Clinical Grade Generation of T-Helper-1-Driven Polyclonal T-Cell Populations for Adoptive Immunotherapy Against Tumor Associated Antigens

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4314-4314
Author(s):  
Simone Kayser ◽  
Cristina Boß ◽  
Vanya Icheva ◽  
Stefan Stevanovic ◽  
Peter Lang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Peripheral Blood ◽  
Adoptive T Cell Therapy ◽  
T Helper ◽  
T Cell Therapy ◽  
Tumor Associated Antigens ◽  
Healthy Donors ◽  
Ifn Γ

Abstract Abstract 4314 Adoptive T cell therapy has been shown an option to treat patients with malignancies. In contrast to vaccinations, T cells for adoptive T-cell therapy are generated ex vivo to be re-infused into the recipient. This enables treatment of immunocompromized hosts and use of allogeneic T cells to exploit graft versus tumor effects. Adoptive T-cell therapy involving CD4+ T-helper cells (Th cells), intends to induce sustained T-cell responses in vivo. The Th1 cytokine interferon-gamma (IFN-γ) has not only an effect in orchestrating cytotoxic T-cell reponses, IFN-γ by itself has antitumor effects. Transferring T cells in a lymphopenic host furthermore eliminates regulatory T cells (Tregs) and offers access to homeostatic cytokines. The aim of our study was the translation of preclinical data into a GMP conform clinical scale protocol to generate specific T cells for adoptive T-cell therapy against tumor associated antigens. Large scale generations of NY-ESO-1 specific T cells was performed according to current GMP regulations in a GMP facility. In brief, peripheral blood mononuclear cells from healthy donors were primed with an overlapping NY-ESO-1 15-mer peptide mix. The priming was done in the presence of IL-7 and IL-2. T cells were enriched using IFN-γ capture technique and expanded for two weeks in autologous culture conditions with IL-7, IL-15 and IL-2. T-cell specificity, function and proliferation capacity was analyzed by flow cytometry. The T-cell products showed high numbers of specifically IFN-γ+, TNF-alpha+ T cells. Tolerance inducing cytokines like IL-10 were absent. Enrichment of Tregs was excluded. Both, CD4+ and CD8+ T cells with an effector memory phenotype proliferated in response to NY-ESO-1. CD107a assays demonstrated cytotoxic capacities of T cells. The T-cell product did not include alloreactive T cells. In summary GMP-conform generation of NY-ESO-1 specific T cells was established. Although tumor associated antigens are potential self antigens, it is possible to induce a functional Th1 response in peripheral blood T cells from healthy donors. Adoptive T-cell therapy against tumor associated antigens could have implications for multiple tumor entities in autologous as well as allogeneic treatment approaches. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Long-Term T Cell Expansion Results in Increased Numbers of Central Memory T Cells with Sustained Functional Properties for Adoptive T Cell Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1943-1943
Author(s):  
Stefanie Herda ◽  
Andreas Heimann ◽  
Stefanie Althoff ◽  
Josefine Ruß ◽  
Lars Bullinger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cell Expansion ◽  
Adoptive T Cell Therapy ◽  
T Cell Therapy ◽  
Human T Cells ◽  
Healthy Donors ◽  
T Cell Expansion

