104 BCMA-targeting CAR-T cells expanded in IL-15 have an improved phenotype for therapeutic use compared to those grown in IL-2 or IL-15/IL-7

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A115-A115
Author(s):  
Anthony Battram ◽  
Mireia Bachiller ◽  
Álvaro Urbano-Ispizua ◽  
Beatriz Martin-Antonio
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Cell Dysfunction ◽  
B Cell Maturation ◽  
Car T ◽  
T Cell Dysfunction

BackgroundChimeric antigen receptor-T (CAR-T) cells that target B cell maturation antigen (BCMA-CARs) have emerged as a promising treatment for multiple myeloma (MM). Despite impressive initial responses to BCMA-CAR therapy in clinical trials, relapse is common, signifying a need to improve the in vivo efficacy and persistence of BCMA-CARs.1 The development of unfavourable differentiation or T cell dysfunction, such as exhaustion and senescence, during the ex vivo expansion of the BCMA-CARs could be limiting their therapeutic potential. For CD19-directed CARs, reduced dysfunction and differentiation and improved anti-tumour responses were achieved by expanding the cells with IL-15 instead of IL-2.2 Therefore, in this study, our aim was to determine whether expanding BCMA-CARs with IL-15 or IL-15/IL-7 instead of IL-2 alters their levels of exhaustion, senescence, differentiation and activity.MethodsT cells stimulated with anti-CD3/anti-CD28-coated beads were supplemented with IL-2, IL-15, IL-15 + IL-7 or no cytokine and transduced with ARI2h, a BCMA-CAR with a 4-1BB co-stimulatory domain produced at our institution.3 Expanded BCMA-CARs were analysed by flow cytometry for markers of T cell dysfunction, or challenged with MM cell line ARP-1 and then tested for cytokine production, cytotoxic ability and activation signals.ResultsBCMA-CARs cultured in IL-15 or IL-15/IL-7 expanded similarly to those grown in IL-2, with comparable CAR transduction efficiencies, CD4:CD8 ratios and proliferation rates. BCMA-CARs grown in IL-15 had low expression of exhaustion marker LAG-3 and high expression of the costimulatory molecule CD27, which is important for T cell survival and persistence, when compared to BCMA-CARs cultured in IL-2. Moreover, BCMA-CARs grown solely in IL-15 were less differentiated than those supplemented with IL-7, and had higher expression of stem cell memory marker CXCR3 within the naïve population than those expanded with IL-2. When challenged with MM cell line ARP-1, IL-15-grown BCMA-CARs upregulated activation marker CD69, exhibited strong cytotoxicity and robust production of IFNγ and IL-2. However, in comparison to BCMA-CARs expanded in IL-2 or IL-15/IL-7, those grown with IL-15 had lower mTORC1 activity and p38 MAPK phosphorylation when activated by ARP-1 cells, suggesting differential regulation of key pathways for T cell metabolism and senescence, respectively.ConclusionsTo summarise, BCMA-CARs expanded with IL-15 alone exhibited the most favourable phenotype for therapeutic use compared those grown with IL-2 or IL-15/IL-7. Future experiments using murine MM models will be critical in understanding the in vivo benefits or drawbacks of culturing BCMA-CARs in IL-15 compared to IL-2 or IL-15/IL-7.Ethics ApprovalResearch involving human material was approved by the Ethical Committee of Clinical Research (Hospital Clinic, Barcelona). Peripheral blood T cells were obtained from healthy donors after informed consent in accordance with the Declaration of Helsinki.ReferencesRoex G, Feys T, Beguin Y, Kerre T, Poiré X, Lewalle P, et al. Chimeric Antigen Receptor-T-Cell Therapy for B-Cell Hematological Malignancies: An Update of the Pivotal Clinical Trial Data. Pharmaceutics [Internet]. 2020;12:1–15. Available from: http://www.ncbi.nlm.nih.gov/pubmed/32102267Alizadeh D, Wong RA, Yang X, Wang D, Pecoraro JR, Kuo CF, et al. IL15 enhances CAR-T cell antitumor activity by reducing mTORC1 activity and preserving their stem cell memory phenotype. Cancer Immunol Res 2019;7:759–72.Perez-Amill L, Suñe G, Antoñana-Vildosola A, Castella M, Najjar A, Bonet J, et al. Preclinical development of a humanized chimeric antigen receptor against B cell maturation antigen for multiple myeloma. Haematologica [Internet]. 2020; Available from: http://www.ncbi.nlm.nih.gov/pubmed/31919085

scholarly journals Murine pre–B-cell ALL induces T-cell dysfunction not fully reversed by introduction of a chimeric antigen receptor

