CD28 Costimulation of Effector T Cells During An Anti-Tumor Attack Sustains Its Repression by Regulatory T Cells Which Can Be Overcome by Preventing Lck Activation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3569-3569
Author(s):  
David M Kofler ◽  
Markus Chmielewski ◽  
Tobias Riet ◽  
Andreas Hombach ◽  
Michael Hallek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Chimeric Antigen Receptor ◽  
Cell Activation ◽  
Antigen Receptor ◽  
Treg Cells ◽  
Effector T Cells ◽  
Cd28 Costimulation ◽  
Signaling Domain

Abstract Abstract 3569 Poster Board III-506 Background Massive infiltrations of the tumor tissue with CD4+CD25highFoxP3+ regulatory T (Treg) cells render a productive T cell anti-tumor response ineffective resulting in unrestricted tumor growth despite the presence of tumor-specific, cytolytic T cells. Methods Using a human/CD1-deficient mouse model we investigated the impact of human regulatory T cells on redirected effector T cells expressing a tumor-specific chimeric antigen receptor. The chimeric antigen receptor consists of an antibody derived binding domain for antigens in the extracellular part and of the TCR/CD3zeta or the combined CD3zeta-CD28 signaling domain in the intracellular part. Upon antigen binding the chimeric antigen receptor transmits an activation signal via the CD3zeta or CD3zeta-CD28 domain to drive T cell activation, resulting in cytokine secretion, T cell proliferation, and cytolytic activity. Results We revealed that effector T cells redirected by a tumor-specific chimeric antigen receptor are more effectively repressed by Treg cells when they are activated through a combined CD3zeta-CD28 signal compared to a CD3zeta signal without CD28 costimulation. Mutations in the CD28 signaling domain of the chimeric antigen receptors resulted in abolished IL-2 secretion by prevention of CD28 mediated lck activation. Abolished IL-2 induction in redirected effector T cells expressing the modified CD3zetaCD28delta antigen receptor increased their in vivo efficacy in an anti-tumor response by reduced sustaining of Treg cell suppression. Conclusions While data indicate the dichotomy of CD28 costimulation in inducing full effector T cell activation and sustaining Treg repression, our findings provide a strategy to improve the efficacy of the T cell anti-tumor attack in the presence of Treg cells for use in adoptive immunotherapy of cancer. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Systemic Xenograft Model of Waldenström’s Macroglobulinemia Demonstrates the Potent Anti-Tumor Effect of Second Generation CD19 Directed Chimeric Antigen Receptor Modified T Cells in This Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4484-4484 ◽  
Author(s):  
Eric L Smith ◽  
Maria Lia Palomba ◽  
Jae H Park ◽  
Renier J. Brentjens
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Activation ◽  
Chimeric Antigen Receptor ◽  
Cell Activation ◽  
Second Generation ◽  
Antigen Receptor ◽  
T Cell Therapy

