Large Scale Expansion of Activated T Cells: Defining Procedures and Cryopreservation Methods for Clinical Use as Adoptive Cellular Therapy Post-Transplant.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5232-5232
Author(s):  
Jia-Yan Wu ◽  
Susan M. Webber ◽  
John M. Hill ◽  
Marc Ernstoff ◽  
Zbigniew Szczepiorkowski ◽  
...  
Keyword(s):  
T Cells ◽  
Large Scale ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
K562 Cells ◽  
Culture Method ◽  
Clinical Use ◽  
Adoptive Cellular Therapy ◽  
Post Transplant ◽  
Large Scale Culture

Abstract We previously demonstrated a laboratory model of ex vivo expansion of mobilized peripheral blood mononuclear cells (PBMNC) that acquire the CD8+CD56+ NKT cell phenotype and aggressively destroy targets through the NKG2D receptor (Cytotherapy.2006;8:141–8). We developed a large scale culture method for clinical use that allows growth, expansion and cryopreservation of these expanded cells for infusion post-transplant. PBMNC were collected by large volume leukapheresis from healthy donors (n=3). Unselected MNC cells (2.5 × 106/ml) were cultured in Life Cell Bags (Nexell Therapeutics) with serum-free AIM V medium (Invitrogen), IL-2 (Chiron, 1000 IU/ml)and OKT3 (Ortho Biotech, 500 ng/ml) at 37° C and 5% CO2. Six bags were arranged from each collection. Fresh cytokines and medium were added on Days 3 and 5, and the cells were harvested on Day 7. The viability of the ex vivo expanded cell populations was 95% +/− 1.8%. Total MNC cell numbers expanded from 2.5 × 106/ml to 6.4 × 106/ml (2.5 +/− 0.5% fold increase). Phenotypic analyses showed CD3 cells increased from 55% (Day 0) to 94% (Day 7; +/−1.42%); the CD4 cells increased from 30% to 58% (+/− 1.48%); the CD8 cells increased from 11% to 48% (+/−3.98%); the CD56 cells increased from 17.2% to 47.8% (+/− 6.2). Cytotoxicity of Day 7 expanded cells was markedly increased at 74% (+/−7.1%) (Targets: K562 cells; E:T ratio 100:1) compared with 33% (+/− 5.4%) at baseline. We also tested if the ex vivo expanded cells could be cryopreserved for future clinical use. After 7 days in liquid nitrogen, cells were promptly thawed, washed and analyzed. The viability was 91% (+/− 2.1%) and the cytotoxicity against K562 cells was only slightly decreased (65%, +/− 5.4%), when compared to non-cryopreserved expanded cells (p = 0.18). The phenotype was not affected by cryopreservation and thawing. This large scale culture method results in growth and expansion of aggressive effector cells that are cytotoxic against tumor cells. The majority of the expanded cells are CD3+ T cells (equally distributed between CD4+ and CD8+ T cells). NK cells (CD56+) also readily expanded. After cryopreservation of the ex vivo expanded cells and a short-term storage, viability and phenotype of the cells did not change. There is minimal decrease in cytotoxicity that is not statistically significant. This large scale expansion model will be used in clinical trials at our institution to expand cells for adoptive cellular therapy following autologous PBSC transplantation in patients with a hematologic malignancy.

scholarly journals IL-15 mediated expansion of rare durable memory T cells following adoptive cellular therapy

2021 ◽  
Vol 9 (5) ◽  
pp. e002232
Author(s):  
Karan Kohli ◽  
Lu Yao ◽  
Theodore Scott Nowicki ◽  
Shihong Zhang ◽  
Ralph Graeme Black ◽  
...  
Keyword(s):  
T Cells ◽  
Disease Progression ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
Single Agent ◽  
Adoptive Cellular Therapy ◽  
Conditioning Treatment ◽  
Ex Vivo Culture

BackgroundSynovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) are ideal solid tumors for the development of adoptive cellular therapy (ACT) targeting NY-ESO-1, as a high frequency of tumors homogeneously express this cancer-testes antigen. Data from early phase clinical trials have shown antitumor activity after the adoptive transfer of NY-ESO-1–specific T cells. In these studies, persistence of NY-ESO-1 specific T cells is highly correlated with response to ACT, but patients often continue to have detectable transferred cells in their peripheral blood following progression.MethodWe performed a phase I clinical trial evaluating the safety of NY-ESO-1–specific endogenous T cells (ETC) following cyclophosphamide conditioning. Peripheral blood mononuclear cells (PBMCs) from treated patients were evaluated by flow cytometry and gene expression analysis as well as through ex vivo culture assays with and without IL-15.ResultsFour patients were treated in a cohort using ETC targeting NY-ESO-1 following cyclophosphamide conditioning. Treatment was well tolerated without significant toxicity, but all patients ultimately had disease progression. In two of four patients, we obtained post-treatment tumor tissue and in both, NY-ESO-1 antigen was retained despite clear detectable persisting NY-ESO-1–specific T cells in the peripheral blood. Despite a memory phenotype, these persisting cells lacked markers of proliferation or activation. However, in ex vivo culture assays, they could be induced to proliferate and kill tumor using IL-15. These results were also seen in PBMCs from two patients who received gene-engineered T-cell receptor–based products at other centers.ConclusionsETC targeting NY-ESO-1 with single-agent cyclophosphamide alone conditioning was well tolerated in patients with SS and those with MRCL. IL-15 can induce proliferation and activity in persisting NY-ESO-1–specific T cells even in patients with disease progression following ACT. These results support future work evaluating whether IL-15 could be incorporated into ACT trials post-infusion or at the time of progression.

scholarly journals T Cells Educated By DC/AML Fusions in the Context of 4-1BB Costimulation As a Potent Strategy for Adoptive Cellular Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2673-2673
Author(s):  
Jessica Liegel ◽  
Dina Stroopinsky ◽  
Haider Ghiasuddin ◽  
Adam Morin ◽  
Marzia Capelletti ◽  
...  
Keyword(s):  
T Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
Advisory Board ◽  
Adoptive Cellular Therapy ◽  
Advisory Committees ◽  
Equity Ownership

Introduction: Our group has developed a novel vaccine using patient-derived acute myeloid leukemia (AML) cells and autologous dendritic cells (DCs), capable of presenting a broad array of leukemia antigens. In a phase I/II clinical trial DC/AML vaccination led to an expansion of leukemia-specific T cells. We hypothesized that the fusion vaccine offered a unique platform for ex vivo expansion of functionally potent leukemia specific T cells with broad specificity targeting shared and tumor specific neoantigens. We postulated that incorporating 4-1BB (CD137) mediated co-stimulation would further enhance activation of antigen specific T cells and the development of a crucial memory response as well as promote survival and persistence. Here we describe therapeutic exploration of the use of 4-1BB to augment vaccine-educated T cells for adoptive cellular therapy in an immunocompetent murine model. Methods: DC/AML fusion vaccine was generated using DCs obtained from C57BL/6J mice and syngeneic C1498 AML cells as previously described. T cells were obtained from splenocytes after magnetic bead isolation and cultured with irradiated DC/AML fusion vaccine in the presence of IL-15 and IL-7. Following co-culture, 4-1BB positive T cells were ligated using agonistic 4-1BB antibody (3H3 clone, BioXCell) and further selected with RatIgG2a magnetic beads (Easy Sep). Subsequently T cells were expanded with anti-CD3/CD28 activation beads (Dynabeads). In vivo, mice underwent retro-orbital inoculation with C1498 and vaccination with irradiated fusion cells the following day. Agonistic mouse anti-4-1BB antibody was injected intraperitoneally on day 4 and day 7. In addition, C1498 cells were transduced with Mcherry/luciferase and a reproducible model of disease progression was established. Results: DC/fusion stimulated T cells showed increased immune activation as measured by multichannel flow cytometric analysis. Compared to unstimulated T cells, there was 5-fold increase in CD4+CD25+CD69+, and a 10-fold and 7-fold increase in 4-1BB and intracellular IFNƔ expression on CD8+ cells respectively. Following agonistic 4-1BB ligation and bead isolation, the proliferation rate was increased in the 4-1BB positive fraction as compared to both 4-1BB negative cells and unstimulated T cells. In addition, the 4-1BB positive fraction demonstrated increased cytotoxicity, as measured by a CTL assay detecting granzyme B with 1:10 tumor to effector cells. A shift from naïve to memory T cell phenotype was also observed. Following DC/fusion stimulation, CD44+CD62L- cells comprised 67% of CD8+ cells versus 20% without stimulation, the latter reflecting the effect of cytokines alone. Following 4-1BB ligation and anti-CD3/CD28 bead expansion, this phenotype was retained with the CD4+ and CD8+ effector memory and central memory compartments comprising the majority of T cells. Such findings are significant as presence of memory T cell populations are a critical component for successful adoptive cell transfer. The effect of agonistic 4-1BB antibody following vaccination was evaluated in vivo in an aggressive immunocompetent murine AML model. The combination of DC/AML fusion vaccine with 4-1BB antibody was associated with increased long-term survival (>120 days) of 40% versus 20% of mice treated with vaccine alone while all controls required euthanasia by 40 days. Conclusion: In the current study we have demonstrated the ability of DC/AML fusion vaccine to stimulate T cells ex-vivo as demonstrated by both early-activation (CD25,CD69), upregulation of antigen-specific markers (CD137) and cytokine secretion. Further enhancement of the cellular product using agonistic 4-1BB ligation and isolation simultaneously enriches for antigen-activated cells, as demonstrated by more potent cytotoxicity, as well as promoting memory phenotype and survival. Use of 4-1BB ligation for antigen-specific selection while providing an agonistic co-stimulatory signal is a potentially novel approach for development of non-engineered T cells. Ongoing experiments evaluating the efficacy of 4-1BB selected vaccine educated T cells using bioluminescence monitoring will be reported as well as in vitro use of patient-derived T cells. Disclosures Kufe: Canbas: Consultancy, Honoraria; Victa BioTherapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genus Oncology: Equity Ownership; Hillstream BioPharma: Equity Ownership; Reata Pharmaceuticals: Consultancy, Equity Ownership, Honoraria; Nanogen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rosenblatt:Dava Oncology: Other: Education; Partner Tx: Other: Advisory Board; Parexel: Consultancy; Celgene: Research Funding; BMS: Research Funding; Amgen: Other: Advisory Board; Merck: Other: Advisory Board; BMS: Other: Advisory Board ; Imaging Endpoint: Consultancy. Avigan:Takeda: Consultancy; Parexel: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy.

