via MHC to these lymphocytes by mononuclear cells and B lymphocytes present in the culture during expansion under the influence of cytokines IL-2 and IL-7. The term “karyotype” as used here refers to the analysis of the diploid chromosome set of a species, representing the total number of 20 chromosomes in a somatic cell. Typically, a human cell has 46 chromosomes. The term “infusion” as used herein refers to the administration of intravenous fluids to patients. The term "product" as used herein refers to the product of the virus-specific lymphocyte expansion method of the present invention, comprising the virus-specific lymphocytes themselves or the composition of the present invention. SELECTION OF STARTING MATERIAL DONOR A) T^RIAGEM W^LINE 30 In the present invention, clinical screening of donors is carried out in compliance with Collegiate Board Resolution - RDC no.214, of February 7, 2018. Briefly, donors must be healthy people and meet all criteria for blood donation: overweight of 55 kg, hemoglobin (Hb) and hematocrit (Ht) (with the minimum for women being Hb=12.5g/dl and Ht=38% and for men the minimum being Hb=13.0g/dl and Ht=39%), blood pressure within limits (blood pressure systolic blood pressure between 120-180 mmHg and diastolic blood pressure between 70-100 mmHg), pulse rate between 50 and 100 bpm, answer the 5 standard blood donation questionnaire that may include one or more health history, including ongoing pregnancy; vaccination history; travel history and exposure to infectious agents, history of transfusion of blood products and use of blood products; history of tissue, cell or organ transplantation and xenotransplantation, etc. Hematocrit (Ht) is the ratio between the erythrocyte volume in relation to the total volume of blood, expressed as a percentage where the reference value for blood donation for an adult man is 39 to 54%, and for an adult woman , from 38 to 48%. Hemoglobin (Hb) can be measured in a blood sample to indicate the blood's ability to carry oxygen to tissues. The Hb reference values are: men: 13 to 18 g/dL and women: 12.5 to 16 g/dL. Reference values may vary according to the parameters and techniques used. Donors eligible for donation and who are donating the platelet collection kit for the CMV study must sign an Informed Consent Form (TCLE). B) SEROLOGICAL TESTS After donating platelets, the donor's blood is evaluated for the presence of markers for infectious diseases transmitted by blood, according to criteria determined in Collegiate Board Resolution - RDC no. 214, of February 7, 2018 and other legislation current conditions: Hepatitis B and C, HIV 1 and 2, HLTV I and II, Syphilis and Chagas and NAT for Hepatitis B and C and HIV 1 and 2. As an inclusion criterion, only donors who present negative serology 25 for such diseases are eligible for cell expansion. The presence of IgM and IgG immunoglobulins for CMV must also be assessed in the blood of these donors and high-resolution HLA and ABO/Rh typing must be performed. Additionally, donors must respond to the questionnaire and clinical assessment to analyze the risk of transmissibility of spongiform encephalopathies (TSE). C) HIGH-RESOLUTION HLA TYPING The term HLA (from the English “Human Leukocyte Antigens”) represents a complex of genes that code for immune system proteins unique to each person, which may comprise distinct HLA I and II loci. The main HLA loci are A, B, C (class I), DR, DP and DQ (class II) among others, and the resolution of typing by molecular biology methods, including sequencing, is related to the depth of the analysis. Low/medium resolution HLA Typing 5 identifies the allelic group to which the gene(s) belong and the result is based on DNA at the level of the digits that make up the first field in the nomenclature. High-resolution typing identifies the specific HLA protein at the level of the digits that make up the second and third fields in the DNA-based nomenclature. HLA typing can be done using blood samples through serology, biochemistry or PCR/DNA sequencing. The high-resolution exam is performed by PCR or DNA sequencing. D) ABO/R TYPING The ABO system is a classification of human blood into four types: A, B, AB and O, while the Rh factor is a classification of human blood according to the presence or absence of a group of D antigens found in red blood cell membranes. ABO/Rh typing is widely known in the art and can be performed using gel agglutination to verify the presence/absence of antigens and antibodies from the ABO system and D antigen from the Rh system. E) Y^{OROLOGY FOR} CMV 20 The presence or absence of immunity to hCMV can be determined by serology or other techniques known in the literature. In the present invention, serology for CMV of platelet donors is preferably carried out by evaluating antibodies of the IgM and IgG isotypes against the CMV virus. Ideally, donors should present results for non-reactive IgM (negative) and reactive IgG 25 (positive). Serology for CMV can be performed from serum, plasma or whole blood samples and through various quantitative and/or qualitative methodologies known in the art, such as immunochromatography, enzyme-linked immunosorbent assay (ELISA) or chemiluminescence. ³⁰ FAN^{ANALYSIS OF} T^{RANSMISSIBILITY OF} AND^{NCEPHALOPATHIES} AND^SPONGIFORMES (EET) The platelet donor samples used in the present invention come from blood donors evaluated through clinical screening who are not at risk for EET in accordance with Collegiate Board Resolutions - RDC no.214 of February 7, 2018, RDC No. 305 of November 14, 2002 and Ordinance 158, February 4, 2016. And^EXAMPLES The following Examples are not intended to limit the scope of the claims of the invention, but are especially intended to be exemplary of certain embodiments. Any variations of the exemplified methods that occur to one skilled in the art are intended to be included within the scope of the present invention. EXAMPLE 1 – PRE-EXPANSION STAGE (I) 1.1^{RIGEM AND} W^{ARACTERIZATION} D^O M^MATERIAL D^AND P^ARTIDA T lymphocytes were obtained from lymphomononuclear cells from the buffy-coat generated in the donation of platelets by apheresis by regular blood donors, submitted to the clinical screening questionnaire, screening assessment and signing of informed consent form, and the samples were submitted to serological tests, high-resolution HLA typing, ABO/Rh typing, CMV serology and EET analysis, as described above. The blood sample, also called starting material, is retained in the center of the collection kit in a central compartment which we call “spinning top”, this compartment accumulates a blood volume of 8 to 10 mL. After platelet donation, the collection kit was sterile sealed and the spinning top was removed without opening the platelet collection system and transported in a waterproof bag inside the rigid thermal suitcase suitable for transporting biological material at room temperature ( 20 to 24 ºC). Platelet donations took place in the Hemotherapy Sector of Hospital Israelita Albert Einstein. 1.2 ANALYSIS OF STARTING MATERIAL After transport, the volume of cells present in the spinning top was removed aseptically with the aid of a syringe and 21G needle in laminar flow. After aspiration using a syringe and needle, the entire volume of cells contained in the spinning top was transferred to a 50 mL conical tube and an aliquot was removed to perform cell counting in the ABX Micros ES60 automatic cell counter. The number of cells present in the compartment was on average 6.6x10⁷/mL and the total number of leukocytes was on average 2x10⁹ cell. with about 79% lymphocytes, as shown in Table 1.

“PION” COMPARTMENT (N=5).