Abstract Chimeric antigen receptor (CAR) modified T-cell therapy consists of ex-vivo genetic manipulation of autologous lymphocytes in order to establish robust T-cell mediated anti-tumor immunity. Our group was the first to design and evaluate a CAR targeted toward the B cell antigen CD19 in mice. Currently we utilize a second generation CAR comprised of a single-chain variable fragment (scFv) derived from an antibody against CD19 fused to the CD3 ζ chain and the CD28 intracellular signaling domain (19-28z) to provide the necessary signal 1 and signal 2 for enhanced T-cell activation and persistence. We have gone on to test the safety and efficacy of 19-28z CAR T-cells in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). We observed rapid complete molecular remissions in the first 14/16 patients treated with relapsed/refractory ALL. We hypothesize that contributing to the enhanced efficacy seen in ALL, when compared to solid tumors or extra-medullary CLL, is the fact that ALL is a bone marrow predominant disease, which may provide a microenvironment more amenable to T-cell therapy. Waldenström’s Macroglobulinemia (WM) is an ideal disease to test 19-28z CAR-modified T-cell therapy, as it is often bone marrow predominant, and WM cells from patient samples typically uniformly express high levels of CD19. Furthermore, despite recent progress made with novel BCR-directed therapy, complete eradication of the WM clone from the bone marrow niche remains elusive, therefore providing an ideal clinical scenario for treatment consolidation via alternative cytotoxic methods such as cellular immunotherapy. Using the human WM cell line, BCWM.1, we evaluated the in vitro efficacy of 19-28z CAR-modified T-cells when compared to mock transduced T cells or T cells transduced with an irrelevant second generation CAR directed towards the ovarian antigen MUC16. In a 4 hour co-culture assay, we observed significant cytotoxicity, even at low effector:target ratios (52% lysis at 1:1; 92% lysis at 10:1; p<0.01). This corresponded to increased secretion of INFγ and IL-2, markers of T-cell activation (p<0.01). We then conducted in vivo studies using sublethally irradiated SCID/beige mice to generate a systemic model of WM via tail vein injection of 1x106 luciferase transduced BCWM.1 cells. This model is characterized by tumor growth in the bone marrow followed by rapid spread to the liver, lungs, kidney, and CNS. Mice were monitored by weekly bioluminescent imaging (BLI) and ultimately were sacrificed when they developed hind leg paralysis. 19-28z CAR modified T-cells administered at day 7, after tumor establishment, when compared to non-treated and irrelevant CAR-modified T cell controls, delayed the progression of disease and doubled the median survival time of the mice after treatment (p=0.001). Taken together, the pre-clinical efficacy demonstrated in this abstract and the clinical features of WM, listed above, provide the rational for testing 19-28z CAR modified T cells clinically for WM. We have now opened a clinical trial for patients with relapsed or refractory WM, in which chemotherapy preconditioning is followed by a single dose of 19-28z CAR modified autologous T-cells (NCT00466531). Disclosures Brentjens: Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

scholarly journals Repurposing endogenous immune pathways to tailor and control chimeric antigen receptor T cell functionality

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Mohit Sachdeva ◽  
Brian W. Busser ◽  
Sonal Temburni ◽  
Billal Jahangiri ◽  
Anne-Sophie Gautron ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Chimeric Antigen Receptor ◽  
Cell Activation ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T ◽  
Immune Pathways ◽  
And Control

Abstract Endowing chimeric antigen receptor (CAR) T cells with additional potent functionalities holds strong potential for improving their antitumor activity. However, because potency could be deleterious without control, these additional features need to be tightly regulated. Immune pathways offer a wide array of tightly regulated genes that can be repurposed to express potent functionalities in a highly controlled manner. Here, we explore this concept by repurposing TCR, CD25 and PD1, three major players of the T cell activation pathway. We insert the CAR into the TCRα gene (TRACCAR), and IL-12P70 into either IL2Rα or PDCD1 genes. This process results in transient, antigen concentration-dependent IL-12P70 secretion, increases TRACCAR T cell cytotoxicity and extends survival of tumor-bearing mice. This gene network repurposing strategy can be extended to other cellular pathways, thus paving the way for generating smart CAR T cells able to integrate biological inputs and to translate them into therapeutic outputs in a highly regulated manner.

Chimeric antigen receptor (CAR+) modified T cells targeting prostate specific membrane antigen (PSMA) in patients (pts) with castrate metastatic prostate cancer (CMPC).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. TPS3115-TPS3115 ◽  
Author(s):  
Susan F. Slovin ◽  
Xiuyan Wang ◽  
Melanie Hullings ◽  
Gabrielle Arauz ◽  
Shirley Bartido ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Chimeric Antigen Receptor ◽  
Copy Number ◽  
Cell Activation ◽  
Ex Vivo ◽  
Antigen Receptor ◽  
Intermittent Fever

