Tumor Specific T Cells Modified to Secrete IL-12 Eradicate Systemic Tumors in the Absence of Prior Toxic Chemotherapy Conditioning Regimens

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3120-3120
Author(s):  
Hollie J. Pegram ◽  
James Lee ◽  
Erik Hayman ◽  
Gavin H Imperato ◽  
Thomas J. Tedder ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Cell Activity ◽  
Adoptive T Cell Therapy ◽  
T Cell Therapy ◽  
Tumor Bearing ◽  
Conditioning Regimens ◽  
Ifn Γ

Abstract Abstract 3120 T cells may be genetically modified to target tumor-associated antigens through the retroviral transduction of genes encoding chimeric antigen receptors (CARs). We have previously generated a series of CARs specific to the CD19 antigen expressed on most B cell tumors. In xenotransplant models of disease we have shown that human T cells expressing CD19 targeted CARs successfully eradicate established B cell tumors in immune compromised SCID-Beige mice. To further our understanding of the in vivo biology of CAR modified T cells, we generated a more clinically relevant syngeneic, immune competent tumor model utilizing CD19 knock out (mCD19−/−) human CD19 knock in (hCD19+/−) C57BL6 transgenic mice (C57BL6(mCD19−/− hCD19+/−)) bearing systemic syngeneic EL4(hCD19+) thymoma tumors. Treatment of tumor bearing mice with syngeneic T cells modified to express the CD19 targeted 19m ζ CAR alone failed to either eradicate tumor or induce predicted B cell aplasias. However, prior lymphodepletion with cyclophosphamide followed by infusion of 19m ζ+ T cells successfully eradicated tumor in 83% of treated mice and induced long term B cell aplasias. Translation of this therapy to the clinic has also revealed that optimal responses require pre-conditioning regimens. Given the toxicity of pre-conditioning treatments, these therapies are largely restricted to younger, healthier patients able to tolerate such intensive regimens. To further understand the mechanisms of action of improved therapy following prior cyclophosphamide therapy, we demonstrated markedly enhanced serum levels of the IL-12 and IFN γ cytokines as well as a marked reduction of endogenous CD4+ regulatory T cells. We postulated that IL-12, which induces IFN γ secretion, may in part explain the enhanced anti-tumor efficacy following prior lymphodepletion, and may potentially obviate the need for toxic conditioning pre-treatments. To address this hypothesis, we modified CAR+ T cells to constitutively secrete IL-12. Subsequent adoptive therapy of EL4(hCD19+) tumor bearing C57BL6(mCD19−/− hCD19+/−) mice with hCD19 targeted IL-12 secreting T cells successfully eradicated disease in 75% of treated mice and induced predicted B cell aplasias in the absence of prior lymphodepletion. Significantly, we found that this enhanced hCD19 targeted T cell activity required the infusion of both CD4+ and CD8+ gene modified T cells, and was further dependent upon autocrine IL-12 stimulation of the modified tumor targeted T cells as well as modified T cell IFN γ secretion and stimulation. To our knowledge, this is the first time adoptive T cell transfer has been demonstrated to successfully eradicate tumor in the absence of prior conditioning regimens. Therefore, these data support the rapid translation of this adoptive T cell therapy to the clinic, as it may enhance the anti-tumor efficacy of this therapy and further expand the patient population eligible for adoptive T cells therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Enabling T Cell Recruitment to Tumours as a Strategy for Improving Adoptive T Cell Therapy

2017 ◽  
Vol 13 (01) ◽  
pp. 66 ◽  
Author(s):  
Bruno Cadilha ◽  
Klara Dorman ◽  
Felicitas Rataj ◽  
Stefan Endres ◽  
Sebastian Kobold ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
B Cell ◽  
Therapeutic Potential ◽  
Cell Activity ◽  
Cell Lineage ◽  
Solid Tumours ◽  
Adoptive T Cell Therapy ◽  
T Cell Therapy

Immunotherapy has successfully been implemented as the standard of care in a number of oncologic indications. A hallmark of cancer immunotherapy is the successful activation of T cells against cancer cells, leading to unparalleled efficacy for some tumour entities. However, current approved approaches are not specific, limiting both their activity and their safety. A more tailored way of using the therapeutic potential of T cells is adoptive T cell therapy, which encompasses ex vivo T cell manipulation and reinfusion to patients suffering from cancer. In haematologic malignancies such as acute lymphatic leukaemia of the B cell lineage, T cells modified with a chimeric antigen receptor against the B cell lineage antigen CD19 induce remissions in a high proportion of patients. In contrast, patients suffering from advanced solid tumours have shown little benefit from cell-based approaches. This is partly due to limited access of T cells to the tumour tissue, consequently restricting T cell activity. In this review, we focus on the limitations of T cell trafficking towards solid tumours. We summarise the existing knowledge on lymphocyte migration to understand how this pathway may be used to open therapeutic approaches for a broader range of indications. We also review new strategies targeting the tumour site that aid naturally occurring or gene-engineered T cells to migrate to solid tumours. Finally, we discuss how guiding T cells towards the tumour might contribute in harnessing their full cytolytic potential.