The volume of blood was diluted 15 times with phosphate buffer saline (PBS; commercially available from ThermoFisher Scientific Gibco)) in the proportion of 1 volume of blood: 15 volumes of diluent, reaching an average volume of 30 mL. 1.3 PROCESSING OF STARTING MATERIAL - SEPARATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS) To eliminate erythrocytes and the majority of granulocytes present in the samples, PBMCs were separated by density gradient with Ficoll-Paque^TM Premium (manufactured by the company Cytiva) in which erythrocytes, granulocytes and dead cells pass through the Ficoll layer, while lymphocytes and monocytes, due to their size/density, accumulate at the interface between plasma and Ficoll-Paque^TM. The diluted cells (30 ml) were placed on a 15 ml layer of Ficoll-Paque^TM Premium in 50 mL conical tubes and centrifuged in a Thermo Scientific Sorvall ST 16R Centrifuge for 40 minutes, 500 G, temperature 20 to 24 ºC, without brake. Approximately four 50 mL conical tubes were needed to separate all the cells. After centrifugation, the mononucleated cells present at the interface between Ficoll-Paque^TM Premium and diluted donor serum were removed using a sterile pipette and transferred to a new 50 mL conical tube. A new aliquot was removed for cell counting on the ABX Micros ES60 equipment and for viability analysis on the Countess equipment. From the material isolated from the spinning top, an average of 1.00x10⁸ leukocytes/mL. For a volume of 10 mL, the total number of leukocytes varied between 0.8x10⁹ and 2.5x10⁹ (n=5) (Table 2). The results of automatic cell counts in a blood count reader showed high concentration of lymphocytes (80% ±13%) and monocytes (10% ±3.0%) and low frequency of granulocytes (13% ± 4%) with efficient elimination of erythrocytes demonstrated by the average hematocrit of 0.78% (Table 2 ). The frequencies are shown in Figure 1. 5 TABLE 2 - GENERAL CHARACTERISTICS OF 5 SAMPLES PROCESSED BY G^RADIENT D^AND D^ENSITY (N=5)

The population of total lymphocytes presented in blood counts performed on automatic cell counters comprises T lymphocytes (helper and cytotoxic), B lymphocytes and Natural Killer (NK) cells. These cells cannot be differentiated 10 in automatic counters because they have similar characteristics of size and granularity. Therefore, to determine the percentage of distinct cells within the total lymphocytes, immunophenotyping was performed by flow cytometry of the populations of leukocytes (CD45+), T lymphocytes (CD3+), helper T lymphocytes (CD3+CD4+), cytotoxic (CD3+ CD8+), B lymphocytes (CD3-CD19+) and NK cells (CD3- 15 CD16+CD56+) present in the sample, as shown in Figure 2. Within the population identified as total lymphocytes, around 60% are T lymphocytes (among the lymphocytes T, there are about 70% helper lymphocytes (CD4+) and 22% cytotoxic T lymphocytes (CD8+), and 35% other cells composed of 20% B lymphocytes and 68% NK cells). 20 These data showed that the selected sample has all the cells necessary for the development of the method and that the separation of PBMCs by Ficoll-Paque^TM contributes to the enrichment of T lymphocytes in the sample. To analyze the cell viability of the samples after separation by a Ficoll-PaqueTM gradient, a 1:1 dilution of PBMCs and Blue Trypan 0.4%, and the presence of blue-stained (dead) and translucent (alive) cells was evaluated by light microscopy. The viability of the isolated cell samples remained above 95% for all samples tested, as shown in Figure 3. Cells that met the appropriate number characteristics (equal to or greater than or equal to 6x10⁸ total cells) and viability (greater than 90%) were centrifuged again for 10 minutes, 500 G, at a temperature of 20 to 24°C. After centrifugation, the supernatant was discarded and approximately 3.0 to 4.5x10⁸ of the initial total cells were resuspended in culture medium at a concentration between 1.0 and 1.5x10⁶/mL (300 mL) to proceed to the expansion phase in the G-REX bioreactor or after validation in the XURI cell expansion system W25 - GE Healthcare bioreactor. 1.4C^FREEZING D^{TO THE} W^CELLS AND^EXCEEDENTS P^ARA R^AND-^STIMULUS Surplus cells that were not expanded were frozen in 5 mL cell freezing tubes, or in a freezing bag using 40% Voluven® 6% (commercially available from Fresenius), 40% human albumin 20% (commercially available from Grifols) and 20% DMSO (CryoPur commercially available from Origen) in a 1:1 ratio to be used later to restimulate lymphocytes in culture. Particularly, a number of total cells equal to or greater than 3.5x10⁸ was frozen. Freezing occurred after centrifuging the cells in a 50 mL conical tube at 500 G for 5 minutes at a temperature between 20 and 24 °C. The supernatant was discarded and the cells were resuspended in the cryoprotectant solution of 40% Voluven® 6%, 40% human albumin 20% and 20% DMSO, in a 1:1 ratio (diluted cells: cryoprotectant solution), respecting the condition of that the concentration of cells for freezing must be 1x10⁷/mL. The cells were transferred to duly identified 5 mL cell cryopreservation tubes, placed in an alcohol-free freezing system (Corning® CoolCell™ 5 mL LX Cell Freezing Container) and placed in a freezer at a temperature between -75 and -90 °C for 24 hours. Afterwards, the tubes were transferred to a properly identified box and could remain frozen for up to 2 years at temperatures between -75 and -90 °C.^EXAMPLE 2 - E^TAPA (II) ^IN AND^{XPANSION IN BIOREACTOR SYSTEM} XURI 2.1 VALIDATION OF THE INITIAL NUMBER OF CELLS AND DOSE OF CYTOKINES FOR CELL EXPANSION The initial number of cells for expansion was defined after standardizing cell expansion with various doses of cytokines and various cell concentrations. For IL-2 (recombinant human IL-2, commercially available from R&D Systems), the minimum concentration was determined as 500 IU/mL, and up to 1,000 IU/mL could be used during the expansion process. For IL-7 (recombinant human IL-7, commercially available from R&D Systems), the ideal concentration was defined as 10 ng/mL. For cell concentration, a minimum of 1x10 was defined⁶ cell./mL and the ideal number of 1.5x10⁶ cell./mL. The results are shown in Figure 4A-C. 2.2 ASEPTIC PREPARATION OF THE XURI BIOREACTOR At least 4 hours before the start of expansion, the Xuri cell bag - 2L was installed in the XURI cell expansion system W25 bioreactor system. The bag was opened under sterile conditions and it was checked that all connections were closed so that the bag could then be installed in the bioreactor (rods, filter heater, pumping tubing). Unicorn software (Cytiva) was started according to the parameters of pocket size, optical density (OD), and pH. The “fast fill” parameter was set to 30 minutes and the CO value^two was adjusted to 5% (v/v). AIM-V culture medium (commercially available from ThermoFisher Scientific (Gibco)) supplemented with 10% Human AB Serum (commercially available from Valley Biomedical), 1% 200 mM L-glutamine (commercially available from ThermoFisher Scientific (Gibco)) , 1% 1 M HEPES (commercially available from ThermoFisher Scientific (Gibco)) and 1% non-essential amino acids (commercially available from ThermoFisher Scientific (Gibco)) was prepared in laminar flow in a sterile transfer bag and connected to the system via connection sterile. The medium necessary for lymphocyte expansion was added to the system so that it was equilibrated by the gas mixture (CO2 and compressed air) for at least 2 hours. In the Unicorn software, the inclination of the stirring platform and the semi-static stirring speed of 2 rpm and an angle of 2º were also determined. The gas flow was maintained at 0.05 Lpm so that this culture was at temperature. 