Blood ◽  
2018 ◽  
Vol 132 (18) ◽  
pp. 1899-1910 ◽  
Author(s):  
Haiying Qin ◽  
Kazusa Ishii ◽  
Sang Nguyen ◽  
Paul P. Su ◽  
Chad R. Burk ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Receptor ◽  
Chimeric Antigen Receptor ◽  
Cell Receptor ◽  
Cell Dysfunction ◽  
Key Points ◽  
T Cell Dysfunction

Key Points Pre–B-cell ALL induces T-cell dysfunction in vivo, mediated in part by a non–T-cell receptor–linked mechanism. Prior exposure of T cells to pre–B-cell ALL in vivo impairs subsequent functionality of CAR-expressing T cells.

CD19-Redirected Chimeric Antigen Receptor T (CART19) Cells Induce a Cytokine Release Syndrome (CRS) and Induction of Treatable Macrophage Activation Syndrome (MAS) That Can Be Managed by the IL-6 Antagonist Tocilizumab (toc).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2604-2604 ◽  
Author(s):  
Stephan A. Grupp ◽  
David L Porter ◽  
David T Teachey ◽  
David M. Barrett ◽  
Anne Chew ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Tumor Lysis Syndrome ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T ◽  
Anti Tumor Activity

Abstract Abstract 2604 We previously reported on CART19 cells expressing a chimeric antigen receptor (CAR) with intracellular activation and costimulatory domains. Infusion of these cells results in 100 to 100,000× in vivo proliferation, tumor lysis syndrome followed by durable antitumor activity, and prolonged persistence in pts with B cell tumors. Here we report that in vivo proliferation of CART19 cells and potent anti-tumor activity is associated with CRS, leading to hemophagocytic lymphohistiocytosis (HLH), also termed MAS. We propose that MAS/HLH is a unique biomarker that is associated with and may be required for potent anti-tumor activity. Autologous T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3-zeta, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into ALL or CLL pts with persistent disease after 2–8 prior treatments. CART19 anti ALL activity was also modeled in a xenograft mouse model with high level of human ALL/human T cell engraftment and simultaneous detection of CAR T cells and ALL using 2-color bioluminescent imaging. We describe updated results of 10 pts who received CART19 cells elsewhere at ASH (Porter, et al), including 9 pts with CLL and 1 pediatric pt with relapsed refractory ALL. 6/9 evaluable pts had a CR or PR, including 4 sustained CRs. While there was no acute infusional toxicity, all responding pts also developed CRS. All had high fevers, as well as grade 3 or 4 hypotension/hypoxia. CRS preceded peak blood expression of CART19 cells, and then increased in intensity until the CART19 cell peak (D10–31 after infusion). The ALL pt experienced the most significant toxicity, with grade 4 hypotension and respiratory failure. Steroid therapy on D6 resulted in no improvement. On D9, noting high levels of TNFa and IL-6 (peak increases above baseline: IFNg at 6040x; IL-6 at 988x; IL-2R at 56x, IL-2 at 163× and TNFa at 17x), we administered TNFa and IL-6 antagonists entanercept and toc. This resulted in resolution of fever and hypotension within 12hr and a rapid wean from ventilator support to room air. These interventions had no apparent impact on CART19 cell expansion or efficacy: peak of CAR T cells (2539 CAR+ cells/uL; 77% of CD3 cells by flow) occurred on D11, and D23 bone marrow showed CR with negative MRD, compared to her initial on-study marrow which showed 65% blasts. Although she had no history of CNS ALL, spinal fluid showed detectable CART19 cells (21 lymphs/mcL; 78% CAR+). At 4mo post infusion, this pt remains in CR, with 17 CART19 cells/uL in the blood and 31% CAR+ CD3 cells in the marrow. Clinical assessment of subsequent responding patients shows all had evidence of MAS/HLH including dramatic elevations of ferritin and histologic evidence of HLH. Peak ferritin levels range from 44,000 to 605,000, preceding and continuing with peak T cell proliferation. Other consistent findings include rapid onset hepatosplenomegaly unrelated to disease and moderate DIC. Subsequently, 3 CLL patients have also been treated with toc, also with prompt and striking resolution of high fevers, hypotension and hypoxia. 1 received toc on D10 and achieved a CR accompanied by CART19 expansion. 1 had rapid resolution of CRS following toc administration on day 9 and follow up for response is too short. A 3rd CLL pt received toc on D3 for early fevers and had no CART-19 proliferation and no response. To model the timing of cytokine blockade, xenografts using bioluminescent primary pediatric ALL were established and then treated with extra cells from the clinical manufacture. The CART19 cells proliferated and resulted in prolonged survival. Cytokine blockade prior to T cell infusion with toc and/or etanercept abrogated disease control with less in vivo proliferation of infused CART19 cells, confirming the result seen in the one pt given early toc (D3). The optimal time and threshold to trigger cytokine blockade is currently being tested in these models. CART19 T cells can produce massive in-vivo expansion, long-term persistence, and anti-tumor efficacy, but can also induce significant CRS with features suggestive of MAS/HLH that responds rapidly to cytokine blockade. Given prior to initiation of significant CART19 proliferation, blockade of TNFa and/or IL-6 may interfere with proliferation and effector function, but if given at a point where cell proliferation is underway, toc may ameliorate the symptoms that we have observed correlate with robust clinical responses. Disclosures: Off Label Use: tocilizumab for cell therapy toxicity. Levine:University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties; TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kalos:University of Pennsylvania: Patents & Royalties. June:Novartis: Research Funding, institution owned patents have been licensed by Novartis, institution owned patents have been licensed by Novartis Patents & Royalties.