scholarly journals IL-21–mediated Foxp3 suppression leads to enhanced generation of antigen-specific CD8+ cytotoxic T lymphocytes

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 229-235 ◽  
Author(s):  
Yongqing Li ◽  
Cassian Yee
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
Human Model ◽  
Adoptive Cellular Therapy ◽  
Specific Ctls ◽  
Regulatory Constraints

Efforts to reproducibly isolate tumor antigen–specific T cells from patients would be facilitated by removing immunoregulatory barriers. Using a human model for eliciting T-cell responses to tumor-associated antigens, we develop a novel strategy that eliminates nearly all Foxp3-expressing cells through the combination of CD25 depletion and IL-21 treatment resulting in a more than 150-fold decrease in Foxp3+ cells to virtually undetectable levels and a more than 200-fold increase in antigen-specific cytotoxic T lymphocytes (CTLs). The extent of Foxp3 elimination and degree of expansion of antigen-specific CTLs shown in this study have not previously been achievable and are unique to IL-21. We demonstrate for the first time a possible mechanism for IL-21–mediated expansion of antigen-specific CTLs that involves suppression of Foxp3-expressing cells and reversal of inhibition to tumor-associated antigen–specific CTL generation in vitro. Taken together, the combination of CD25 depletion and IL-21 exposure, by releasing regulatory constraints, leads to markedly enhanced CTL induction and represents a robust strategy for the ex vivo generation of antigen-specific T cells for adoptive cellular therapy.

Phase I adoptive cellular therapy trial with ex-vivo stimulated autologous CD8+ T-cells against multiple targets (ACTolog IMA101) in patients with relapsed and/or refractory solid cancers.

2018 ◽  
Vol 36 (5_suppl) ◽  
pp. TPS77-TPS77
Author(s):  
Apostolia Maria Tsimberidou ◽  
Chad Stewart ◽  
Carsten Reinhardt ◽  
Hong Ma ◽  
Steffen Walter ◽  
...  
Keyword(s):  
T Cells ◽  
Phase I ◽  
Mononuclear Cells ◽  
Tumor Antigens ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
Performance Status ◽  
Conditioning Regimen ◽  
Tumor Escape ◽  
Adoptive Cellular Therapy