37 °C and the percentage of CO2 at 5%. After at least 2 hours of equilibration of the culture medium in the system, the pH of the culture medium was measured and added as a parameter in the Unicorn software. The pH must be maintained between 7.0 and 7.5 and the minimum volume to start lymphocyte expansion in the XURI Bioreactor was 300 mL containing a minimum of 1.0x10⁶ cell./mL, the maximum being 1.5x10⁶ cell./mL. 2.3 CELL EXPANSIONS IN XURI BIOREACTOR WITH STIMULATION OF CYTOKINES AND CMV-SPECIFIC PEPTIDES After stabilizing the XURI cell expansion system W25 as described above, cells were added at a minimum concentration of 1x10⁶ cell./mL. The cytokines IL-2 (500 IU/mL) and IL-7 (10 ng/mL) were used in supplemented AIM-V medium and 1 μg of the hCMV virus capsid peptide pp65/mL (Peptivator pp65) (commercially available from Miltenyi Biotec). Stimulation with the peptide occurred in a 150 mL transfer bag containing the final volume of 100 mL of cells diluted in culture medium, remaining for 2 hours in the CO2 oven and after this period, the volume was transferred via sterile connection to the XURI bag, which already contained 200 mL of previously balanced medium. The culture lasted from 14 to 21 days. In the first 3 days, culture conditions were maintained as semi-static (2 rpm and 2º angle) and gas flow of 0.05 Lpm. In order for this culture to remain undisturbed, no samples were removed in the first 3 days of culture. From day 5 onwards, the culture conditions were changed to a shaking speed of 4 rpm and a shaking angle of 4º. On the seventh day, the culture returned to semi-static condition (2 rpm and 2° angle) and the cells were restimulated with 1 μg of the capsid peptide of the hCMV pp65/mL virus and PBMC from the same sample that was previously frozen, as per described in the item “freezing excess cells for restimulation” (Example 1-1.4). The cell culture restimulation procedure for the XURI system is described below (item 2.4). The culture was supplemented with fresh culture medium always in accordance with the dosage of metabolites in the culture supernatant (glucose and lactate), measured routinely by a technician in the field. Was added the volume of medium suitable for the cell concentration to remain at 1x10⁶ cell./mL via transfer bag and sterile connection to the XURI bioreactor inlet route. The culture should not exceed 21 days and, at the end of the culture, the number of T lymphocytes and cell viability were evaluated. At this stage, expansion whose final number of lymphocytes was at least twice the initial number of lymphocytes was considered adequate. 2.4 RESTIMULATION OF CELL CULTURE FOR THE 10 minutes. After centrifugation, the supernatant was discarded and the pellet was resuspended in supplemented AIM-V culture medium. Cell viability was checked according to the protocols described here, which must be above 80% and the concentration can vary between 1:1 and 1:10 of new cells for each cell in culture depending on the number of cells available. For example, if there are about 5x10⁸ cells in culture, restimulation can vary between 5x10⁷ and 5x10⁸ viable cells. After verifying that the cells were suitable for restimulation, the cells were transferred to a 150 mL transfer bag with the aid of a sterile access and a syringe and diluted in 40 mL of supplemented AIM-V culture medium. 1 μg of the hCMV pp65/mL virus capsid peptide was also added and the bag was left in a CO oven.^two for 2 hours. After this period, it was transferred to the XURI bioreactor using a sterile connection system, where the culture continued for another 7 to 14 days. 2.5 F^{COMPLETION OF EXPANSION AND REDUCTION OF CULTURE VOLUME IN} B^IOREACTOR Xuri For bags that did not contain a 0.3 μM filter, the reduction was performed manually, where the entire volume contained in the XURI bioreactor was transferred to 600 mL transfer bags using the sterile connection system or by inversion of the feed system. For cases in which the volume was already reduced after transferring the volume from the bioreactor bag to a transfer bag in a closed system, preparation for the selection stage continued. For cases where the volume exceeded 150 mL, the bags were centrifuged for 10 minutes at 700 G for 10 minutes. At the end of the centrifugation, the 600 mL bag was placed in a plasma extractor and the culture supernatant was transferred to another transfer bag, to perform quality control on cell manipulation. Cells were gathered into a single bag by sterile connection system and cell counting and cell viability determination were performed. The maximum number of cells that can proceed to the selection step is 1x10⁹ cells with viability greater than 80%. 1x10 were transferred⁹ cells into a 250 mL bag and the concentration was adjusted to approximately 1x10⁷ cell./mL, that is, in a final volume of 100 mL. Samples were taken for cell counting, viability and karyotype analysis and proceeded to step (III) of lymphocyte selection for the production of INF-γ in a Miltenyi Biotec capture system or in CliniMACS PRODIGY equipment. The reserved cell supernatant was sent for sterility testing and sterile processing quality control. EXAMPLE 3 – STAGE (II) IN G-REX 3.1 E BIOREACTOR SYSTEM^{CELL EXPANSIONS IN} B^IOREACTOR G-REX ^{WITH CYTOKINE STIMULATION AND} CMV-SPECIFIC PEPTIDES As an alternative to the XURI bioreactor, lymphocytes were expanded in a G-REX bioreactor. Cells were placed at a minimum concentration of 1x10⁶ cell./mL and maximum of 1.5x10⁶ cell./mL. The cytokines IL-2 (500 IU/mL) and IL-7 (10 ng/mL) were used in AIM-V medium supplemented after sterilizing filtration and 1 μg/mL of the hCMV virus capsid peptide pp65/mL. Stimulation with the peptide occurred in a final volume of 100 mL of cells diluted in culture medium for 2 hours and, after this period, the volume was adjusted to a final volume of 300 mL. The culture lasted from 12 to 21 days in a CO greenhouse^two. After the start of culture, from the fifth day of cultivation, samples were removed every other day to check the number of cells in culture, pH, lactate and glucose. On the seventh day of culture, cells were restimulated with 1 μg of HCMV pp65/mL virus capsid peptide and PBMC from the same sample that was previously frozen, as described below. The culture was supplemented with fresh culture medium always according to the dosage of metabolites in the culture supernatant (glucose and lactate) and the number of cells/mL. The appropriate pH must be between 7.0 and 7.5, the glucose concentration in the culture supernatant must be greater than 150 g/dL, the lactate dosage ¹⁰ must be less than 150 mmol/dL and the number of cells must be between 1.0 and 1.5x10⁶ cell./mL. For cases in which the G-REX bottle reached its maximum volume capacity (500 mL) and there was a need for supplementation due to a change in the dosage of metabolites, it was necessary to change the culture medium by removing at least 15 mL of culture medium into two 50 mL tubes. These 2 tubes were centrifuged at 500 G for 10 minutes. After centrifugation, the medium contained in the tubes was discarded and new medium was added as well as the cytokines IL-2 (500 IU/mL) and IL-7 (10 ng/mL). Then, the new culture medium was transferred to the G-REX bioreactor. For cases where the number of cells exceeded the capacity of the culture flask, the culture was divided into two flasks. The maximum culture maintenance time for cell expansion was 21 days. During expansion and at the end of this period, the total number of cells, cell viability and the number of lymphocytes were evaluated. The expansion was completed when reaching 1x10⁹ cells or when the number of T lymphocytes was doubled 25 with viability greater than 80%. 