TPS3115 Background: A phase I dose-escalating study to assess safety, dose and targeting efficiency of genetically modified autologous human T cells targeted to PSMA was initiated. Preclinical models demonstrated anti-tumor activity and accumulation, migration, and persistence of these cells to tumor. The autologous PSMA-targeted T cells utilizes the P28z second generation chimeric antigen receptor following iv cyclophosphamide (Cy). For safety, the herpes simplex virus-1 thymidine kinase (hsvtk) gene is co-expressed with the P28z receptor, rendering T cells sensitive to ganciclovir for immediate T cell elimination. The expression of hsvtk enables PET imaging using radiolabeled FIAU to localize these T cells Methods: Autologous T cells are activated from a leukapheresis product using anti-CD3/CD28 Dynabeads. Release criteria include mean vector copy number by Q-PCR and vector identity by Southern blot, absence of Replication Competent Retrovirus and residual Dynabeads. Pts were dosed from 107 to 3 x 107 CAR+ T cells/kg.All 7 pts received 300mg/m2 of Cy one day before infusion. Baseline and post treatment imaging included FDG, FDHT and 18F-FIAU PET scans. Results: Three pts in cohort 1 received 1 x 107 CAR+ T cells/kg safely. A fourth pt received the same dose with a modified vector with higher copy number. One pt had stable disease for > 6 months; a second pt has stable scans for > 20 months; the third and fourth patients progressed. Of 3 pts in cohort 2, one received 1.5 x 107 CAR+ T cells/kg and 2 received 3 x 107 CAR+ cell/kg. All 3 had intermittent fever spikes up to 39oC associated with increased levels of IL-4, IL-8, IP-10, sIL-2ra and IL-6 suggesting T cell activation. CAR+ cells persisted in the circulation for up to 2 weeks. Scans with 18F-FIAU labeling suggests that imaging may be cell dose dependent. Conclusions: We have shown that pts can be safely treated with an ex vivo transduction, expansion and therapeutic protocol for the generation of PSMA targeted T cells. Cytokine production suggests in vivo activation and persistence of T cells in blood for up to 2 weeks. Ongoing imaging with 18F may be suboptimal; a second cohort of pts will be studied with 124I-FIAU. Clinical trial information: NCTO1140373.

Chimeric antigen receptor (CAR+) modified T cells targeting prostate-specific membrane antigen (PSMA) in patients (pts) with castrate metastatic prostate cancer (CMPC).

2013 ◽  
Vol 31 (6_suppl) ◽  
pp. 72-72 ◽  
Author(s):  
Susan F. Slovin ◽  
Xiuyan Wang ◽  
Melanie Hullings ◽  
Gabrielle Arauz ◽  
Shirley Bartido ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Chimeric Antigen Receptor ◽  
Copy Number ◽  
Cell Activation ◽  
Ex Vivo ◽  
Antigen Receptor ◽  
Intermittent Fever ◽  
Herpes Simplex Virus 1

72 Background: A phase I dose-escalating study to assess safety, dose and targeting efficiency of genetically modified autologous human T cells targeted to PSMA was initiated. Preclinical models demonstrated anti-tumor activity and accumulation, migration, and persistence of these cells to tumor. The autologous PSMA-targeted T cells utilizes the P28z second generation chimeric antigen receptor following iv cyclophosphamide (Cy). For safety, the herpes simplex virus-1 thymidine kinase (hsvtk) gene is co-expressed with the P28z receptor, rendering T cells sensitive to ganciclovir for immediate T cell elimination. The expression of hsvtk enables PET imaging using radiolabeled FIAU to localize these T cells. Methods: Autologous T cells are activated from a leukapheresis product using anti-CD3/CD28 Dynabeads. Release criteria include mean vector copy number by Q-PCR and vector identity by Southern blot, absence of Replication Competent Retrovirus and residual Dynabeads. Pts were dosed from 107 to 3 x 107 CAR+ T cells/kg. All 7 pts received 300mg/m2 of Cy one day before infusion. Baseline and post treatment imaging included FDG, FDHT and 18F-FIAU PET scans. Results: Three pts in cohort 1 received 1 x 107 CAR+ T cells/kg safely. A fourth pt received the same dose with a modified vector with higher copy number. One pt had stable disease for > 6 months; a second pt has stable scans for > 16 months; the third and fourth patients progressed. Of 3 pts in cohort 2, one received 1.5 x 107 CAR+ T cells/kg and 2 received 3 x 107 CAR+ cell/kg. All 3 had intermittent fever spikes up to 39oC associated with increased levels of IL-4, IL-8, IP-10, sIL-2ra and IL-6 suggesting T cell activation. CAR+ cells persisted in the circulation for up to 2 weeks. Scans with 18F-FIAU labeling suggests that imaging may be cell dose dependent. Conclusions: We have shown that pts can be safetly treated with an ex vivo transduction, expansion and therapeutic protocol for the generation of PSMA targeted T cells. Cytokine production suggests activation of these T cells with their persistence in blood for up to 2 weeks. If imaging with FIAU is suboptimal, a second cohort of pts will be studied with 124I- FIAU. Clinical trial information: NCT01140373.