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.

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.

scholarly journals The Effect of Antigen Dose and Antigen Presenting Process on T Cell Stimulation: A Method for Enrichment of TB10.4 Antigen-specific T-cell Clones

2021 ◽  
Author(s):  
Mahdieh Motiee ◽  
Ahmad Zavaran Hosseini ◽  
Sara Soudi ◽  
Seyed Mehdi Hassanzadeh
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Immune Responses ◽  
Interleukin 2 ◽  
Adoptive Cell Transfer ◽  
Adoptive T Cell Therapy ◽  
T Cell Therapy ◽  
Average Cell ◽  
Ifn Γ

T-lymphocytes have critical functions in the immune responses against viral and intracellular bacterial infections as well as cancers. Antigen (Ag)-specific T-lymphocyte clones enriched and expanded in vitro are valuable tools in the study of immune responses in animal models and adoptive T-cell therapy of patients with cancer or infection. We described a method for inducing, enriching, and replicating Ag-specific poly-clonal T-cells from BALB/c mice infected with live Bacillus Calmette Guérin (BCG) bacterium. During a 7-8 days procedure, T-lymphocytes were purified from immune cells of lymph nodes stimulated with immunodominant Ag of BCG, TB10.4, and expanded by interleukin -2 cytokine. We evaluated the effect of Ag doses (1, 10, and 100 µg/mL) and exposure method of Ag presenting cells (APCs) to T-cells, on T-cells’ proliferation, viability, and Interferon-gamma (IFN-γ) secretion at 2, 5, and 7 days after Ag stimulation. Increasing Ag concentration increased the average cell division, but at the highest dose of Ag (100 µg/mL), T-cell viability is decreased. Only clones induced by 10 µg/mL Ag produced a desirable amount of IFN-γ. Incubation of Ag and APCs, 24 h before T-lymphocytes addition, increased the proliferation and viability of cells. T cells are in a more favorable condition around day 5 of Ag stimulation in terms of proliferation and survival, and it is the desired time for T cell restimulation. For optimal preparation of specific T-cells for adoptive cell transfer, optimization of Ag dose, the order of APCs and T-cells exposure with Ag, and the duration of initial Ag stimulation, as well as the time for restimulation, is essential.

Stromal Cross-Presentation and Host Interferon-γ Signaling and Are Required for Elimination of Antigen-Loss Variants of High-Grade B Cell Lymphomas: Implications for Adoptive T Cell Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3927-3927
Author(s):  
Armin Gerbitz ◽  
Madhusudhanan Sukumar ◽  
Florian Helm ◽  
Andrea Wilke ◽  
Christian Friese ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Antigen Expression ◽  
High Grade ◽  
B Cell Lymphomas ◽  
Lymphoma Cells ◽  
T Cell Therapy ◽  
Ifn Γ ◽  
Antigen Loss