scholarly journals The roles of T cell competition and stochastic extinction events in chimeric antigen receptor T cell therapy

2021 ◽  
Vol 288 (1947) ◽  
Author(s):  
Gregory J. Kimmel ◽  
Frederick L. Locke ◽  
Philipp M. Altrock
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chimeric antigen receptor (CAR) T cell therapy is a remarkably effective immunotherapy that relies on in vivo expansion of engineered CAR T cells, after lymphodepletion (LD) by chemotherapy. The quantitative laws underlying this expansion and subsequent tumour eradication remain unknown. We develop a mathematical model of T cell–tumour cell interactions and demonstrate that expansion can be explained by immune reconstitution dynamics after LD and competition among T cells. CAR T cells rapidly grow and engage tumour cells but experience an emerging growth rate disadvantage compared to normal T cells. Since tumour eradication is deterministically unstable in our model, we define cure as a stochastic event, which, even when likely, can occur at variable times. However, we show that variability in timing is largely determined by patient variability. While cure events impacted by these fluctuations occur early and are narrowly distributed, progression events occur late and are more widely distributed in time. We parameterized our model using population-level CAR T cell and tumour data over time and compare our predictions with progression-free survival rates. We find that therapy could be improved by optimizing the tumour-killing rate and the CAR T cells' ability to adapt, as quantified by their carrying capacity. Our tumour extinction model can be leveraged to examine why therapy works in some patients but not others, and to better understand the interplay of deterministic and stochastic effects on outcomes. For example, our model implies that LD before a second CAR T injection is necessary.

scholarly journals p16INK4a Regulates Cellular Senescence in PD-1-Expressing Human T Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Valérie Janelle ◽  
Mathieu Neault ◽  
Marie-Ève Lebel ◽  
Dave Maurice De Sousa ◽  
Salix Boulet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cellular Senescence ◽  
Ex Vivo ◽  
Cell Dysfunction ◽  
Human T Cells ◽  
Car T ◽  
P38mapk Signaling ◽  
T Cell Dysfunction

T-cell dysfunction arising upon repeated antigen exposure prevents effective immunity and immunotherapy. Using various clinically and physiologically relevant systems, we show that a prominent feature of PD-1-expressing exhausted T cells is the development of cellular senescence features both in vivo and ex vivo. This is associated with p16INK4a expression and an impaired cell cycle G1 to S-phase transition in repeatedly stimulated T cells. We show that these T cells accumulate DNA damage and activate the p38MAPK signaling pathway, which preferentially leads to p16INK4a upregulation. However, in highly dysfunctional T cells, p38MAPK inhibition does not restore functionality despite attenuating senescence features. In contrast, p16INK4a targeting can improve T-cell functionality in exhausted CAR T cells. Collectively, this work provides insights into the development of T-cell dysfunction and identifies T-cell senescence as a potential target in immunotherapy.

