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Phase I Clinical Trial of 4-1BB-based Adoptive T-Cell Therapy for Epstein-Barr Virus (EBV)-positive Tumors.

Eom HS, Choi BK, Lee Y, Lee H, Yun T, Kim YH, Lee JJ, Kwon BS - J. Immunother. (2016)

Bottom Line: Although adoptive cell therapy using Ag-specific T cells has been tested successfully in the clinic, the production of these T cells has been challenging.By applying our simple and practical 4-1BB-based method for the generation of Ag-specific CD8 T cells, here we determined the maximum tolerated dose, toxicity profile, immunologic responses, and clinical efficacy of autologous Epstein-Barr virus (EBV)/LMP2A-specific CD8 T cells (EBV-induced Natural T cell; EBViNT) in patients with relapsed/refractory EBV-positive tumors.The strength of the second wave was related to the efficacy of the treatment.

View Article: PubMed Central - PubMed

Affiliation: *Hematologic Oncology Clinic, Center for Specific Organs Cancer, National Cancer Center, Korea †Center for Clinical Trial, National Cancer Center, Korea ‡Cancer Immunology Branch, Division of Cancer Biology §Immune Cell Production Unit, Program for Immunotherapeutic Research, Research Institute and Hospital, National Cancer Center, Ilsandong-gu, Goyang, Gyeonggi ∥Department of Hematology-Oncology, Chonnam National University Medical School, Gwangju, Korea ¶Department of Medicine, Tulane University Health Sciences Center, New Orleans, LA.

ABSTRACT
Although adoptive cell therapy using Ag-specific T cells has been tested successfully in the clinic, the production of these T cells has been challenging. By applying our simple and practical 4-1BB-based method for the generation of Ag-specific CD8 T cells, here we determined the maximum tolerated dose, toxicity profile, immunologic responses, and clinical efficacy of autologous Epstein-Barr virus (EBV)/LMP2A-specific CD8 T cells (EBV-induced Natural T cell; EBViNT) in patients with relapsed/refractory EBV-positive tumors. This was a single-center, phase I, dose-escalation trial study evaluating 4 escalating dosing schedules of single injected EBViNT. CD8 T-cell responses against different LMP2A peptides in each patient were determined, and the most effective peptides were used to produce EBViNT. The produced autologous EBViNTs were single infused to patients with EBV-associated malignancy who had failed to standard treatments and were of HLA-A02 or A24 type. Of 11 patients enrolled, 8 patients received a single infusion of EBViNT: 4 with nasopharyngeal carcinomas, 1 with Hodgkin lymphoma, 2 with extranodal NK/T lymphomas, and 1 with diffuse large B-cell lymphoma. Single infusion of EBViNT was well tolerated by all the patients and generated objective antitumor responses in 3 of them. EBViNT infusion induced 2 waves of interferon-γ response: 1 approximately 1 week and the other 4-8 weeks after the treatment. The strength of the second wave was related to the efficacy of the treatment. The current trial shows that EBViNT therapy is safe and may provide a new option for treating EBV-positive recurrent cancer patients resistant to conventional therapy.

No MeSH data available.