3.2 R^ESTIMULUS D^A W^ULTURE D^AND W^CELLS P^ARA YOU^SYSTEM G-REX To restimulate the culture, the PBMCs that were previously frozen were thawed in laminar flow, resuspended in supplemented AIM-V culture medium and centrifuged in a Thermo Scientific Sorvall 30 ST 16R centrifuge at 500 G for 10 minutes. After centrifugation, the supernatant was discarded and the cell-containing pellet was resuspended in supplemented AIM-V culture medium. Cell viability was checked according to the protocols here described and must be above 80% and the concentration can vary between 1:1 and 1:10 of new cells for each cell in culture depending on the number of cells available. For example, if there are about 5x10⁸ cells in culture, restimulation can vary between 5x10⁷ and 5x10⁸ viable cells. After checking that the cells were suitable for restimulation, they were transferred to a 150 mL transfer bag with the aid of a sterile access and a syringe and diluted in 20 to 40 mL of supplemented AIM-V culture medium. 1 μg of the HCMV pp65/mL virus capsid peptide was also added and the bag was left in a CO2 oven for 2 to 3 hours. After this period, it was transferred to the G-REX bioreactor using a sterile connection system, where the culture continued for another 7 to 14 days. 3.3 COMPLETION OF EXPANSION AND REDUCTION OF CULTURE VOLUME IN THE G-REX BIOREACTOR. At the end of the culture, the entire volume contained in the G-REX was transferred to 50 mL tubes in laminar flow. Next, the tubes were centrifuged at 500 G for 10 minutes in the Thermo Scientific Sorvall ST 16R centrifuge and the culture supernatant was transferred to perform quality control for sterile manipulation of the cells. The cells were gathered in a single tube and counted using the protocols described here. The maximum number of cells that can proceed to the selection step is 1x10⁹ cells. Thus, 1x10 were transferred⁹ cells into a 250 mL bag and the concentration was adjusted to about 1x10⁷ cell./mL, that is, in a final volume of 100 mL. The volume for processing on the CliniMACS PRODIGY equipment can vary between 100 and 150 mL and the number of cells must not exceed 1x10⁹ cells. Samples were taken for cell counting, viability and karyotype analysis. Then, we proceeded to step (III) of lymphocyte selection given the production of INF-γ in a Miltenyi Biotec capture system or in CliniMACS PRODIGY equipment. The reserved cell supernatant was sent for sterility testing and sterile processing quality control. EXAMPLE 4 – COMPARATIVE VALIDATION OF LYMPHOCYTE EXPANSION IN THE G-REX BIOREACTOR ^AND X^URI 4.1 COMPARISON OF THE CELL EXPANSION PATTERN AND CONTROL OF PH, GLUCOSE AND LACTATE IN THE XURI AND G-REX BIOREACTORS To compare the expansion bioreactors, samples were used in both platforms with the same volume and initial cell concentration, but which were derived from different donors. The expansion pattern between the 2 bioreactors was quite similar, with a drop in the number of total leukocytes in the first days of expansion and, after 8 days in culture, the cells in culture began to proliferate on a larger scale (Figure 5A). Variations in pH and glucose and lactate levels occurred around the eighth day of expansion, with a decrease in pH and glucose and an increase in lactate production, consistent with the log phase of cell expansion (Figure 5B-D). According to the glucose and lactate level, the culture medium was replaced/changed. Material collection for analysis was always carried out before replacing/changing the culture medium, which occurred twice a week. 4.2 COMPARISON OF LYMPHOCYTE PROFILE DURING EXPANSION IN G-R BIOREACTORS^{EX E} X^URI The lymphocyte profile during expansion for 15 days on both platforms also maintained the same pattern. An increase in the percentage of CD3+ T lymphocytes and results above 80% of these cells was observed after 15 days of expansion on both platforms (Figure 6A). An increase in CD3+CD8+ T lymphocytes was also observed on both platforms, with results above 50% of CD8 T lymphocytes after 15 days of expansion (Figure 6B). There was also an increase in the percentage of memory T lymphocytes (CD3+CD45RO+CD45RA-) and a decrease in naïve T lymphocytes (CD3+CD45RA+CD45RO-) during the 15 days of culture on both platforms. AND^EXAMPLE 5 – E^TAPA (III) ^{SELECTION OF CELLS PRODUCING} IFN-γ. 5.1 METHOD FOR CAPTURE OF CELLS PRODUCING IFN-γ IN AN OPEN AND MANUAL SYSTEM (LS COLUMN - MYLTENYI BIOTEC). After expansion of CMV-specific cells in a bioreactor, IFN-γ-producing cells were isolated using the IFN-γ-secreting cell detection and enrichment kit (MACS Miltenyi Biotec). The PBS/EDTA buffer was topped up with 0.5% human albumin before use. Non-adherent cells were transferred to conical tubes in the ratio of 10⁸ cells for each 100 mL of ice-cold buffer (CliniMACS PBS/EDTA) and centrifuged at 300 G at 4°C for 10 minutes and the supernatant was discarded. Subsequently, cells were resuspended in 800 μL of ice-cold buffer (CliniMACS PBS/EDTA) and 200 μL of CliniMACS Capture IFN-γ for every 10⁸ of cells and incubated on ice for 5 minutes. 100 mL of heated TexMacs culture medium (commercially available from Miltenyi Biotec) was added for every 10⁸ cells, which remained for 45 minutes in a CO25% incubator at 37°C, under periodic shaking. The cell suspension was centrifuged again under the same conditions as above in ice-cold buffer, then incubated at 4°C for 15 minutes with 800 μL of ice-cold buffer and 200 μL of anti-IFN-γ for every 10⁸ cells. After this period, the cells were washed again at a rate of 10⁸ cells for every 100 mL of ice-cold buffer, centrifuged under the same conditions as above, then resuspended at 10⁸ cell./mL of ice-cold buffer. Meanwhile, the LS columns were washed 3 times with 1 mL of ice-cold buffer (CliniMACS PBS/EDTA) and the cell suspension was applied to the column, which was washed again 3 times to remove any IFN-γ-negative cells. The column was placed in a conical tube and quickly washed with 3 mL of buffer using a plunger to remove cells retained in the column. Separation was performed on another column to ensure purity and enrichment of cells positive for IFN-γ. The result of positive selection was called the TCB fraction and the unselected cells were in the NTCB fraction. Finally, cellular immunophenotyping was performed by flow cytometry for IFN-γ expression to verify the isolated and depleted cell populations. At the end of the selection, there must be above 80% of IFN-γ positive cells and above 80% of T lymphocytes with viability also above 80% in the TCB. The supernatant of the NTCB fraction was sent for aerobic, anaerobic, fungal and special microorganism cultures (25 mL), for endotoxin testing (1 mL) and for sterility testing at the BCQ Laboratory (50 mL). The supernatant of the TCB fraction (15 mL) was sent for NAT serology for HIV, HCV, HBV and for PCR for CMV, EBV, Parvovirus B19 and Mycoplasma pneumoniae, according to protocols described in the present invention. Furthermore, approximately 1 mL of the cells were sent for NAT HTLV-I serology. The remainder of the TCB supernatant was frozen for later impurity tests (determination of IL-2 and IL-7 concentration). 5 At the end of the step, the TCB fraction was frozen. The number of cells selected varied according to the number of initial cells and due to biological variation between individuals. Table 3 summarizes all the critical points of the open system selection process and the decision making to be carried out. ¹⁰ T^{THE BEAUTY} 3 - P^{CRITICAL PROCESS OUTCOMES AND ACCEPTABLE RESULTS AT THE END OF THE PROCESS} PROCESS USING LS COLUMN