The Effects of Co-Stimulatory Endodomains on the Fate of T Cells Expressing a Tumor Directed Chimeric Antigen Receptor (CAR) In Human Subjects with B Cell Malignancies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3949-3949
Author(s):  
Barbara Savoldo ◽  
Carlos A. Ramos ◽  
Catherine M. Bollard ◽  
Enli Liu ◽  
Martha Mims ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Chimeric Antigen Receptor ◽  
Human Subjects ◽  
Antigen Receptor ◽  
B Cell Malignancies ◽  
Molecular Signals ◽  
Level 2

Abstract Abstract 3949 B cell lymphomas have been effectively treated by immunotherapy, including monoclonal antibodies (MAbs) and adoptive T cell transfer. To extend this success, investigators have genetically modified T cells to express a B cell specific antibody incorporated in an artificial chimeric antigen receptor (CAR), essentially combining antibody and cell-based approaches. Early B cell directed CARs combined the antigen binding domains of the variable regions of a CD19 or CD20 MAb (scFv) with the CD3ζ endodomain of the TCR/CD3 complex. Although such CARs confer potent cytotoxic function to T cells, initial clinical trials showed that T cells modified to express CARs engineered in this way had limited in vivo persistence, apparently receiving insufficient costimulation following CAR engagement. To overcome the above limitations, a multiplicity of costimulatory endodomains, including CD28, 41BB or OX40, have been incorporated into the CAR molecule. Because of patient heterogeneity, it has proved difficult to draw definitive conclusions about the relative expansion, persistence and effectiveness of cells with each modification, so that their comparative value in human subjects remains speculative. We therefore designed a phase I, dose escalation clinical trial in which patients with refractory/relapsed B cell malignancies were simultaneously infused with two autologous T cell products. Both express a CAR with an identical CD19-specific exodomain, but one CAR also has a CD28-ζ endodomain while the other expresses only a ζ endodomain. With this study design, each patient acts as a “self-control”, allowing us to directly discover the consequences of CD28 costimulation for the fate of the T cells in vivo. We enrolled two patients at each of the three cell dose levels (1st level = 2×107/m2 of each product; 2nd level = 1×108/m2; 3rd level = 2×108/m2). End points of the study were safety, persistence of each of the two generations of CAR-modified T cells, and assessment of antitumor activity. T cell products were generated by activation of autologous PBMC with immobilized OKT3 and gene modified with retroviral vectors encoding either CAR.19ζ or CAR.19-28ζ. After transduction, T cells were expanded ex vivo for a median of 14 days (range 6–18) in the presence of IL-2. CAR expression was 42%±18% and 49%±16% for CAR.19ζ and CAR.19-28ζ, respectively. This corresponded to 51,246±16,795 and 18,283±9,484 transgene copy numbers/μg DNA, respectively, as measured by Q-PCR. Products contained both CD8+ cells (CAR.19ζ = 49%±22%, CAR.19-28ζ = 48%±22%). Few naïve T cells were present in either transduced population (CD45RA+ = 6%±5% and 6%±6%, respectively), and memory T cells predominated in both (CD45R0+CD62L+ = 50%±24% and 47%±66%, respectively). Both T cell components equally and specifically targeted CD19+ tumors in vitro as assessed by 51Cr release assays (specific lysis was 53%±10% for CAR.19ζ and 65%±19% for CAR.19-28ζ at a 20:1 E:T ratio). All infusions were well tolerated in all patients. Persistence of CAR+ T cell was assessed in peripheral blood by Q-PCR assays specific for CAR.19ζ and CAR.19-28ζ. Molecular signals for CAR.19-28ζ began at a low level after infusion, but progressively increased (7 to 63 fold) to peak at 1–2 weeks post infusion, before declining to background levels over the ensuing 8 to 13 weeks. By contrast, molecular signals corresponding to CAR.19ζ+ cells were barely detectable after infusion, showed no expansion, and rapidly disappeared. Currently 4 patients are evaluable for disease response; 2 had stable disease for up to 6 months and 2 had progressive disease. Hence, infusion of both CAR.19ζ and CD19-28ζ T cells has been safe at current doses. Direct comparison of each cell product in individual patients indicates that inclusion of the CD28 costimulatory endodomain (2nd generation CAR) enhances expansion and persistence. Nonetheless, both the limited expansion and persistence and the modest clinical effects suggest that additional modifications will need to be made to CAR endodomains to optimize the benefits of this therapy. We suggest our approach will allow these modifications to be evaluated systematically and directly even in small-scale clinical studies. Disclosures: Off Label Use: T cell products in studies conducted under INDs.

scholarly journals A CAR RNA FISH assay reveals functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue

2020 ◽  
Author(s):  
Karsten Eichholz ◽  
Alvason Zhenhua Li ◽  
Kurt Diem ◽  
Semih U. Tareen ◽  
Michael C. Jensen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T Cell ◽  
Rna Fish ◽  
Car T

AbstractChimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer and HIV sanctuary sites. Here, we describe a CAR RNA FISH histocytometry platform combined with a dnnRRS image analysis algorithm to quantitate spatial distribution and in vivo functional ability of a CAR T cell population at a single cell resolution. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions engineered cells in situ with their target cells in tissues with high statistical power and thus, as an important tool for preclinical and potentially clinical assessment of CAR T cell effectiveness.One Sentence SummaryWe developed a CAR RNA FISH assay to study chimeric antigen receptor T cell trafficking and function in human and mouse tissue.Impact statementWe developed an imaging platform and analysis pipeline to study large populations of engineered cells on a single cell level in situ.

scholarly journals Allogeneic CAR-T Cells: More than Ease of Access?

Cells ◽  
2018 ◽  
Vol 7 (10) ◽  
pp. 155 ◽  
Author(s):  
Charlotte Graham ◽  
Agnieszka Jozwik ◽  
Andrea Pepper ◽  
Reuben Benjamin
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Healthy Donor ◽  
B Cell Lymphoma ◽  
Cell Dysfunction ◽  
Car T Cells ◽  
Car T ◽  
T Cell Dysfunction ◽  
Ease Of Access

Patient derived anti-CD19 chimeric antigen receptor-T (CAR-T) cells are a powerful tool in achieving a complete remission in a range of B-cell malignancies, most notably B-acute lymphoblastic leukaemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL). However, there are limitations, including inability to manufacture CAR-T cells from the patient’s own T cells, disease progression and death prior to return of engineered cells. T cell dysfunction is known to occur in cancer patients, and several groups have recently described differences in CAR-T cells generated from chronic lymphocytic leukaemia (CLL) patients compared with those from a healthy donor. This is thought to contribute to the low response rate in this disease group. Healthy donor, gene-edited CAR-T cells which do not require human leucocyte antigen (HLA) matching have the potential to provide an ‘off the shelf’ product, overcoming the manufacturing difficulties of producing CAR-T cells for each individual patient. They may also provide a more functional, potent product for malignancies such as CLL, where T cell dysfunction is common and frequently cannot be fully reversed during the manufacturing process. Here we review the potential benefits and obstacles for healthy donor, allogeneic CAR-T cells.

scholarly journals High-Affinity Chimeric Antigen Receptor With Cross-Reactive scFv to Clinically Relevant EGFR Oncogenic Isoforms

2021 ◽  
Vol 11 ◽  
Author(s):  
Radhika Thokala ◽  
Zev A. Binder ◽  
Yibo Yin ◽  
Logan Zhang ◽  
Jiasi Vicky Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Tumor Escape ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Tumor heterogeneity is a key reason for therapeutic failure and tumor recurrence in glioblastoma (GBM). Our chimeric antigen receptor (CAR) T cell (2173 CAR T cells) clinical trial (NCT02209376) against epidermal growth factor receptor (EGFR) variant III (EGFRvIII) demonstrated successful trafficking of T cells across the blood–brain barrier into GBM active tumor sites. However, CAR T cell infiltration was associated only with a selective loss of EGFRvIII+ tumor, demonstrating little to no effect on EGFRvIII- tumor cells. Post-CAR T-treated tumor specimens showed continued presence of EGFR amplification and oncogenic EGFR extracellular domain (ECD) missense mutations, despite loss of EGFRvIII. To address tumor escape, we generated an EGFR-specific CAR by fusing monoclonal antibody (mAb) 806 to a 4-1BB co-stimulatory domain. The resulting construct was compared to 2173 CAR T cells in GBM, using in vitro and in vivo models. 806 CAR T cells specifically lysed tumor cells and secreted cytokines in response to amplified EGFR, EGFRvIII, and EGFR-ECD mutations in U87MG cells, GBM neurosphere-derived cell lines, and patient-derived GBM organoids. 806 CAR T cells did not lyse fetal brain astrocytes or primary keratinocytes to a significant degree. They also exhibited superior antitumor activity in vivo when compared to 2173 CAR T cells. The broad specificity of 806 CAR T cells to EGFR alterations gives us the potential to target multiple clones within a tumor and reduce opportunities for tumor escape via antigen loss.

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