Related in: MedlinePlus

Manufacturing of EBV-induced Natural T cell (EBViNT). A, Epitope screening to select Epstein Barr virus (EBV)/LMP2A peptides. B, Primary amplification of LMP2A-specific CD8+ T cells from peripheral blood mononuclear cells (PBMCs) of cancer patient using the selected LMP2A peptides for 14 days. 4-1BB-expressing CD8+ T cells were assessed on day 9 as in-process control. C, The cultured PBMCs were restimulated with the same LMP2A peptides for 24 hours to induce 4-1BB on CD8+ T cells. 4-1BB-expressing CD8+ T cells were sorted using anti-4-1BB mAb-coated plate on day 15. D, Rapid expansion method is as follows: the selected 4-1BB-expressing CD8+ T cells (5×105 cells) were mixed with 30 ng/mL anti-CD3 mAb, 1000 IU/mL IL-2, and a >200-fold of irradiated allogeneic PBMCs in 50 mL medium, injected into a 1 L culture bag (Nipro Inc.) and cultured for 14 days with routine addition of fresh medium containing 1000 IU/mL interleukin (IL)-2. LAMP1 assay and intracellular interferon (IFN)-γ staining were performed on day 28 (−3D analysis), and phenotyping, TCRvβ typing, and quality control tests were performed on day 31 (final product assay).
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Figure 1: Manufacturing of EBV-induced Natural T cell (EBViNT). A, Epitope screening to select Epstein Barr virus (EBV)/LMP2A peptides. B, Primary amplification of LMP2A-specific CD8+ T cells from peripheral blood mononuclear cells (PBMCs) of cancer patient using the selected LMP2A peptides for 14 days. 4-1BB-expressing CD8+ T cells were assessed on day 9 as in-process control. C, The cultured PBMCs were restimulated with the same LMP2A peptides for 24 hours to induce 4-1BB on CD8+ T cells. 4-1BB-expressing CD8+ T cells were sorted using anti-4-1BB mAb-coated plate on day 15. D, Rapid expansion method is as follows: the selected 4-1BB-expressing CD8+ T cells (5×105 cells) were mixed with 30 ng/mL anti-CD3 mAb, 1000 IU/mL IL-2, and a >200-fold of irradiated allogeneic PBMCs in 50 mL medium, injected into a 1 L culture bag (Nipro Inc.) and cultured for 14 days with routine addition of fresh medium containing 1000 IU/mL interleukin (IL)-2. LAMP1 assay and intracellular interferon (IFN)-γ staining were performed on day 28 (−3D analysis), and phenotyping, TCRvβ typing, and quality control tests were performed on day 31 (final product assay).

Mentions: EBViNTs were routinely produced from EBV-related cancer patients as described in Figure 1. The best LMP2A peptide for CD8+ T-cell production was selected for each patient based on the epitope screening (Fig. 1A), and EBV LMP2A-specific CD8+ T cells were produced as previously described.11 In brief, PBMCs were isolated from 50 mL of blood and suspended in RPMI1640 medium (WelGENE) supplemented with 4 mM l-glutamine, 12.5 mM HEPES, 50 μM 2-mercaptoethanol, and 3% autologous serum (CTL medium; CM) at 1×106 cells/mL density. The PBMCs were cultured in 14 mL round-bottomed tubes (BD Biosciences) and EBV/LMP2A-specific CD8+ T cells were amplified by incubating them with 2 μg/mL EBV/LMP2A peptide for 14 days (Fig. 1B). On day 14, the cells were harvested, washed, and restimulated with 5 μg/mL of EBV/LMP2A peptide and 100 IU/mL interleukin (IL)-2. After 24 hours, 4-1BB+CD8+ T cells were sorted by placing the cells in anti-4-1BB mAb-coated 6-well plates for 10 minutes in a 37°C CO2 incubator (Fig. 1C). The plate-bound cells were maintained in CM containing 1000 IU/mL IL-2 for 2 days and then rapidly expanded as follows: the sorted cells (5×105 cells) were mixed with 30 ng/mL anti-CD3 mAb, 1000 IU/mL IL-2, and a >200-fold of irradiated allogeneic PBMCs in 50 mL ALyS505N medium (CSTI). The mixture was injected into a 1 L culture bag (Nipro Inc.) and cultured in a CO2 incubator for 14 days with routine addition of fresh CM containing 1000 IU/mL IL-2 (Fig. 1D). As in-process control, 4-1BB expression by the CD8+ T cells was monitored by flow cytometry on days 9, 14, and 15. IFN-γ and LAMP1 assays were performed on day 28 and all the cells were recovered from the culture bag on day 31. Some were used in quality control tests.