Success of adoptive T cell therapy (ATT) is dependent on sufficient numbers of T cells and the characteristics of the final T cell product. In several studies, clinical grade CD19 CAR T cell products could not be generated from about 6-30% patients, particularly if they were isolated from older or heavily pretreated diffuse large B cell lymphoma (DLBCL) patients. In cyclophosphamide/fludarabine-lymphodepleted patients with persistent or progressive disease a sequential second dose of T cells has been shown to be effective resulting in tumor regression. Here we investigated to what extend T cell numbers could be increased via prolonged expansion with standard cytokines IL-7/IL-15 and how transcriptome and function of central memory T cells (Tcm) longitudinally change during culture. Method: Murine and human T cells were cultured with the cytokine combination IL-7/IL-15. Short-term expanded (ST, one week) and long-term expanded (LT) CD8+ (4 weeks) and CD4+ (3 weeks) T cells were compared for proliferation capacity (CFSE), extent of apoptosis (AnnexinV), up-regulation of T cell inhibitory receptors (TIRs) and cytokine expression pattern after in vitro re-stimulation upon anti-CD3/CD28 stimulation. Further, RNA sequencing of ST and LT expanded murine CD8+ and CD4+ Tcm followed by unsupervised hierarchical clustering, principal component analysis (PCA) and differential expression analysis was performed. In vivo mouse models were used to analyze engraftment, persistence and anti-tumor capacity applying our bioluminescent dual-luciferase reporter mouse (BLITC - bioluminescent imaging of T cells) allowing us to monitor migration, expansion (RLuc luciferase) and activation (NFAT-driven Click-beetle luciferase) of adoptively transferred T cells in vivo. Finally, we analyzed the expansion and in vitro properties of T cells from healthy donors and DLBCL patients. Results: There was a 50-fold increase of T cells in LT vs. ST culture, the Tcmproportion was extended and stem cell markers were comparable or even higher expressed in LT expanded T cells. Differential analysis revealed 2786 (CD8) and 912 (CD4) with statistically significant expression alterations with generally only moderate effect size when comparing LT and ST expanded T cells. Interestingly, the dynamically modified genes largely overlapped for CD8 and CD4 T cells suggesting culture-associated changes. Comparable RLuc signals and T cells counts in peripheral lymph nodes (LN) and spleen indicate similar engraftment (4 weeks post ATT) and persistence capacities (up to 6 months post ATT) of transferred ST and LT T cells. SV40-TAg+ tumor bearing mice were treated with TCR-I retrovirally transduced CD8+ BLITC T cells, which were ST or LT expanded. The T cells infiltrated rapidly in the tumor where they got similarly activated resulting in a complete tumor rejection in all recipient mice. Finally, we analyzed the expansion and in vitro properties of T cells from healthy donors (n=3-5) and DLBCL patients (n=3) who were eligible for CAR T cell therapy. LT T cell expansion from healthy donors resulted in a 10.000-fold increase of CD8+CD45RO+CCR7+ T cells. In vitro assays showed comparable apoptosis and expression of TIRs between ST and LT CD8 T cells and stable expression of IFN-g and TNF-a within the first 3 weeks. The CD8+CD45RO+CCR7+ T cell expansion from DLBCL patients was weaker in comparison to healthy donors. The extent of cell death and up-regulation of TIRs after re-stimulation was comparable between ST and LT T cells, whereas cytokine expression varied individually. Conclusion: Our data suggest that it is feasible to expand CD8+ and CD4+ murine and human T cells up to a month, thereby increasing numbers of T cells with Tcm/Tscm properties and with sustained function for murine and human T cells from healthy donors, whereas there seems to be a high individual variance for DLBCL patients, which warrants further investigation in larger patient cohorts. Disclosures Bullinger: Bayer: Other: Financing of scientific research; Abbvie: Honoraria; Seattle Genetics: Honoraria; Sanofi: Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Astellas: Honoraria; Amgen: Honoraria.

PD-1 Expression Is Markedly Upregulated on Intratumoral CD4+ and CD8+ T Cells in Follicular Lymphoma and Is Associated with T-Cell Exhaustion.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2749-2749 ◽  
Author(s):  
Durgha Nattamai ◽  
Sattva S. Neelapu
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Follicular Lymphoma ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cell Function ◽  
Adoptive T Cell Therapy ◽  
T Cell Therapy ◽  
T Cell Function

Abstract Follicular lymphoma is one of the most immune-responsive of all human malignancies. However, immunoregulatory mechanisms in the tumor microenvironment may impair the efficacy of immunotherapies such as vaccines and adoptive T-cell therapy. The inhibitory receptor programmed death 1 (PD1), a negative regulator of activated T cells was recently shown to be upregulated on the surface of HIV-specific CD4+ and CD8+ T cells in humans and was associated with impaired T-cell function. Blockade of the immunoregulatory PD-1/PD-ligand 1 (PD-L1) pathway with antibodies against the PD-L1 augmented the function of HIV-specific CD4+ and CD8+ T cells (Day CL et al, Nature, 2006). To investigate the role of PD-1 in lymphoma, we examined PD-1 expression on peripheral blood mononuclear cells (PBMC) and intratumoral T cells in patients with follicular lymphoma prior to therapy. We observed that PD-1 expression is significantly upregulated on peripheral blood and intratumoral CD4+ and CD8+ T cells in patients with follicular lymphoma as compared with normal donor PBMC. Furthermore, PD-1 expression was significantly higher on intratumoral (mean 61%, range 34% to 86%) compared with peripheral blood CD4+ T cells (mean 25%, range 9 to 40%). Likewise, PD-1 expression was significantly higher on intratumoral (mean 44%, range 31% to 69%) compared with peripheral blood CD8+ T cells (mean 16%, range 9 to 31%). PD-1 expression on CD4+ and CD8+ T cells was associated with impaired type 1 cytokine production (IL-2, TNFa, and IFNg) and blockade of the PD-1/PD-ligand pathway with antibodies against PD-1 significantly enhanced T-cell function. These data indicate that the immunoregulatory PD-1/PD-ligand pathway is operative in patients with follicular lymphoma. Moreover, this pathway of reversible T-cell impairment provides a potential target for enhancing the function of exhausted T-cells in follicular lymphoma in combination with other immunomodulatory strategies such as vaccines and adoptive T-cell therapy.