scholarly journals Optimizing Manufacturing Protocols of Chimeric Antigen Receptor T Cells for Improved Anticancer Immunotherapy

2019 ◽  
Vol 20 (24) ◽  
pp. 6223 ◽  
Author(s):  
Sophia Stock ◽  
Michael Schmitt ◽  
Leopold Sellner
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Chimeric Antigen Receptor ◽  
Cell Activation ◽  
Ex Vivo ◽  
Antigen Receptor ◽  
Tumor Control ◽  
Therapeutic Success ◽  
T Cell Therapy

Chimeric antigen receptor (CAR) T cell therapy can achieve outstanding response rates in heavily pretreated patients with hematological malignancies. However, relapses occur and they limit the efficacy of this promising treatment approach. The cellular composition and immunophenotype of the administered CART cells play a crucial role for therapeutic success. Less differentiated CART cells are associated with improved expansion, long-term in vivo persistence, and prolonged anti-tumor control. Furthermore, the ratio between CD4+ and CD8+ T cells has an effect on the anti-tumor activity of CART cells. The composition of the final cell product is not only influenced by the CART cell construct, but also by the culturing conditions during ex vivo T cell expansion. This includes different T cell activation strategies, cytokine supplementation, and specific pathway inhibition for the differentiation blockade. The optimal production process is not yet defined. In this review, we will discuss the use of different CART cell production strategies and the molecular background for the generation of improved CART cells in detail.

scholarly journals Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR)

2019 ◽  
Author(s):  
Rui Dong ◽  
Kendra A. Libby ◽  
Franziska Blaeschke ◽  
Alexander Marson ◽  
Ronald D. Vale ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Chimeric Antigen Receptor ◽  
Intracellular Signaling ◽  
Cell Activation ◽  
Antigen Receptor ◽  
Downstream Signaling ◽  
Car T Cells ◽  
Car T

AbstractThe chimeric antigen receptor (CAR) directs T cells to target and kill specific cancer cells. Despite the success of CAR T therapy in clinics, the intracellular signaling pathways that lead to CAR T cell activation remain unclear. Using CD19 CAR as a model, we report that, similar to the endogenous T cell receptor (TCR), antigen-engagement triggers the formation of CAR microclusters that transduce downstream signaling. However, CAR microclusters do not coalesce into a stable central supramolecular activation cluster (cSMAC). Moreover, LAT, an essential scaffold protein for TCR signaling, is not required for microcluster formation, immunological synapse formation, and actin remodeling following CAR activation. Meanwhile, CAR T cells still require LAT for the normal production of the cytokine IL-2. Together, these data show that CAR T cells can bypass LAT for a subset of downstream signaling outputs, thus revealing a rewired signaling pathway as compared to native T cells.