Phase I Clinical Trial of 4-1BB-based Adoptive T-Cell Therapy for Epstein-Barr Virus (EBV)-positive Tumors.

Eom HS, Choi BK, Lee Y, Lee H, Yun T, Kim YH, Lee JJ, Kwon BS - J. Immunother. (2016)

Manufacturing of EBV-induced Natural T cell (EBViNT). A, Epitope screening to select Epstein Barr virus (EBV)/LMP2A peptides. B, Primary amplification of LMP2A-specific CD8+ T cells from peripheral blood mononuclear cells (PBMCs) of cancer patient using the selected LMP2A peptides for 14 days. 4-1BB-expressing CD8+ T cells were assessed on day 9 as in-process control. C, The cultured PBMCs were restimulated with the same LMP2A peptides for 24 hours to induce 4-1BB on CD8+ T cells. 4-1BB-expressing CD8+ T cells were sorted using anti-4-1BB mAb-coated plate on day 15. D, Rapid expansion method is as follows: the selected 4-1BB-expressing CD8+ T cells (5×105 cells) were mixed with 30 ng/mL anti-CD3 mAb, 1000 IU/mL IL-2, and a >200-fold of irradiated allogeneic PBMCs in 50 mL medium, injected into a 1 L culture bag (Nipro Inc.) and cultured for 14 days with routine addition of fresh medium containing 1000 IU/mL interleukin (IL)-2. LAMP1 assay and intracellular interferon (IFN)-γ staining were performed on day 28 (−3D analysis), and phenotyping, TCRvβ typing, and quality control tests were performed on day 31 (final product assay).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834812&req=5

Figure 1: Manufacturing of EBV-induced Natural T cell (EBViNT). A, Epitope screening to select Epstein Barr virus (EBV)/LMP2A peptides. B, Primary amplification of LMP2A-specific CD8+ T cells from peripheral blood mononuclear cells (PBMCs) of cancer patient using the selected LMP2A peptides for 14 days. 4-1BB-expressing CD8+ T cells were assessed on day 9 as in-process control. C, The cultured PBMCs were restimulated with the same LMP2A peptides for 24 hours to induce 4-1BB on CD8+ T cells. 4-1BB-expressing CD8+ T cells were sorted using anti-4-1BB mAb-coated plate on day 15. D, Rapid expansion method is as follows: the selected 4-1BB-expressing CD8+ T cells (5×105 cells) were mixed with 30 ng/mL anti-CD3 mAb, 1000 IU/mL IL-2, and a >200-fold of irradiated allogeneic PBMCs in 50 mL medium, injected into a 1 L culture bag (Nipro Inc.) and cultured for 14 days with routine addition of fresh medium containing 1000 IU/mL interleukin (IL)-2. LAMP1 assay and intracellular interferon (IFN)-γ staining were performed on day 28 (−3D analysis), and phenotyping, TCRvβ typing, and quality control tests were performed on day 31 (final product assay).
Mentions: EBViNTs were routinely produced from EBV-related cancer patients as described in Figure 1. The best LMP2A peptide for CD8+ T-cell production was selected for each patient based on the epitope screening (Fig. 1A), and EBV LMP2A-specific CD8+ T cells were produced as previously described.11 In brief, PBMCs were isolated from 50 mL of blood and suspended in RPMI1640 medium (WelGENE) supplemented with 4 mM l-glutamine, 12.5 mM HEPES, 50 μM 2-mercaptoethanol, and 3% autologous serum (CTL medium; CM) at 1×106 cells/mL density. The PBMCs were cultured in 14 mL round-bottomed tubes (BD Biosciences) and EBV/LMP2A-specific CD8+ T cells were amplified by incubating them with 2 μg/mL EBV/LMP2A peptide for 14 days (Fig. 1B). On day 14, the cells were harvested, washed, and restimulated with 5 μg/mL of EBV/LMP2A peptide and 100 IU/mL interleukin (IL)-2. After 24 hours, 4-1BB+CD8+ T cells were sorted by placing the cells in anti-4-1BB mAb-coated 6-well plates for 10 minutes in a 37°C CO2 incubator (Fig. 1C). The plate-bound cells were maintained in CM containing 1000 IU/mL IL-2 for 2 days and then rapidly expanded as follows: the sorted cells (5×105 cells) were mixed with 30 ng/mL anti-CD3 mAb, 1000 IU/mL IL-2, and a >200-fold of irradiated allogeneic PBMCs in 50 mL ALyS505N medium (CSTI). The mixture was injected into a 1 L culture bag (Nipro Inc.) and cultured in a CO2 incubator for 14 days with routine addition of fresh CM containing 1000 IU/mL IL-2 (Fig. 1D). As in-process control, 4-1BB expression by the CD8+ T cells was monitored by flow cytometry on days 9, 14, and 15. IFN-γ and LAMP1 assays were performed on day 28 and all the cells were recovered from the culture bag on day 31. Some were used in quality control tests.