A^AUTOMATIC (W^LINIMACS PRODIGY) To capture cells in a closed and automatic system, the CliniMACS Cytokine Capture System (INF-γ) and the CliniMACS 5 PRODIGY equipment were used; for cell selection, the “CCS_INF- γ_Enrichment” program was used. according to the manufacturer's guidelines. The bag containing a maximum of 1x10⁹ Cells for selection were connected using the sterile connection system with the sample bag (100 to 150 mL), the TS500 10 disposable tube system (Miltenyi Biotec), TexMacs culture medium, PBS/EDTA were also connected to the equipment plus human albumin solution (0.5%), cytokine capture system (INF-γ), 0.9% saline solution plus human albumin solution (2.5%). The process starts and continues automatically until the separation of 02 bags, one containing the selected cells (TCB) and one containing the discarded material (NTCB). At the end of processing, the bag with the discarded cell fraction (NTCB) and the bag with the enriched cells (TCB) were sealed and two aliquots per fraction were collected for analysis by flow cytometry, while the rest of the cells were stored. at 4°C. One aliquot was used to determine the number of cells and the other to verify the yield of the entire process. 20 The criterion to determine whether the selection was adequate was above 80% of IFN-γ positive cells and above 80% of T lymphocytes with viability also above 80% in the TCB. The supernatant of the NTCB fraction (25 mL) was sent for culture aerobic, anaerobic, fungal and special microorganisms, 1 mL was sent for endotoxin testing, 50 mL for sterility testing at the BCQ Laboratory, 10 ml for Mycoplasma sp. in the Invitrocell Laboratory. The supernatant of the TCB fraction (15 mL) was sent for NAT 5 serology for HIV, HCV, HBV and for PCR for CMV, EBV, Parvovirus B19 and Mycoplasma pneumoniae. Approximately 1 mL of the cells were sent for NAT HTLV-I serology and the remainder of the TCB supernatant was frozen for later impurity and potency tests. Table 4 summarizes all the critical points of the selection process in 10 closed system and the decision making to be carried out. TABLE 4 - CRITICAL POINTS OF THE PROCESS AND ACCEPTABLE RESULTS AT THE END OF THE PROCESS USING CLINIMACS PRODIGY

5.3 COMPARATIVE VALIDATION BETWEEN SELECTION IN OPEN COLUMN SYSTEM AND S^{CLOSED COLUMN SYSTEM} (W^LINIMACS PRODIGY) Cells from the same donor were expanded in the G-REX bioreactor, and after expansion, 2x10⁹ cells were used for both selections, i.e. 1x10⁹ for each selection. After selection, the final number of selected cells (TCB) in the 2 systems was quite similar (Figure 7A): 2.7x10⁷ in the PRODIGY system and 3.0x10⁷ in the LS Column system. The percentage and number of T lymphocytes in the 2 systems was also quite similar (Figure 7B): 93% in the PRODIGY system and 97% in the LS Column system, both having above 90% T lymphocytes. 5.4 COMPARISON OF SELECTION PURITY IN SYSTEMS OPEN AND CLOSED For this comparison, cells from the same donor were used, which were expanded in the G-REX bioreactor. After expansion, 1x10⁹ cells were used for both selections. After selection, the final number of selected cells (TCB) in the 2 systems was quite similar, the purity was above 80%, being 80.5% in the manual column system and 80.6% in the PRODIGY system (Figure 8) . Additionally, the CliniMACS PRODIGY equipment was evaluated and validated by 3 selections. The average number of T lymphocytes (CD3) in the depleted fraction – NTCB was on average 73.8±7.84%, while in the selected fraction – TCB it was on average 91±1.9%. Regarding CD3 T lymphocytes positive for IFN-γ in the depleted fraction – NTCB, the average was 36.7±7.08%, while in the selected cells it was an average of 83.63±2.20%, as shown in Figure 9. 5.5V^{ERIFICATION OF THE PERFORMANCE OF THE SELECTION PROCESS BY CELL CAPTURE} INF-Y BY THE RESEARCH LABORATORY OF THE HOSPITAL ISRAELITA ALBERT EINSTEIN. The process yield was performed by flow cytometry (FORTESSA), in which cells from the NTCB and TCB pockets were labeled with monoclonal antibodies to: CD45-PerCP-Cy5.5, CD3-PE-CF594, CD4 APC-Cy7, CD8-Alexa 700 from BD Pharmingen and the anti-INF-γ-PE provided by the kit (Milteniy Biotec). In addition, possible contaminating cells were identified and named other CD3- cells, using CD14-BV421 (BD Pharmingen) to identify monocytes, CD19-FITC (BD Pharmingen) to identify B lymphocytes and 5 CD16-APC (BD Pharmingen) to identify identify NK cells. For labeling with antibodies, an aliquot of 1x10 was separated⁵ cells (TCB and NTCB) and previously titrated amounts of antibodies were added to each tube. To control autofluorescence, the FMO tube was used, with labeling with all antibodies except 1. In this tube, the cutoff between the positive and negative population was determined for each fluorescence that was not added to the tube. After labeling, the samples were incubated for 30 minutes in the dark. Then, 1 mL of flow cytometry buffer was added and the samples were centrifuged for 10 minutes at room temperature (22 °C). The supernatant was discarded and the pellet was resuspended in 200 µL of cytometry buffer. The acquisition was carried out on the template prepared for this marking and the analysis considered the first selection in FSC vs. SSC. Next, positive CD3 cells, T lymphocytes and IFN-γ staining were analyzed, as well as the CD4 and CD8 subpopulations. For the CD3 negative population, the 20 populations CD14 (monocytes), CD19 (B lymphocytes) and CD16 (NK cells) were analyzed. Figure 10A-D shows the analysis of the cell fraction discarded by the process (NTCB) where it was observed that only 78% of the cells were T lymphocytes, which were composed of 48% CD4 T lymphocytes and 50% CD8 T lymphocytes. Of these T lymphocytes, less than 1% secreted IFN-γ. It was also observed that 25% more than 20% of other cells were present, of which, within this 20%, 30% were monocytes, 36% NK and 21% B lymphocytes. In the cell fraction enriched by the process (TCB) it was observed that 92% of the cells were T lymphocytes, which were composed of 34% CD4 T lymphocytes and 60% CD8 T lymphocytes. Of these T lymphocytes, almost 100% secreted IFN-γ. 30 It was also observed that only 6% of other cells were present, and within this 6%, 32% were monocytes, 13% NK cells and 31% B lymphocytes. An aliquot was also sent to the Pathology Laboratory Clinic from Hospital Israelita Albert Einstein to determine viability, (%) CD3 cells and (%) positive CD3/IFN-γ cells (purity of selection). 