Abstract Abstract 3927 The incidence of high-grade B cell lymphomas has been increasing over the last decades in western countries for unclear reasons. Relapse after conventional chemotherapy especially in high-grade B cell lymphomas remains a very difficult clinical issue. Contrary to CML and AML, the benefit of allogeneic SCT for treatment of high-grade lymphomas is not well established. Several studies suggested a potential graft versus lymphoma (GvL) effect for acute lymphoblastic leukemia (ALL) and several types of non-Hodgkin lymphomas. To study mechanisms involved in T cell-mediated rejection of B cell lymphomas, we have developed a murine lymphoma model in which three antigens, human c-MYC protein, chicken ovalbumin (OVA) and GFP, serve as foreign antigens for rejection. Lymphomas expressing all three antigens were rejected in 60 to 70% of animals after transfer into wild type mice, whereas lymphomas expressing only human c-MYC protein were not rejected. Outgrowing OVA-expressing lymphomas were infiltrated by T cells, showed MHC class I and II upregulation and loss of antigen expression, indicating immune escape. In contrast to wild type recipients of OVA-expressing lymphomas, 80 to 100% of recipient STAT1-, IFN-γ-, or IFN-γ receptor-deficient mice died due to lymphoma growth. Remarkably, lymphomas arising in IFN-γ- and IFN-γ-receptor-deficient mice also invariably showed lost antigen expression. Thus, poor overall survival of IFN-γ- and IFN-γ-receptor-deficient recipient mice is not due to a lack of antigen-specific T cell killing but due to inefficient eradication of antigen-negative variants of the lymphoma. In order to address the role of the stroma in eradication of lymphoma cells we made use of B6bm1 animals that do not present the immunodominant OVA derived peptide SIINFEKL in the context of MHC class I. Since the wildtype MHC represents an allo-antigen in B6bm1 mice, B6bm1 and B6 wildtype control recipients were T-cell depleted by 30H12 anti CD90.2 antibody prior to transfer of lymphoma cells. Anti OVA immunity was restored by adoptive transfer of 1 Mio. primed CD90.1+ OT-I-T-cells one day after lymphoma transfer. T-cell depletion was continued for 28 days biweekly. Lymphoma growth was faster in bm1 recipients and disease free survival significantly reduced (A). In addition, T-cell expansion was significantly reduced (B) in bm1 recipients as analyzed by pentamer staining of OT-I-T-cells in peripheral blood (day 21 0.84%±0.2 vs. 3.53%±0.2 of lymphocytes, p=0.001) indicating an important role of stromal crosspresentation for the rejection of lymphoma cells. Our data show that mechanisms established for solid tumors hold true also for hematologic neoplasias such as B cell lymphomas. Antigen-dependent eradication of tumor antigen-loss variants makes antigen-specific T cell therapy particularly attractive as a novel therapeutic treatment option. Disclosures: No relevant conflicts of interest to declare.

The Exhausted Intratumoral T Cell Population in B-Cell Non-Hodgkin Lymphoma Is Defined By LAG-3, PD-1 andtim-3 Expression

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2661-2661 ◽  
Author(s):  
Zhi-Zhang Yang ◽  
Tammy Price-troska ◽  
Anne J Novak ◽  
Stephen M Ansell
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Conflicts Of Interest ◽  
Early Time ◽  
Effector Memory ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Ifn Γ

Abstract T-cell exhaustion plays an important role in attenuating the function of immune cells in B-cell non-Hodgkin's lymphoma (NHL) and PD-1 expression is typically used to identify exhausted T-cells. We have however previously shown that not all PD-1+ cells are exhausted and that PD-1 is differentially expressed on two distinct T-cell subpopulations, with high expression on T follicular helper cells and dim expression on exhausted T cells. Other markers are therefore needed to more clearly identify exhausted intratumoral T cells. To further define exhaustion of intratumoral T cells, we determined the co-expression, regulation and function of PD-1, TIM-3 and LAG-3 on CD4+ or CD8+ T cells by flow cytometry. Using biopsy specimens from follicular B-cell NHL, we found that the percentages of PD-1+ and TIM-3+ T cells were 53.1% (range: 17.2-81.2%, n=32) and 34.5% (range: 14.9-62.6%, n=34) in CD4+ T cells and 46.8% (range: 12.8-81.7%, n=32) and 40.4% (range: 15.0-78.4%, n=34) in CD8+ T cells, respectively. We observed that TIM-3 was predominantly expressed on PD-1dim T cells and TIM-3+ cells accounted for 40% of CD4+ PD-1dim or 45% of CD8+ PD-1dim T cells. Similarly, LAG-3 was variably expressed on intratumoral T cells from B-cell NHL. A median of 9.54% (range: 3.01-15.46, n=6) of CD4+ or 20.48% (7.93-33.9, n=8) of CD8+ T cells express LAG-3. We found that LAG-3+ T cells almost exclusively came from PD-1+ TIM-3+ cells, forming a defined population of intratumoral PD-1+ TIM-3+ LAG-3+ CD4+ or CD8+ T cells. While the majority of LAG-3+ T cells were effector memory T cells (CD45RA- CCR7-), some LAG-3-expressing T cells displayed a phenotype of terminally-differentiated T cells (CD45RA+ CCR7-). Functionally, the intratumoral TIM-3+ LAG-3+ T cells exhibited reduced capacity to produce cytokines (IL-2, IFN-γ) and granules (perforin, granzyme B). Similar to TIM-3, LAG-3 expression was strongly up-regulated on CD4+ or CD8+ T cells by IL-12, a cytokine that has been shown to induce T-cell exhaustion. Interestingly, we observed that while expression of TIM-3 on CD8+ T cells was upregulated by IL-12 at an early time point (day 1), LAG-3 was only induced after TIM-3 up-regulation (day 3) and almost exclusively on TIM-3+ T cells. Furthermore, we found that blockade of both TIM-3 and LAG-3 signaling was able to reverse the exhausted phenotype of CD8+ T cells resulting in increased IFN-γ and IL-2 production. This effect was further enhanced when CD8+ T cells were treated with both anti-TIM-3 and anti-LAG-3 Abs. Taken together, these results suggest that PD-1, TIM-3 and LAG-3 were involved in the induction of exhaustion of T cells in B-cell NHL. We find that PD-1, TIM-3 and LAG-3 are expressed on the same T cells and that blocking TIM-3 and LAG-3 can reverse T-cell exhaustion signaling. These results suggest that PD-1, TIM-3 and LAG-3 play a synergistic role in the development of T cell exhaustion in NHL. Disclosures No relevant conflicts of interest to declare.