scholarly journals A modular and controllable T cell therapy platform for acute myeloid leukemia

Leukemia ◽  
2021 ◽  
Author(s):  
Mohamed-Reda Benmebarek ◽  
Bruno L. Cadilha ◽  
Monika Herrmann ◽  
Stefanie Lesch ◽  
Saskia Schmitt ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Cell Therapy ◽  
Myeloid Leukemia ◽  
Tumor Antigens ◽  
Adoptive T Cell Therapy ◽  
Single Chain ◽  
T Cell Therapy ◽  
Acute Myeloid

AbstractTargeted T cell therapy is highly effective in disease settings where tumor antigens are uniformly expressed on malignant cells and where off-tumor on-target-associated toxicity is manageable. Although acute myeloid leukemia (AML) has in principle been shown to be a T cell-sensitive disease by the graft-versus-leukemia activity of allogeneic stem cell transplantation, T cell therapy has so far failed in this setting. This is largely due to the lack of target structures both sufficiently selective and uniformly expressed on AML, causing unacceptable myeloid cell toxicity. To address this, we developed a modular and controllable MHC-unrestricted adoptive T cell therapy platform tailored to AML. This platform combines synthetic agonistic receptor (SAR) -transduced T cells with AML-targeting tandem single chain variable fragment (scFv) constructs. Construct exchange allows SAR T cells to be redirected toward alternative targets, a process enabled by the short half-life and controllability of these antibody fragments. Combining SAR-transduced T cells with the scFv constructs resulted in selective killing of CD33+ and CD123+ AML cell lines, as well as of patient-derived AML blasts. Durable responses and persistence of SAR-transduced T cells could also be demonstrated in AML xenograft models. Together these results warrant further translation of this novel platform for AML treatment.

scholarly journals Engineering adoptive T cell therapy to co-opt Fas ligand-mediated death signaling in ovarian cancer enhances therapeutic efficacy

2021 ◽  
Author(s):  
Kristin G. Anderson ◽  
Shannon K. Oda ◽  
Breanna M. Bates ◽  
Madison G. Burnett ◽  
Magdalia Rodgers Suarez ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Cell Therapy ◽  
Tumor Vasculature ◽  
Adoptive T Cell Therapy ◽  
T Cell Therapy ◽  
Tumor Bearing ◽  
Infiltrating Lymphocytes

Background: In the U.S., more than 50% of ovarian cancer patients die within 5 years of diagnosis, highlighting the need for innovations such as engineered T cell therapies. Mesothelin (Msln) is an attractive immunotherapy target for this cancer, as it is overexpressed by the tumor and contributes to malignant and invasive phenotypes, making antigen loss disadvantageous to the tumor. We previously showed that adoptively transferred T cells engineered to be Msln-specific (TCR1045) preferentially accumulate within established ovarian tumors, delay tumor growth and significantly prolong survival in the ID8VEGF mouse model. However, T cell persistence and anti-tumor activity were not sustained, and we and others have previously detected FasL in the tumor vasculature and the tumor microenvironment (TME) of human and murine ovarian cancers, which can induce apoptosis in infiltrating lymphocytes expressing Fas receptor (Fas). Methods: To concurrently overcome this mechanism for potential immune evasion and enhance T cell responses, we generated an immunomodulatory fusion protein (IFP) containing the Fas extracellular binding domain fused to a 4-1BB co-stimulatory domain, rather than the natural death domain. T cells engineered to express TCR1045 alone or in combination with the IFP were transferred into ID8VEGF-tumor bearing mice and evaluated for persistence, proliferation, anti-tumor cytokine production, and therapeutic efficacy. Results: Relative to T cells modified only to express TCR1045, T cells engineered to express both TCR1045 and a Fas IFP preferentially persisted in the TME of tumor-bearing mice due to improved T cell proliferation and survival. Moreover, adoptive immunotherapy with IFP+ T cells significantly prolonged survival in tumor-bearing mice, relative to TCR1045 T cells lacking the IFP. Conclusions: Fas/FasL signaling can mediate T cell death in the ovarian cancer microenvironment, as well as induce activation-induced cell death, an apoptotic mechanism responsible for regulating T cell expansion. Upregulation of FasL by tumor cells and tumor vasculature represents a mechanism for protecting growing tumors from attack by tumor-infiltrating lymphocytes. As many solid tumors overexpress FasL, an IFP that converts the Fas-mediated death signal into pro-survival and proliferative signals may provide an opportunity to enhance engineered adoptive T cell therapy against many malignancies.