Influence of Retronectin-Mediated T-Cell Activation on Expansion and Phenotype of CD19-Specific Chimeric Antigen Receptor T Cells

2018 ◽  
Vol 29 (10) ◽  
pp. 1167-1182 ◽  
Author(s):  
Sophia Stock ◽  
Jean-Marc Hoffmann ◽  
Maria-Luisa Schubert ◽  
Lei Wang ◽  
Sanmei Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Chimeric Antigen Receptor ◽  
Cell Activation ◽  
Antigen Receptor

Tumor-Specific Chimeric Receptor-Mediated T Cell Activation Is Enhanced by 2B4 Signaling Via an NFκB-Independent Mechanism.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5199-5199
Author(s):  
Bianca Altvater ◽  
Sibylle Pscherer ◽  
Silke Landmeier ◽  
Heribert Juergens ◽  
Claudia Rossig
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Tumor Antigen ◽  
Cell Activation ◽  
Effector T Cells ◽  
Chimeric Receptor ◽  
Signaling Domain ◽  
Ifn Γ

Abstract Genetic modification of polyclonal T cells with tumor antigen-specific chimeric receptors (chRec) specifically redirects their effector functions towards tumor cells. However, the therapeutic value of chRec-gene-modified T cells is limited, since T cell activation by chRec fails to mediate proliferative responses, probably due to the lack of costimulatory T cell signaling in response to tumor cells. The costimulatory signaling domain of CD28 has been shown to enhance chimeric-receptor mediated proliferation in polyclonal T cell populations but not in CD8+ effector memory CTL, which represent the primary mediators of effective antitumor immunity. Indeed, alternative costimulatory molecules are now recognised as having complementary roles in the activation of antigen-experienced effector T cells. The SLAM-related receptor 2B4 (CD244), which is expressed on NK cells and on CD8+ cytotoxic T cells, is known to positively regulate T-cell-mediated cytotoxicity. We hypothesized that inclusion of the 2B4 endodomain into the chRec would enhance tumor-specific activation and proliferation of in vitro expanded effector T cells. We focused our studies on a GD2-specific scFv, which is currently being used in clinical trials for the treatment of neuroblastoma, and generated chRecs containing either the 2B4 signaling domain alone (14.G2a-2B4) or combined with TCR (14.G2a-2B4ζ). These chRecs were expressed in activated polyclonal human peripheral blood T cells using retroviral gene transfer. As controls, T cells were transduced with 14.G2aζ. High chRec surface expression was obtained for all three constructs, with 51±12% for 14.G2a-ζ, 70±15% for 14.G2a-2B4, and 53±7% for 14.G2a-2B4ζ. Immunphenotypes were dominated by a CD3+CD8+ population in all cell cultures. 51Cr-release assays showed efficient and comparable lysis of GD2+ tumor targets by T cells transduced with 14.G2a-ζ (44±10%) and 14.G2a-2B4ζ (44±3%) at an effector-to-target ratio of 40:1, whereas 2B4 alone failed to mediate specific tumor cell lysis. Intracellular cytokine secretion by chRec+ T cells was induced in response to GD2+ tumor targets by 14.G2a-ζ (mean 18.3%, range 12.1–29.7% IFN-γ+ CD3+ T cells) and 14.G2a-2B4ζ (mean 7.7%, range 6.0–11.3%). In contrast, 14.G2a-2B4 transduced T cells failed to induce IFN-γ secretion (mean 0.2%, range 0.06–0.47%). Weekly stimulations with tumor cells resulted in substantially superior expansion of T cells transduced with 14.G2a-2B4ζ (8–45fold) compared to T cells transduced with either 14.G2a-ζ (3.5–18fold) or 14.G2a-2B4 (2–4fold) over a 5 week period. It is known that T cell costimulation by CD28 receptor family members is mediated by downstream signaling involving the NFκB pathway. However in our studies, luciferase reporter gene assays failed to show a significant difference in tumor antigen-specific NFκB recruitment between T cells transduced with 14.G2aζ (240,000±5,000 RLU) and 14.G2a-2B4ζ (180,000±7,000 RLU). Although the nature of the signaling pathway mediating 2B4-induced T cell stimulation is still to be defined, our data support a costimulatory role for the activating NK cell receptor 2B4 in peripheral blood T cells. Therefore, 2B4-containing chRec may be a powerful new tool for adoptive immunotherapy of cancer.

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