5.6A^{ANALYSIS BY FLOW CYTOMETRY IN THE} L^ABORATORY D^AND P^ATOLOGY W^{LINE OF} HOSPITAL ISRAELITA ALBERT EINSTEIN The performance of the process was carried out by flow cytometry (FACS CANTO), in which cells from the NTCB and TCB pockets were labeled with monoclonal antibodies to: CD45-KO, CD3-PE-CF594, CD4 APC-Cy7, CD8-Alexa 700 from BD Pharmingen and the anti-INF-γ-PE provided by the kit (Milteniy Biotec). Viability was determined by cells that were not labeled with 7-AAD, considered viable cells. In addition, possible contaminating cells were identified and named other CD3- cells. For this analysis, CD11b-BV421 (BD Pharmingen) was used to identify contamination with myeloid cells. For labeling with antibodies, aliquots of 1x10⁵ Cells from the TCB and NTCB fractions were separated and the previously titrated amounts of antibodies were added to each tube. To control autofluorescence, an FMO tube was created, in which there was labeling with all antibodies except 1. In this tube, the cutoff between the positive and negative population was determined for each fluorescence that was not added to the tube. After labeling, the samples were incubated for 30 minutes in the dark. Then, 1 mL of cytometry buffer was added and the samples were centrifuged for 10 minutes at room temperature (22 °C). The supernatant was discarded and the pellet containing the cells was resuspended in 200 µL of cytometry buffer. The acquisition was carried out on the template prepared for this marking. For the analysis, the first selection in FSC vs. SSC. Next, the following were analyzed sequentially: CD45 positive cells, viable 7AAD negative cells and CD3 positive cells (T lymphocytes), and staining for IFN-γ, CD4 and CD8 subpopulations. For the CD3 negative population, the CD11b populations (myeloid cells) were analyzed. The result of the analysis performed by the Clinical Pathology Laboratory is illustrated in Figure 11A-F. EXAMPLE 6 - STAGE (IV) - FREEZING OF CELLS 6.1 C^{FREEZING OF H CELLS}CMV-^SPECIFIC (TCB) To freeze the hCMV-specific cells (TCB), the bag containing the lymphocytes was placed in laminar flow and a volume of approximately 6 mL was diluted to a volume of 50 mL. An aliquot was removed for cell counting. Next, the cells were centrifuged and the supernatant removed to perform viability, sterility and serology analyses, according to the protocols described here. Cells were frozen in amounts of 1x10⁶ per dose/bag. To do this, they were transferred to a 250 mL freezing bag and each 1x10⁶ Cells were diluted in 15 mL of 0.9% saline solution containing 2.5% human albumin used in cell selection. This volume (15 mL) containing 1x10⁶ Cells were transferred to each freezing bag. The bags containing the cells in 15 mL were placed in the refrigerator for 15 minutes and the freezing medium for the same volume of cells to be frozen was also prepared and added to the refrigerator for the same period as the cells (40% Voluven ® 6%, 40% human albumin 6% and 20% DMSO). Next, one bag at a time was removed from the refrigerator and transferred to laminar flow to add the cryoprotectant solution, using a 50 mL syringe and bag adapter. After adding the cryoprotectant solution, the bag was sealed and kept in a compartment with 4 fragments that can be removed without changing the status of the frozen bag for periodic cell viability. After sealing the bag tube, it was placed on a metal support and then placed in a horizontal position in a mechanical freezer at -80 °C for 24 h. After this period, the bags were stored in a vertical position. A bag was thawed to perform ELISPOT assays (INF-γ release) against peptide stimulation and was also used for co-cultivation assay with control MCR-5 fibroblasts and those infected with CMV strain AD169 (potency). The assays and their results are described later in the present invention. 6.2 C^{FREEZING OF UNSELECTED CELLS} (NTCB) NTCB (about 230 mL) was transferred to 50 mL tubes and centrifuged. The supernatant was removed for viability, sterility and serology analyses, according to the protocols described here. NTCB cells were frozen in 5 mL freezing tubes and were used for quality control. AND^EXAMPLE 7 – E^TAPA (V) ^IN D^FREEZING 7.1 SELECTION OF CMV-SPECIFIC CELLS FOR PATIENT INFUSION HLA COMPATIBILITY AND NUMBER OF CELLS PER Kilogram The minimum compatibility between lymphocytes and the recipient was 1 in 6 alleles (high resolution), considering class I alleles - HLA loci- A, HLA-B and HLA-C. Preference was given to compatibility with the HLA class I alleles listed below: A*01:01, A*02:01, A*03:01, A*11:01, A*23:01, A* 24:02, B*07:02, B*08:01, B*15:01, B*18:01, B*35:01, B*44:02 and B*44:03. The number of cells to be infused into each patient was also evaluated, so that it did not exceed the value of 1 to 2x10⁴ cell./kg of receiver weight. Table 5 below summarizes the critical analysis performed for the release of T lymphocytes (TCB) for cell infusion. After analysis, suitable cells were thawed in laminar flow at 37 °C. 7.2 STABILITY AFTER THAWING By analyzing cell death with propidium iodide using flow cytometry, the stability of lymphocytes after thawing was analyzed. 3 batches of cells from different donors were tested. The Donor 15IVN9083 cells tested had been frozen for approximately 7 days when they were thawed for testing. The cells from Donor 18FS4945 had been frozen for approximately 6 months when they were thawed for the test and the cells from Donor 24MAPM692 had been frozen for approximately 11 months when they were thawed for the test. The stability of lymphocytes after thawing kept between 2 and 6 °C showed that viability varies according to the thawing time. Even after 2 hours of thawing, over 80% of the cells remained viable (Figure 12). T^{THE BEAUTY} 5 - ^{CRITICAL ANALYSIS PERFORMED FOR THE RELEASE OF LYMPHOCYTES} T (TCB) FOR CELL INFUSION

EXAMPLE 8 – IN VITRO STUDY TO SIMULATE THE RESPONSE TO STIMULUS AND^SPECIFIC. To evaluate the function of hCMV-specific T lymphocytes, the ELISPOT test was performed for IFN production in response to the specific stimulus. These tests 5 were carried out on 03 expanded and selected batches of product (TCB) on the CliniMACS PRODIGY equipment. Thawed cells from the following products/donors were used: product: B300121394945-donor: 18FSH4945 (Figure 13), product: B300121391223-donor: 59TPAA1223 (Figure 14) and product: B300121327279-donor: 60GDD2727 (Figure 15). The results of these assays were evaluated in the automatic spot counter (AID Advanced Imaging Devices GmbH Ebinger Str. 4 D72479 Strassberg Germany). The assays were also carried out using 03 different amounts of cells (1x10³/100 μL, 5x10³/100 μL and 1x10⁴/100 μL) in the product of selected cells (TCB) and also in discarded cells (NCTB). All assays were performed in triplicates. In this assay, thawed CMV-specific T lymphocytes were stimulated for approximately 2 hours in the presence of the viral peptide (pp65) and it was observed that approximately 10% of the cells were capable of secreting IFN-γ during this period, being more accurate in lower concentrations of cells. The ability to secrete IFN-γ in more than 50 lymphocytes for every 10 lymphocytes was defined as adequate.