An Update on Adoptive T-Cell Therapy and Neoantigen Vaccines

2019 ◽  
pp. e70-e78 ◽  
Author(s):  
Patrick A. Ott ◽  
Gianpietro Dotti ◽  
Cassian Yee ◽  
Stephanie L. Goff
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Adoptive Cell Therapy ◽  
Adoptive T Cell Therapy ◽  
Clinical Activity ◽  
Single Chain ◽  
T Cell Therapy ◽  
Substantial Impact ◽  
Mhc Molecules

Adoptive T-cell therapy using tumor-infiltrating lymphocytes (TILs) has demonstrated long-lasting antitumor activity in select patients with advanced melanoma. Cancer vaccines have been used for many decades and have shown some promise but overall relatively modest clinical activity across cancers. Technological advances in genome sequencing capabilities and T-cell engineering have had substantial impact on both adoptive cell therapy and the cancer vaccine field. The ability to identify neoantigens—a class of tumor antigens that is truly tumor specific and encoded by tumor mutations through rapid and relatively inexpensive next-generation sequencing—has already demonstrated the critical importance of these antigens as targets of antitumor-specific T-cell responses in the context of immune checkpoint blockade and other immunotherapies. Therapeutically targeting these antigens with either adoptive T-cell therapy or vaccine approaches has demonstrated early promise in the clinic in patients with advanced solid tumors. Chimeric antigen receptor (CAR) T cells, which are engineered by fusing an antigen-specific, single-chain antibody (scFv) with signaling molecules of the T-cell receptor (TCR)/CD3 complex creating an antibody-like structure on T cells that recognizes antigens independently of major histocompatibility complex (MHC) molecules, have demonstrated remarkable clinical activity in patients with advanced B-cell malignancies, leading to several approvals by the U.S. Food and Drug Administration (FDA).

Adoptive T Cell Therapy of Human CMV Infection in Immunocompromised Hosts: In Vitro Generation of CMV-Specific T Cells from Naïve Precursors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 206-206 ◽  
Author(s):  
Sonja Schmucker ◽  
Mario Assenmacher ◽  
Jurgen Schmitz ◽  
Anne Richter
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Peptide Pool ◽  
Adoptive T Cell Therapy ◽  
T Cell Therapy ◽  
A Cell ◽  
Ifn Γ

Abstract Adoptive transfer of virus-specific T cells is a promising therapy for the treatment of infections in immunocompromised patients. Virus-specific T cells can readily be obtained from antigen-experienced, but not naïve donors. In this study we describe a cell culture system for the in vitro generation of CMV-specific T cells from naive T cells derived from CMV-seronegative donors. We isolated naïve T cells by magnetic depletion of non-T cells, CD25+ regulatory T cells, and CD45RO+ effector and memory T cells from peripheral blood mononuclear cells (PBMC) of CMV-seronegative donors. These naïve T cells were co-cultured with autologous mature monocyte-derived DC (MoDC) loaded with a pool of overlapping peptides from the CMV protein pp65. CD3-depleted autologous PBMC were used as feeder cells and CD28 antibody, IL-2, IL-7, and IL-15 were added to the culture. Already only 9–13 days after starting the priming culture, frequencies of 0.0024% and 0.009% pp65495–503/A2-tetramer+ cells among CD8+ T cells were found for 2 HLA-A2+ blood donors. In contrast pp65495–503/A2-tetramer+ T cells were not detectable when naive T cells were cultured with unpulsed MoDC. Tetramers are suitable tools for the identification of antigen-specific T cells but are restricted to single epitopes of mainly CD8+ T cells. To analyze primed CD4+ T cells as well as CD8+ T cells having specificities other than for the peptide pp65495–503, we looked for upregulation of the activation marker CD137 after a second stimulation and found increased frequencies of CD137+ CD4+ T cells as well as CD137+ CD8+ T cells in the pp65-primed cell cultures only when restimulated with the peptide pool of pp65. Because IFN-γ is important for the control of CMV infection, we studied the capability of the in vitro primed pp65-specific CD4+ and CD8+ T cells to produce this cytokine. Restimulation of the T cells with pp65 peptide pool induced IFN-γ secretion in up to 3.9% of the CD8+ T cells and up to 3.8% of the CD4+ T cells in each of six donors tested. No specific IFN-γ production was detected after restimulation with an irrelevant IE-1 peptide pool. As expected the frequency of pp65-specific T cells in the priming cultures is low. For generation of T cell lines, we magnetically enrich pp65- specific T cells according to their IFN-γ secretion using the cytokine secretion assay technology. After further cultivation for 2 weeks the antigen-specificity of the expanded T cells was again evaluated. Only if restimulated with the pp65 peptide pool 56.6% of the CD4+ T cells showed upregulated expression of the activation marker CD154 (CD40L). Cytokine analysis of the cells revealed IFN-γ production in 40.2% of the CD4+ T cells, of which 36% co-expressed IL-2, indicating the functionality of the in vitro primed and expanded T cells. In conclusion, we established a cell culture system for in vitro priming of CMV-specific CD4+ and CD8+ T cells derived from peripheral blood of donors not infected by CMV. This should extend the application of adoptive T cell therapy to patients for whom immune donors are not available.