Impact of the Conditioning Chemotherapy On Outcomes in Adoptive T Cell Therapy: Results From a Phase I Clinical Trial of Autologous CD19-Targeted T Cells for Patients with Relapsed CLL

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1797-1797 ◽  
Author(s):  
Jae H. Park ◽  
Isabelle Rivière ◽  
Xiuyan Wang ◽  
Jolanta Stefanski ◽  
Qing He ◽  
...  
Keyword(s):  
Clinical Trial ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Phase I ◽  
B Cell ◽  
Tumor Burden ◽  
Adoptive T Cell Therapy ◽  
T Cell Therapy ◽  
Conditioning Therapy

Abstract Abstract 1797 Patient T cells may be genetically modified to express chimeric antigen receptors (CARs) targeted to antigens expressed on tumor cells. We have previously reported initial results from a phase I clinical trial treating patients with chemotherapy refractory chronic lymphocytic leukemia (CLL) with autologous T cells modified to express the 19–28z CAR targeted to the CD19 antigen expressed on most B cell malignancies (Brentjens RJ, Rivière I et al., Blood, 2011;118(18):4817-28). In the previous reported cohorts of 8 patients, CAR-modified T cells were infused in the setting of rapidly progressive and chemotherapy refractory disease. Although prior conditioning therapy with cyclophosphamide enhanced in vivo persistence of the modified T cells, all patients had cyclophosphamide-resistant disease and none experienced objective remissions or significant hematologic recovery. We hypothesized that suboptimal clinical response observed in the study was because of a large tumor burden at the time of T cell infusion and refractoriness to conditioning therapy. On the basis of these findings, we have modified the protocol to allow prior cytoreductive therapy and conditioning with chemotherapeutic agents based on predicted chemosensitivity. Since these protocol modifications, two patients have been treated. Both had relapsed disease with unfavorable disease phenotype following previous treatments with various chemotherapy and biologic regimens. Of the two patients treated to date, one achieved partial remission (PR) and the other attained minimal residual disease (MRD)-negative complete remission (CR) according to standard international criteria. The first patient experienced reduction in peripheral lymphocytosis and obtained stable disease with persistent anemia and thrombocytopenia after two cycles of bendamustine and rituximab (BR). Following the bendamustine conditioning and modified T cell infusion, PR was achieved with complete hematologic recovery, lasting for more than 8 months at the time of this report. The second patient achieved PR following two cycles of BR and subsequently attained MRD-negative CR with concomitant development of B cell aplasia after receiving the bendamustine conditioning and CAR-modified T cells. At the time of this report, the response has been sustained for more than 5 months. Notably, this patient has long-term persistence of the CAR-modified T cells, detected at 12 weeks following the T cell infusion. No significant toxicities were observed in the two patients, except for fevers lasting 3–4 days and transient grade 2 hypoxia. While the number of treated patients on the revised protocol is too small to draw a definitive conclusion, our findings of a significant improvement in the degree and depth of response with the bendamustine conditioning compared to our previous cohorts of cyclophosphamide-refractory CLL who received cyclophosphamide as their conditioning suggest a potentially greater effect of conditioning regimens through tumor burden reduction than the induction of a supportive cytokine response or lymphocyte depletion. In light of these initial observations, the role of the conditioning chemotherapy regimen given prior to adoptive T cell therapy needs to be carefully evaluated since not all regimens may ultimately be equally effective with respect to clinical outcomes. Disclosures: Lamanna: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