TPS77 Background: Adoptive cellular therapy (ACT) has dramatically changed the landscape of immunotherapy; however, only a small proportion of solid tumor patients have benefited from these advances due to i) heterogeneity of tumor antigen expression, ii) tumor escape (e.g. only one target is addressed), or iii) off-target toxicities (e.g. expression of targets on normal tissues). The ACTolog concept, utilizing antigen specific T cells (IMA101) against targets identified by the Immatics’ proprietary XPRESIDENT technology, is intended to overcome these limitations by addressing multiple novel relevant tumor antigens per patient. ACTolog is a personalized, multi-targeted ACT approach in which autologous T-cell products are manufactured against the most relevant tumor target peptides for individual patients whose tumors are positive against a predefined target warehouse. Methods: This study is an open-label first-in-human phase I trial in patients with relapsed or refractory solid tumors expressing at least one target from a warehouse of 8 cancer targets. Key eligibility criteria include: HLA-A*02:01 phenotype, qPCR expression of warehouse target(s), prior established lines of therapy, RECIST v1.1 measurable lesions, and ECOG performance status 0 or 1. At baseline, patients will undergo leukapheresis to collect mononuclear cells for manufacturing of IMA101 cells. Patients will receive their last line of established therapy during the production phase of IMA101. IMA101 will be infused after a pre-conditioning regimen (lymphodepletion) followed by LD-IL2. The primary objective is to assess safety and tolerability of IMA101. Secondary endpoints include overall response rate (RECIST and irRC), PFS and OS. The translational objective is to assess the in vivo persistence and ex vivo functionality of transferred T cells in addition to evaluation of target expression in tumors. Enrollment to the study is currently ongoing. Clinical trial information: NCT02876510 .

scholarly journals Adoptive Cellular Therapy for Solid Tumors

2021 ◽  
pp. 57-65
Author(s):  
Adham S. Bear ◽  
Joseph A. Fraietta ◽  
Vivek K. Narayan ◽  
Mark O’Hara ◽  
Naomi B. Haas
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Gene Editing ◽  
Cellular Therapy ◽  
Immune Cell ◽  
Ex Vivo ◽  
Oncolytic Viruses ◽  
Adoptive Cellular Therapy

Cancer immunotherapy tools include antibodies, vaccines, cytokines, oncolytic viruses, bispecific molecules, and cellular therapies. This review will focus on adoptive cellular therapy, which involves the isolation of a patient’s own immune cells followed by their ex vivo expansion and reinfusion. The majority of adoptive cellular therapy strategies utilize T cells isolated from tumor or peripheral blood, but may utilize other immune cell subsets. T-cell therapies in the form of tumor-infiltrating lymphocytes, T-cell receptor T cells, and CAR T cells may act as “living drugs” as these infused cells expand, engraft, and persist in vivo, allowing adaptability over time and enabling durable remissions in subsets of patients. Adoptive cellular therapy has been less successful in the management of solid tumors because of poor homing, proliferation, and survival of transferred cells. Strategies are discussed, including expression of transgenes to address these hurdles. Additionally, advances in gene editing using CRISPR/Cas9 and similar technologies are described, which allow for clinically translatable gene-editing strategies to enhance the antitumor activity and to surmount the hostilities advanced by the host and the tumor. Finally, the common toxicities and approaches to mitigate these are reviewed.

scholarly journals Augmented expansion of Treg cells from healthy and autoimmune subjects via adult progenitor cell co-culture

2020 ◽  
Author(s):  
JL Reading ◽  
VD Roobrouck ◽  
CM Hull ◽  
PD Becker ◽  
J Beyens ◽  
...  
Keyword(s):  
T Cell ◽  
Treg Cell ◽  
Regulatory T Cell ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
Fold Increase ◽  
Adoptive Cellular Therapy ◽  
T Cell Therapy

AbstractRecent clinical experience has demonstrated that adoptive regulatory T cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPCⓇ) promote regulatory T cell differentiation in vitro, suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a GMP compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is linked with a distinct Treg cell-intrinsic transcriptional program, characterized by diminished levels of core exhaustion (BATF, ID2, PRDM1, LAYN, DUSP1), and quiescence (TOB1, TSC22D3) related genes, coupled to elevated expression of cell-cycle and proliferation loci (MKI67, CDK1, AURKA, AURKB). In addition, MulTreg display a unique gut homing (CCR7lo β7hi) phenotype and importantly, are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or Th1-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 TSDR demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno graft vs host disease (xGvHD) in vivo. These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product.

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