CD4+Foxp3+ Regulatory T Cells Persist in High Numbers in Peripheral Blood after Induction of Clinical Remission with Standard Chemotherapy and Suppress Tumor-Specific Effector T Cells in Patients with Follicular Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3778-3778
Author(s):  
Myriam Foglietta ◽  
David Delgado ◽  
Durgha Nattamai ◽  
Larry W Kwak ◽  
Sattva Neelapu
Keyword(s):  
T Cells ◽  
Cell Therapy ◽  
Peripheral Blood ◽  
Clinical Remission ◽  
Chemotherapy Regimen ◽  
Effector T Cells ◽  
Adoptive T Cell Therapy ◽  
Therapeutic Vaccines ◽  
T Cell Therapy ◽  
Specific Effector

Abstract Follicular lymphoma (FL) is one of the most immune responsive of all human malignancies. However, immunoregulatory mechanisms in the tumor microenvironment may impair the efficacy of endogenous immunity as well as therapeutic vaccines and adoptive T-cell therapy. CD4+CD25+CD127loFoxp3+ regulatory T cells (Tregs) are one of the most potent suppressors of effector T cells and were recently shown to be increased in peripheral blood and tumors of patients with various malignancies. Here, we determined the percentage of Tregs, in the tumors and the peripheral blood mononuclear cells (PBMC) of patients with FL (n = 13) at the time of initial diagnosis and after induction of clinical remission with a uniform cyclophosphamide and doxorubicin containing chemotherapy regimen, PACE (Modified ProMACE without Methotrexate; Prednisone, Doxorubicin, Cyclophosphamide, and Etoposide). We observed that Tregs are markedly increased in number both in the tumors (median 19.45%, range 13.4%–44.5%) and PBMC (median 17.7%, range 7.9%–64.5%) of patients with FL at the time of initial diagnosis as compared with PBMC from normal donors (median 3.33%, range 1.88%–6.3%). Unexpectedly, Tregs persisted in high numbers in peripheral blood after induction of clinical remission with a cyclophosphamide and doxorubicin containing chemotherapy regimen (median 19.95%, range 7.6%–67.2%). To determine the immunosuppressive effects of Tregs, we assessed the proliferation of CFSE-labeled effector T cells following polyclonal stimulation with anti-CD3 and anti-CD28 antibodies. The presence of high numbers of Tregs was associated with significantly decreased proliferation of peripheral blood CD4+ T cells in patients with FL both at baseline (median 24.9%, range 3.7%–40.7%) and after chemotherapy induced clinical remission (median 15.8%, range 1.5%–23.5%) as compared with normal donors (median 38%, range 11.7%–58.8%). A similar decrease in proliferation was also noted in CD8+ T cells in the presence of high numbers of Tregs both at baseline and after induction of clinical remission in patients with FL as compared with normal donors. More importantly, intratumoral and peripheral blood Tregs significantly inhibited effector T cells proliferating in response to autologous tumor cell stimulation. In conclusion, these results suggest that CD4+CD25+CD127loFoxp3+ Tregs that inhibit the function of tumor-specific effector T cells are markedly increased in number in both tumor and peripheral blood of patients with FL and persist in high numbers despite induction of clinical remission with standard cyclophosphamide and doxorubicin containing chemotherapy. This observation may potentially explain the failure of two recent Phase III therapeutic vaccine trials in patients with FL. Furthermore, these results suggest that a state of peripheral tolerance continues to be present after induction of clinical remission and strategies that deplete or block function of Tregs may be necessary to enhance the efficacy of therapeutic vaccines and adoptive T-cell therapy in patients with follicular and possibly other non-Hodgkin’s lymphomas.

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