Bottom Line: Although adoptive cell therapy using Ag-specific T cells has been tested successfully in the clinic, the production of these T cells has been challenging.By applying our simple and practical 4-1BB-based method for the generation of Ag-specific CD8 T cells, here we determined the maximum tolerated dose, toxicity profile, immunologic responses, and clinical efficacy of autologous Epstein-Barr virus (EBV)/LMP2A-specific CD8 T cells (EBV-induced Natural T cell; EBViNT) in patients with relapsed/refractory EBV-positive tumors.The strength of the second wave was related to the efficacy of the treatment.

View Article: PubMed Central - PubMed

Affiliation: *Hematologic Oncology Clinic, Center for Specific Organs Cancer, National Cancer Center, Korea †Center for Clinical Trial, National Cancer Center, Korea ‡Cancer Immunology Branch, Division of Cancer Biology §Immune Cell Production Unit, Program for Immunotherapeutic Research, Research Institute and Hospital, National Cancer Center, Ilsandong-gu, Goyang, Gyeonggi ∥Department of Hematology-Oncology, Chonnam National University Medical School, Gwangju, Korea ¶Department of Medicine, Tulane University Health Sciences Center, New Orleans, LA.

ABSTRACT
Although adoptive cell therapy using Ag-specific T cells has been tested successfully in the clinic, the production of these T cells has been challenging. By applying our simple and practical 4-1BB-based method for the generation of Ag-specific CD8 T cells, here we determined the maximum tolerated dose, toxicity profile, immunologic responses, and clinical efficacy of autologous Epstein-Barr virus (EBV)/LMP2A-specific CD8 T cells (EBV-induced Natural T cell; EBViNT) in patients with relapsed/refractory EBV-positive tumors. This was a single-center, phase I, dose-escalation trial study evaluating 4 escalating dosing schedules of single injected EBViNT. CD8 T-cell responses against different LMP2A peptides in each patient were determined, and the most effective peptides were used to produce EBViNT. The produced autologous EBViNTs were single infused to patients with EBV-associated malignancy who had failed to standard treatments and were of HLA-A02 or A24 type. Of 11 patients enrolled, 8 patients received a single infusion of EBViNT: 4 with nasopharyngeal carcinomas, 1 with Hodgkin lymphoma, 2 with extranodal NK/T lymphomas, and 1 with diffuse large B-cell lymphoma. Single infusion of EBViNT was well tolerated by all the patients and generated objective antitumor responses in 3 of them. EBViNT infusion induced 2 waves of interferon-γ response: 1 approximately 1 week and the other 4-8 weeks after the treatment. The strength of the second wave was related to the efficacy of the treatment. The current trial shows that EBViNT therapy is safe and may provide a new option for treating EBV-positive recurrent cancer patients resistant to conventional therapy.

No MeSH data available.


Related in: MedlinePlus