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WO2024145700A1 - Méthode d'expansion de lymphocytes virus-spécifiques, composition et utilisation d'une composition - Google Patents

Méthode d'expansion de lymphocytes virus-spécifiques, composition et utilisation d'une composition Download PDF

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Publication number
WO2024145700A1
WO2024145700A1 PCT/BR2023/050384 BR2023050384W WO2024145700A1 WO 2024145700 A1 WO2024145700 A1 WO 2024145700A1 BR 2023050384 W BR2023050384 W BR 2023050384W WO 2024145700 A1 WO2024145700 A1 WO 2024145700A1
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cells
lymphocytes
cell
virus
culture
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English (en)
Portuguese (pt)
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Fernanda AGOSTINI ROCHE
Luciana CAVALHEIRO MARTI
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Sociedade Beneficente Israelita Brasileira Hospital Albert Einstein
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Sociedade Beneficente Israelita Brasileira Hospital Albert Einstein
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • CD8 T lymphocytes also known as CTL
  • CD8 T lymphocytes are 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.
  • the cells were infused into 14 patients via central catheter in 4 doses staggered weekly 3.3x10 7 , 1x10 8 , 3.3x10 8 and 10 9 cell./m two 4.1x10 5 , 3.3x10 5 and 1x10 6 /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.
  • 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 1x10 7 cell./m two (equivalent to 1x10 4 /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 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 10 5 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.
  • the cells were infused intravenously and the average dose of cells infused was 10 2.1x10 4 CD3/kg (ranging between 1x103 and 1.3x10 5 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.
  • the pool of 138 synthetic peptides from the hCMV viral capsid 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).
  • CCS automatic cell capture system
  • the dose of cells infused intravenously was on average 4x10 5 /kg for CD8 and 3x10 3 /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.
  • 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 1x10 4 /Kg intravenously and compatibility was partial up to 1 in 6 alleles for HLA A, B and C.
  • 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- ⁇ , IL-2 or TNF- ⁇ 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).
  • 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).
  • 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.
  • FIG. 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- na ⁇ ve lymphocytes.
  • Figure 7A-B shows the comparison between the selected cells (TCB, target cell bag) in the column systems and PRODIGY system.
  • TB 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- ⁇ 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- ⁇ capture assay. NTCB - Analysis of the cellular fraction discarded in the capture assay. (A) Selection of cells by SSC vs. FSC.
  • 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.
  • 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.
  • 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- ⁇ in the co-culture supernatant of hCMV-specific lymphocytes and hCMV-infected fibroblasts.
  • 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”.
  • the amplification and detection of nucleic acids can be carried out by polymerase chain reaction (PCR).
  • serological test 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 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) .
  • 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%.
  • CD3 T lymphocytes positive for IFN- ⁇ 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.
  • each 1x10 6 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 1x10 6 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).
  • 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.
  • 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 (1x10 3 /100 ⁇ L, 5x10 3 /100 ⁇ L and 1x10 4 /100 ⁇ L) in the product of selected cells (TCB) and also in discarded cells (NCTB).
  • MCR-5 fibroblasts were seeded at a concentration of 2.84x10 4 /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 (2.84x10 3 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.
  • 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 lactate dehydrogenase
  • 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).

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Abstract

La présente invention concerne une méthode d'expansion de lymphocytes virus-spécifiques. La présente invention concerne également une composition comprenant des lymphocytes virus-spécifiques et l'utilisation de cette composition pour la fabrication d'une thérapie cellulaire.
PCT/BR2023/050384 2023-01-02 2023-11-10 Méthode d'expansion de lymphocytes virus-spécifiques, composition et utilisation d'une composition Ceased WO2024145700A1 (fr)

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BR102023000028-2A BR102023000028A2 (pt) 2023-01-02 Método de expansão de linfócitos vírus-específicos, composição e uso de uma composição
BR1020230000282 2023-01-02

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009053109A1 (fr) * 2007-10-24 2009-04-30 Anne Letsch Préparations de lymphocytes t spécifiques d'un antigène à partir de la moelle osseuse
US20150044258A1 (en) * 2011-12-12 2015-02-12 Cell Medica Limited Process for t cell expansion
WO2022125746A2 (fr) * 2020-12-09 2022-06-16 Tevogen Bio Inc. Lymphocytes t spécifiques d'un virus et procédés de traitement et de prévention d'infections virales

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009053109A1 (fr) * 2007-10-24 2009-04-30 Anne Letsch Préparations de lymphocytes t spécifiques d'un antigène à partir de la moelle osseuse
US20150044258A1 (en) * 2011-12-12 2015-02-12 Cell Medica Limited Process for t cell expansion
WO2022125746A2 (fr) * 2020-12-09 2022-06-16 Tevogen Bio Inc. Lymphocytes t spécifiques d'un virus et procédés de traitement et de prévention d'infections virales

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LENNART BISSINGER ALFRED, RAUSER GEORG, HEBART HOLGER, FRANK FRIEDERIKE, JAHN GERHARD, EINSELE HERMANN: "Isolation and expansion of human cytomegalovirusspecific cytotoxic T lymphocytes using interferon-␥ secretion assay", EXPERIMENTAL HEMATOLOGY, vol. 30, no. 10, 1 October 2002 (2002-10-01), pages 1178 - 1184, XP093189023 *
PAINE ANANTA, OELKE MATHIAS, BLASCZYK RAINER, EIZ‐VESPER BRITTA: "Expansion of human cytomegalovirus‐specific T lymphocytes from unfractionated peripheral blood mononuclear cells with artificial antigen‐presenting cells", TRANSFUSION, AMERICAN ASSOCIATION OF BLOOD BANKS, BETHESDA, MD., US, vol. 47, no. 11, 1 November 2007 (2007-11-01), US , pages 2143 - 2152, XP093189025, ISSN: 0041-1132, DOI: 10.1111/j.1537-2995.2007.01439.x *
VAZ-SANTIAGO JOCELYN, LULÉ JACQUELINE, ROHRLICH PIERRE, JACQUIER CÉLINE, GIBERT NICOLAS, LE ROY EMMANUELLE, BETBEDER DIDIER, DAV: "Ex Vivo Stimulation and Expansion of both CD4 + and CD8 + T Cells from Peripheral Blood Mononuclear Cells of Human Cytomegalovirus-Seropositive Blood Donors by Using a Soluble Recombinant Chimeric Protein, IE1-pp65", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 75, no. 17, 1 September 2001 (2001-09-01), US , pages 7840 - 7847, XP093189027, ISSN: 0022-538X, DOI: 10.1128/JVI.75.17.7840-7847.2001 *

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