831 E3 ubiquitin ligase Cbl-b deficient CD8+ T cells overcome Treg cell-mediated suppression through IFN-γ and induce robust anti-tumor immunity

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A883-A883
Author(s):  
SeongJun Han ◽  
Zhe Qi Liu ◽  
Douglas Chung ◽  
Michael St Paul ◽  
Carlos Garcia-Batres ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Treg Cell ◽  
Ubiquitin Ligase ◽  
Cd8 T Cells ◽  
Treg Cells ◽  
Adoptive T Cell Therapy ◽  
T Cell Therapy ◽  
Ifn Γ

BackgroundAdoptive T cell therapy (ACT) is reaching its potential in multiple malignancies. However, anti-tumor T cell responses can be attenuated by suppressive cells in the tumor microenvironment, such as CD4+FoxP3+ regulatory T (Treg) cells. Depletion of Treg cells can be technically challenging in ACT and may be associated with unwanted adverse effects. Alternatively, studies suggest that specific modifications in T cell signaling network may render T cells resistant to regulation by Treg cells. Here, we investigated the role of Casitas B- Lineage Lymphoma-b (Cbl-b), an E3 ubiquitin ligase and a negative regulator of TCR signaling pathways, in rendering CD8+ T cells resistant to the effects of Treg cells to bolster ACT.MethodsIn vitro stimulated Cbl-b+/+ or Cbl-b-/- Thy1.1+ P14 TCR-transgenic CD8+ T cells were adoptively transferred into B16-gp33 melanoma-bearing Thy1.2+ FoxP3-GFP/DTR transgenic mice treated with or without diphtheria toxin (n = 15). Tumor size and overall survival were measured. Congenically labelled T cells from tumor, draining lymph node, and spleen were comprehensively profiled using flow cytometry. To further examine the biological mechanism of Treg resistance, we performed in vitro Treg suppression assays and RNA-sequencing.ResultsAdoptively transferred tumor-specific Cbl-b-/- effector CD8+ T cells mediated superior control over tumor growth and increased overall survival in comparison to the wild-type counterpart. Depletion of FoxP3+ cells increased the quantity and percentage of CD25+ 4-1BB+ expressing P14 Thy1.1+ CD8+ T cells in the tumor, whereas the effect of FoxP3+ cell depletion was negligible with Cbl-b deficient CD8+ T cells. Cbl-b deficiency also attenuated sensitivity to Treg cell-mediated suppression in vitro. Transcriptomic analyses suggested that Cbl-b regulates pathways associated with cytokine production and cellular proliferation. Specifically, hyper-secretion of IFN-γ by Cbl-b deficient CD8+ T cells attenuated suppression by Treg cells. In murine models of adoptive T cell therapy, Cbl-b deficient CD8+ T cells were less susceptible to suppression by Treg cells in the tumor through the effects of IFN-γ.ConclusionsWe demonstrate that adoptively transferred effector CD8+ T cells are susceptible to regulation by Treg cells in the tumor, and that ablation of Cbl-b abrogates Treg cell-mediated suppression. We highlight the therapeutic implications of targeting Cbl-b in the context of ACT.AcknowledgementsWe would like to thank Dr. Tak Mak and Dr. Naoto Hirano for their suggestions and insights for this project.

scholarly journals Inducible gene switches with memory in human T cells for cellular immunotherapy

2018 ◽  
Author(s):  
Deboki Chakravarti ◽  
Leidy D Caraballo ◽  
Benjamin H. Weinberg ◽  
Wilson W. Wong
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Activity ◽  
Adoptive T Cell Therapy ◽  
Cellular Immunotherapy ◽  
Antigen Receptors ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Gene Switches ◽  
Engineered T Cells

AbstractCell-based therapies that employ engineered T cells—including the expression of chimeric antigen receptors (CARs)—to target cancer cells have demonstrated promising responses in clinical trials. However, engineered T cell responses must be regulated to prevent severe side effects such as cytokine storms and off-target responses. Here we present a class of recombinase-based gene circuits that will enable inducible switching between two states of adoptive T cell therapy using an FDA-approved drug, creating a generalizable platform that can be used to control when and how strongly a gene is expressed. These circuits exhibit memory such that induced T cells will maintain any changes made even when the drug inducer is removed. This memory feature avoids prolonged drug inducer exposure, thus reducing the complexity and potential side effect associated with the drug inducer. We have utilized these circuits to control the expression of an anti-Her2-CAR, demonstrating the ability of these circuits to regulate CAR expression and T cell activity. We envision this platform can be extended to regulate other genes in T cell behavior for various adoptive T cell therapies.

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.

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