³ lymphocytes, considering the stimulation time and the low quantity of antigen-presenting cells in the product. This assay showed that laboratory-generated cells are functional. EXAMPLE 9 - SIMULATION OF RESPONSE AMONG PARTIALLY COMPATIBLE INDIVIDUALS TO A SPECIFIC STIMULUS For the in vitro safety assessment, an assay of IFN-γ release by TCB lymphocytes was developed in the presence of commercial cells (MCR-5 lung fibroblast) infected by hCMV . 9.1 INFECTION OF MCR-5 FIBROBLASTS WITH THE COMMERCIAL STRAIN OF CYTOMEGALOVIRUS - AD169 (CMVAD169). For infection of MCR-5 fibroblasts with CMV-AD169 strain (ATCC# VR-538) for viral load expansion, MCR-5 fibroblasts were seeded at a concentration of 2.84x10⁴/well in 24-well culture plate in NB2 culture room. After 24 h of culturing MCR-5 cells, the culture medium was removed and the cells were adsorbed with 0.1 MOI of strain CMV-AD169 for each cell in culture (2.84x10³ MOIs) for 8 h. After this period, the supernatant was removed and new culture medium was added. After 4 days of culture, it was possible to observe cytopathic changes in MCR-5. After 10 days of cultivation, the supernatant was removed and frozen with a solution of 7% DMSO in liquid nitrogen. To titrate the CMV-AD169 frozen, the supernatant was used in serial dilutions to reinfect MCR-5. In a 24-well culture plate, cells were maintained without infection (CTRL) and also infected in triplicates at viral dilutions of 10^-1.10^-two, 10^-3, 10^-4, 10^-5, 10^-6 and 10^-7. After 2 and 3 days of infection, staining with the anti-CMV antibody (green) was evaluated by microscopy and cytometry, confirming the infection of 17% of MCR-5 fibroblasts with a viral concentration of 10^-1, and 7.5% with a viral concentration of 10^-two. It was also possible to verify the infection up to a concentration of 10^-3 (Figure 16). Therefore, for co-culture experiments with virus-specific T lymphocytes, only viral dilutions of 10 were used.^-1 and 10^-two. 9.2C^O-^{CULTURE OF FIBROBLASTS} MCR-5 ^{INFECTED BY} CMV-AD169 ^WITH SPECIFIC HCMV T LYMPHOCYTES FOR ANALYSIS OF IFN SECRETION For co-cultivation, fibroblasts were seeded at a concentration of 1x10⁴/well and after 24 hours of culture, infection was carried out for 8 h. After this period, the culture medium was changed. After 3 days of cultivation, co-cultures were carried out for 24 h and 48 h with specific hCMV T lymphocytes generated in a clean room in proportions (1x10⁴ Lymphocytes: 1x10⁴ Fibroblasts), (5x10³ Lymphocytes: 1x10⁴ Fibroblasts), (1x10³ Lymphocytes: 1x10⁴ Fibroblasts), i.e., 1 effector:1 target, 1 effector:2 targets and 1 effector:4 targets with controls of fibroblast without lymphocyte and only lymphocyte without the fibroblast. The culture supernatant was obtained at 24 h and 48 h for INF-γ measurement. Class I MHC compatibility between fibroblasts and hCMV-specific T lymphocytes was 3:6 as shown in Table 6. TABLE 6 - DESCRIPTION OF FIBROBLAST CLASS I HLA TYPING (MCR-5) AND C^{H CELLS}CMV-^{SPECIFICATIONS GENERATED FROM} P^RODUCT/D^OADOR B300121394945/18FSH4945 RESPECTIVELY

The measurement of IFN-γ from lymphocytes without fibroblasts and from fibroblasts without lymphocytes was called spontaneous release. The dosage of IFN-γ in uninfected control fibroblasts and CMV-infected fibroblasts in concentrations 10^-1 and 10^-two for 24 and 48 h, spontaneous release was decreased. It was observed that for the product: B300121394945-donor: 18FSH4945 in co-culture with MCR-5 fibroblast in the proportion (1:1) the secretion of IFN-γ was: In 24 h of co-culture: - 4.0 pg /mL at a viral concentration of 10^-two; - 34.2 pg/mL at viral concentration of 10^-1. In 48 h of co-cultivation: - 43.5 pg/mL at viral concentration of 10^-two; - 45.0 pg/mL at viral concentration of 10^-1. No IFN-γ secretion was observed in control MCR-5 fibroblasts not infected with the viral strain. A summary of these results is presented in Table 7 and Figure 17, where UI is equivalent to international units. T^{THE BEAUTY} 7 - C^{ONCENTRATIONS OF} IFN-γ ^{IN CO}-^{CULTIVATION OF} 24 ^HE 48 ^H.

9.3 CO-CULTURE OF MCR-5 FIBROBLASTS INFECTED BY CMV-AD169 WITH L^INFOCYTES T ^HCMV ^{SPECIFIC FOR DEATH ANALYSIS OF} MCR-5 For safety assessment, a cell death test (cytotoxicity) of MCR-5 cells infected by hCMV by lymphocytes of the product was also carried out. The cytotoxic potential of hCMV-specific T lymphocytes, isolated by the CliniMACs PRODIGY equipment, was analyzed by culturing MRC-5 fibroblasts (ATCC CCL-171) infected with CMV (ATCC VR-538) at concentration 10^-1 in the presence of different concentrations of CMV-specific lymphocytes (1:1 and 2:1) from 3 different donors/TCBs, with class I HLA compatibility between lymphocyte donors and MCR-5 cells varying as indicated below: - Product : B300121394945 - Donor: 18FSH4945 (HLA 3:6 compatibility); - Product: B300121391223 - Donor: 59TPAA1223 (HLA 1:6 compatibility); - Product: B300121327279 - Donor: 60GDD2727 (HLA compatibility 1:6). Co-cultures were evaluated after 24 and 48 hours. Triplicate supernatants from cocultures of hCMV-specific T lymphocytes with hCMV-infected MRC-5 fibroblasts were collected and the assay was performed using the CytoTox 96® Non-Radio kit (Promega Cat.: G1780; Lot.:430121). Specifically, this assay measures the concentration of LDH (lactate dehydrogenase), the enzyme released after cell lysis. The concentration of LDH is modified when cell death occurs, and as a death control, co-culture of lymphocytes with uninfected fibroblasts was used. LDH released into culture supernatants was measured in a 30-minute enzymatic assay, which results in the conversion of a tetrazolium salt (violet iodonitrotetrazolium; INT) into a red formazan product, altering the optical density. Optical densities were obtained by colorimetric reading and their variation is proportional to cell lysis. The protocol follows the manufacturer's guidelines and optical densities were obtained using a spectrophotometer (Varioskan LUX - Thermo Scientific). To evaluate the percentage of lymphocyte cytotoxicity in each culture condition, the formula provided by the manufacturer below was applied: % ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ = ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ − ^^ ^^ ^^ ^^ ^^ ^^â ^^ ^^ ^^ ^^ ^ ^ ^^ ^^ ^^ ^^ − ^^ ^^ ^^ ^^ ^^ ^^â ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^á ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ − ^^á ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^â ^^ ^^ ^^ ^^ 100 The results of the cytotoxicity of cells from 3 donors in 48 h of co-cultivation with infected fibroblasts was on average 3% when the cell concentration is lower (1:2), and on average 13% when the cell concentration is higher (1:1), as shown in Table 8 and in Figure 18. In addition, it was observed that the percentage of MCR-5 fibroblasts infected at the viral concentration of 10^-1 it was about 17% in 48 h. Therefore, it was inferred that at the lowest lymphocyte concentration (1:2), the death of hCMV-infected cells was on average 20%; and at the highest cell concentration (1:1), the death of hCMV-infected cells averaged 75%. TABLE 8 - SUMMARY OF THE CYTOTOXICITY OF CMV-SPECIFIC T LYMPHOCYTES AGAINST MCR-5 FIBROBLASTS INFECTED WITH HCMV-AD169.

In conclusion, this experiment demonstrated that in vitro there is no nonspecific death of MCR-5 fibroblasts by CMV-specific T lymphocytes even in HLA class I matches (1:6). In co-cultures, where fibroblasts were not infected, there was no detection of death at the highest cell concentration (1:1). 5 Depending on the concentration of T lymphocytes, the specific death of infected fibroblasts was on average 17% for low concentrations (1:2) and 75% for higher concentrations (1:1), indicating the safety and efficacy of the product in vitro. R^EFERENCES 10 Düver F., Weißbrich B., Eyrich M., Wölfl M., Schlegel P.G., Wiegering V. Viral reactivations following hematopoietic stem cell transplantation in pediatric patients - A single center 11-year analysis. PLoS One. 2020;15(2): e0228451. Published on February 4, 2020. doi: 10.1371/journal.pone.0228451. Blümel J.V., Burger R., et al. Human Cytomegalovirus (HCMV) - Revised. 15 Transfus Med Hemother.2010;37(6):365-375. doi:10.1159/000322141. Zuhair M, Smit GSA, Wallis G, et al. Estimation of the worldwide seroprevalence of cytomegalovirus: A systematic review and meta-analysis. Rev Med Virol.2019;29(3): e2034. doi:10.1002/rmv.2034. Teira P., Battiwalla M., Ramanathan M., et al. Early cytomegalovirus reactivation remains associated with increased transplant-related mortality in the current era: a CIBMTR analysis. Blood.2016; 127(20):2427-2438. doi:10.1182/blood- 5 2015-11-679639. Asano-Mori Y., Oshima K., Sakata-Yanagimoto M., et al. High-grade cytomegalovirus antigenemia after hematopoietic stem cell transplantation. Bone Marrow Transplant, 2005; 36: 813-819. Frere P, Baron F, Bonnet C, et al. Infections after allogeneic 10 hematopoietic stem cell transplantation with a nonmyeloablative conditioning regimen. Bone Marrow Transplant.2006; 37:411-418. Einsele H., Roosnek E., Rufer N., et al. Infusion of cytomegalovirus (CMV) specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy, Blood 2002:99; 3916-3922. 15 Hanley P.J., Melenhorst J.J., Nikiforow S., et al. CMV-specific T cells generated from naive T cells recognize atypical epitopes and may be protective in vivo. Science translational medicine.2015;7(285):285ra63. Blyth E., Clancy L., Simms R., Ma C.K., Burgess J., Deo S., et al. Donor- derived CMV-specific T cells reduce the requirement for CMV-directed 20 pharmacotherapy after allogeneic stem cell transplantation. Blood. 2013;121(18):3745-58. Leen A.M., Myers G.D., Sili U., et al. Monoculture-derived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals. Nat Med.2006;12(10):1160-6. 25 Leen A.M., Bollard C.M., Mendizabal A.M., Shpall E.J., Szabolcs P., Antin J.H., Kapoor N., Pai S.Y., Rowley S.D., Kebriaei P., et al. Multicenter study of banked third-party virus- specific T cells to treat severe viral infections after hematopoietic stem cell transplantation. Blood.2013; 121:5113-5123. Leen A. M., Heslop H. E., Brenner M. K. Antiviral T-cell therapy. Immunol 30 Rev.2014 Mar.; 258(1):12-29. Arasaratnam R.J., Leen A.M. Adoptive T cell therapy for the treatment of viral infections. Ann Transl Med.2015 Oct;3(18):278. Feuchtinger T., Opherk K., Bethge W. A., et al. Adoptive transfer of pp65- specific T cells for the treatment of chemorefractory cytomegalovirus disease or reactivation after haploidentical and matched unrelated stem cell transplantation. Blood.2010;116(20):4360-7. Riddell SR, Watanabe KS, Goodrich JM, Li CR, Agha ME, Greenberg 5 PD. Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science.1992;257(5067):238-41. Walter E.A., Greenberg P.D., Gilbert M.J., Finch R.J., Watanabe K.S., Thomas E.D., Riddell S.R. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N 10 Engl J Med.1995 Oct 19;333(16):1038-44. Peggs K.S., Verfuerth S., Pizzey A., et al. Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem cell transplantation with virus specific T cell lines. Lancet 2003 Oct 25:3621375-1377. Peggs K.S., Thomson K., Samuel E., Dyer G., Armoogum J., Chakraverty 15 R., et al. Directly selected cytomegalovirus-reactive donor T cells confers rapid and safe systemic reconstitution of virus specific immunity following stem cell transplantation. Clin Infect Dis.2011;52(1):49-57. Meij P., Jedema I., VanVleet M.L., van der Heiden P.L., van de Meent M., van Egmond H.M., et al. Effective treatment of refractory CMV reactivation after 20 allogeneic stem cell transplantation with in vitro-generated CMV pp65-specific CD8+ T-cell lines. J Immunother.2012;35(8):621. Pei X.Y., et al. Cytomegalovirus-Specific T-Cell Transfer for Refractory Cytomegalovirus Infection After Haploidentical Stem Cell Transplantation: The Quantitative and Qualitative Immune Recovery for Cytomegalovirus. J Infect Dis.2017 25 Nov 15;216(8):945-956. Withers B., Blyth E., Clancy L. E., Yong A., Fraser C., Burgess J., et al. Long-term control of recurrent or refractory viral infections after allogeneic HSCT with third-party virus-specific T cells. Blood Adv.2017;1(24):2193-205. Withers B., Clancy L., Burgess J., Simms R., Brown R., Micklethwaite K., 30 et al. Establishment and operation of a third-party virus-specific T cell Bank within an allogeneic stem cell transplant program. Biol Blood Marrow Transplant. 2018;24(12):2433-42. Kallay K., Kassa C., Reti M., Karaszi E., Sinko J., Goda V., et al. Early experience with CliniMACS PRODIGY CCS (IFN-gamma) system in selection of virus-specific T cells from third-party donors for pediatric patients with severe viral infections after hematopoietic stem cell transplantation. J Immunother.2018;41(3):158-63.

Claims

CLAIMS 1. VIRUS-SPECIFIC LYMPHOCYTE EXPANSION METHOD, characterized by comprising the steps of: (I) pre-expansion from a sample of lymphomononuclear cells, which comprises the steps of: (a) separation of mononuclear cells from the blood peripheral (PBMCs); (b) centrifugation and discarding the supernatant; and (c) resuspension of PBMCs; 0 (II) expansion of cells in vitro, (III) selection of lymphocytes positive for INF-γ. 2. METHOD, according to claim 1, characterized by additionally comprising the step of (IV) freezing at -80 °C. 3. METHOD, according to claim 1, characterized in that the sample of lymphomononuclear cells is obtained from the buffy-coat (cell concentrate) generated in the donation of platelets by apheresis, which is retained in the center of the collection kit. 4. METHOD, according to claim 1, characterized in that step (a) is carried out by density gradient. 5. METHOD, according to claim 1, characterized in that it comprises a step of counting cells and checking the viability of cells after step (a), wherein the sample acceptance criteria are a number of at least 6 x 10 ⁸ total cells and at least 90% viability. 6. METHOD, according to claim 1, characterized in that part of the sample in step (c) is resuspended in culture medium, and the remainder of the sample is resuspended in a cryoprotectant solution for freezing between -75 °C and -95 °C . 7. METHOD, according to claim 1, characterized by step (II) comprising the steps of: (d) inoculating the cells obtained in step (I) in a bioreactor comprising culture medium comprising IL-2 and IL-7 at a temperature of 37+1 ºC, pH between 7.0 and 7.5 and 5% (v/v) CO2; (e) stimulation of cells with the hCMV virus capsid peptide; It is (f) restimulation of cells with the hCMV virus capsid peptide and PBMC cells from the same sample previously frozen in step (c); Preferably, the method further comprises the step of: (g) adding culture medium in a volume suitable for maintaining cell concentration; more preferably, step (II) further comprises the step(s) of: (h) evaluating the number of T lymphocytes and the viability of the cells, in which expansion is considered adequate when the final number of lymphocytes is at least at least twice the initial number of lymphocytes and the viability is at least 80%;0 and/or (i) reduction of the culture volume, in which the reduction is preferably carried out by filtration, centrifugation and/or plasma extractor. 8. METHOD, according to claim 1, characterized by step (III) comprising the steps of: 5 (j) capturing cells producing INF-γ in a capture system; (k) optionally, enrichment of INF-γ-positive cells; and (l) cellular immunophenotyping by flow cytometry. 9. METHOD, according to claim 1, characterized in that lymphocytes are specific for phosphoprotein 65 of the human cytomegalovirus0 capsid. 10. COMPOSITION, characterized in that it comprises virus-specific T lymphocytes and one or more additional components, wherein the virus-specific T lymphocytes are obtained by the method, as defined in claim 1. 11. COMPOSITION, according to claim 11, characterized5 by the one or more additional components being selected from a pharmaceutically acceptable carrier, a mixture of hydroxyethyl starch and sodium chloride, human albumin, dimethyl sulfoxide (DSMO) and mixtures thereof. 12. USE OF A COMPOSITION, as defined in any one of claims 10 to 11, characterized in that it is for the manufacture of a cell therapy to treat viral infections, in bone marrow transplant patients who do not respond to conventional therapies. 13. USE, according to claim 12, characterized in that the viral infection is caused by human cytomegalovirus (hCMV), adenovirus, Epstein- barr (EBV), HHV-6 or polyomavirus (BK virus).